Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi … › bitstream › 1807 › 71313 › ... · 2016-02-19 · balance, and promoted excess lipid storage, as well asbody-weight

Metabolic Reprogramming by Hexosamine Biosynthetic and

Golgi N-Glycan Branching Pathways

By

Michael Christopher Ryczko

A thesis submitted in conformity with the requirements

for the Degree of Doctor of Philosophy

Graduate Department of Molecular Genetics

University of Toronto

© Copyright by Michael Ryczko 2015




Doctor of Philosophy

Department of Molecular Genetics


2015

Abstract

Most mammalian growth factor receptors and solute transporters are co-translationally N-

glycosylated. N-glycans are branched and elongated in the Golgi, and their interaction with

lectins regulates cell surface residency and activity of transmembrane glycoproteins. The Golgi

branching N-acetylglucosaminyltransferases, Mgat1, 2, 4, 5 and 6, require a common donor

substrate uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) generated de novo by the

hexosamine biosynthetic pathway (HBP) from glucose, glutamine and acetyl-CoA, as well as

from GlcNAc salvage pathway. In this thesis I describe evidence for cell-autonomous regulation

of cellular metabolism by the Golgi N-glycan branching pathway. Induced expression of Mgat1,

Mgat5 or Mgat6, and GlcNAc supplementation to the HBP, increased central metabolites in an

additive manner. I show that UDP-GlcNAc levels are also sensitive to dietary GlcNAc

supplementation in vivo, increasing nutrient uptake and promoting anabolic metabolism via the

Golgi N-glycan branching pathway. Chronic oral GlcNAc supplementation in C57BL/6 mice

ii

increased hepatic UDP-GlcNAc and N-glycan branching on liver glycoproteins. Furthermore,

GlcNAc supplementation altered levels of numerous hepatic metabolites, insulin and glucagon

balance, and promoted excess lipid storage, as well as body-weight increase, without affecting

food intake or energy expenditure. In cultured cells, GlcNAc enhanced uptake of glucose,

glutamine and fatty acids, and elevated fatty acid synthesis and storage in an N-glycan-dependent

manner. Mgat5-/- mice exhibit a lean phenotype, and oral GlcNAc rescued fat accumulation,

consistent with functional redundancy of N-glycan branches. However, fat accumulation in this

context was rescued at the expense of lean mass, suggesting that Mgat5 plays a role in lean to fat

body composition. These results suggest that GlcNAc reprograms cellular metabolism by

enhancing nutrient uptake and lipid storage through the HBP and UDP-GlcNAc supply to the

Golgi N-glycan branching pathway.

iii

There are more things in heaven and earth, Horatio,

Than are dreamt of in our philosophy.

Hamlet Act 1, Scene 5

William Shakespeare

iv

Acknowledgments

First I would like to thank my supervisor Jim Dennis for his support and motivation

during completion of this thesis. Jim’s dedication and enthusiasm for science is remarkable.

I would like to thank all members of the Dennis and Swallow labs, both past and present,

who have helped me during my graduate odyssey. In particular, I would like to acknowledge

Judy Pawling for her moral support and excellent technical contributions. Special thanks also

goes to Ryan Williams, Anita Johswich, Wendy Johnston, Carol Swallow, and Roland Xu for

their friendship, moral support and advice during my graduate tenure.

Since science is a collaborative endeavor, I also want to thank my collaborators: Judy

Pawling, Anas M. Abdel Rahman, Rui Chan (from Daniel Figeys’ lab), Miyako Nakano (from

Naoyuki Taniguchi’s lab), Kevin Yau, Anita Johswich, Tania Rodrigues, Aldis Krizus and

Cunjie Zhang, who contributed to work presented in chapters 2 and 3.

I also want to thank Sean Egan and Anne-Claude Gingras for serving as my supervisory

committee members. I am grateful for your input and guidance throughout my Ph.D.

Last but not least, I would like to thank my beloved wife, Rubeena Khan, my dearest

mom, Wandziunia Ryczko, our little potlu Goldie, and the rest of my family for unconditional

love and understanding, unwavering support, and personal sacrifice. Thank you for always being

there for me. I am grateful for having you in my life and I would not have been able to reach this

stage without you.

v

Table of Contents

Abstract ............................................................................................................................... ii

Acknowledgments................................................................................................................v

Table of Contents ............................................................................................................... vi

List of Figures .................................................................................................................... xi

List of Tables .................................................................................................................... xii

List of Abbreviations ....................................................................................................... xiii

Chapter 1. Introduction.................................................................................................................1

1.1. Glycobiology............................................................................................................2

1.1.1. Glycans ........................................................................................................2

1.1.2. Glycans Synthesis and Function ..................................................................3

1.1.3. N-Glycosylation ...........................................................................................5

1.1.3.1. N-Glycan Branching and Glycan-Galectin Lattice .....................................7

1.1.3.2. Golgi N-Glycan Branching Pathway ..........................................................9

1.1.3.2.1. UDP-GlcNAc ...............................................................................................9

1.1.3.2.2. Mgat Branching Enzymes .........................................................................12

1.1.3.2.2.1. Mgat1 in N-Glycan Branching ..................................................................14



1.1.3.2.2.4. Mgat5 and β1,6-linked GlcNAc Branching ..............................................17


1.1.3.2.2.6. Functional Redundancy of N-Glycan Branches ........................................20

vi

1.1.4. HBP and UDP-GlcNAc Formation ...........................................................21

1.1.4.1. de novo UDP-GlcNAc Biosynthesis .........................................................21

1.1.4.2. Fate of UDP-GlcNAc ................................................................................22

1.1.4.3. Precursor Metabolites for UDP-GlcNAc Biosynthesis ..............................24

1.1.4.4. HBP and UDP-GlcNAc in Insulin Resistance and Diabetes .....................25

1.1.4.5. UDP-GlcNAc from Salvage Pathway ........................................................26

1.1.4.5.1. GlcNAc Supplementation in vitro and in vivo ...........................................26

1.1.4.5.1.1. GlcNAc Increases UDP-GlcNAc and β1,6-Branched N-Glycans .............27

1.1.4.5.1.2. GlcNAc Supplementation for N-Glycan Compensation ............................29

1.1.4.5.1.3. GlcNAc Safety and Efficacy ......................................................................30

1.1.4.5.2. Dietary Monosaccharide Supplementation as Therapy .............................31

1.2. Metabolism ........................................................................................................................32

1.2.1. Glycolysis and the Tricarboxylic Acid (TCA) Cycle ...........................................33

1.2.2. Insulin, Glucagon and Liver Function in Glucose Homeostasis ...........................34

1.2.2.1. Liver Glycogen .....................................................................................................35

1.2.3. Feeding and Fasting ..............................................................................................36

1.2.4. Glycoprotein Receptors for Glucagon and Insulin, and Glucose Transporters ....39

1.2.4.1. Glucagon Receptor (Gcgr) ....................................................................................39

1.2.4.2. Insulin Receptor (Insr) ..........................................................................................40

1.2.4.3. Glucose Transporters (Gluts)................................................................................40

1.2.5. Nutrient Sensing and Signaling Pathways ............................................................41

1.2.5.1. mTOR ...................................................................................................................41

1.2.5.2. AMPK ...................................................................................................................43

1.2.6. Fatty Acid Synthesis .............................................................................................44

vii

1.2.7. Lipid Storage and Breakdown ..............................................................................44

1.3. Body Weight and Obesity ..................................................................................................46

1.3.1. Body Weight Regulation .......................................................................................47

1.3.1.1. Leptin ....................................................................................................................48

1.3.2. Determinants of Body Weight and Obesity ..........................................................49

1.3.2.1. Energy Balance .....................................................................................................50

1.3.2.2. The Carbohydrate Hypothesis of Obesity ............................................................50

1.4. Rationale, Objectives and Summary ..................................................................................53

Chapter 2. Golgi N-Glycan Branching N-Acetylglucosaminyltransferases I, V and VI

Promote Nutrient Uptake and Metabolism ...............................................................................55

2.1. Summary ............................................................................................................................56

2.2. Introduction ........................................................................................................................57

2.3. Materials and Methods .......................................................................................................60

2.3.1. Materials and Chemicals ........................................................................................60

2.3.2. Cell Culture ............................................................................................................61

2.3.3. Western Blotting ....................................................................................................61

2.3.4. Enzyme Assays ......................................................................................................62

2.3.5. Quantitative Lectin Fluorescence Imaging ............................................................63

2.3.6. Cell Viability Assay ...............................................................................................63

2.3.7. Cell Membrane Preparation for N-Glycan Profiling .............................................64

2.3.8. Enzymatic Release and Purification of N-Glycans ................................................65

2.3.9. LC-ESI MS for Analysis of N-Glycan Alditols .....................................................66

2.3.10. Metabolite Analysis by LC-MS/MS ......................................................................67

2.3.11. LC-MS/MS Measurement of 15N15N-Glutamine Uptake and Metabolism ............70

2.4. Results ................................................................................................................................70

viii

2.4.1. Tet-Inducible Expression of Mgat1, Mgat5 and Mgat6 in Human Cells ..............70

2.4.2. Tet-Inducible Mgat1, Mgat5 and Mgat6 N-Glycan Branching ............................72

2.4.3. Regulation of Cellular Metabolite Levels by Mgat1, Mgat5, Mgat6 and HBP .....73

2.4.4. Tet-Inducible Mgat5 Enhances Amino Acid Uptake and Growth in Nutrient-Poor

Conditions ..............................................................................................................75

2.5. Discussion ..........................................................................................................................77

2.5.1. Mgat6 Enhances Functionality of N-Glycan Branching in Mammalian cells .......78

2.5.2. Mgat5 Enhances Metabolism Under Glutamine-Deprived Conditions .................79

Chapter 3. Metabolic Reprogramming by the Hexosamine Biosynthetic Pathway and Golgi

N-Glycan Branching ....................................................................................................................95

3.1. Summary ............................................................................................................................96

3.2. Introduction ........................................................................................................................97

3.3. Materials and Methods .....................................................................................................100

3.3.1. Chemicals and Materials ......................................................................................100

3.3.2. Mice .....................................................................................................................101

3.3.3. Phenotyping in vivo..............................................................................................102

3.3.4. Targeted Metabolomics .......................................................................................103

3.3.5. Biochemical Studies and Histology .....................................................................104

3.3.6. Site-Specific Characterization of Hepatic N-Glycosylation ................................105

3.3.7. Cell Culture ..........................................................................................................106

3.3.8. Statistical Analysis ...............................................................................................108

3.4. Results ..............................................................................................................................109

3.4.1. Oral GlcNAc Supplementation Alters Liver Metabolism ...................................110

3.4.2. Oral GlcNAc Increases Body-Weight Without Increasing Food Consumption ..111

3.4.3. Oral GlcNAc Increases Lipid Accumulation and Catabolism .............................112

ix

3.4.4. Oral GlcNAc Does Not Alter Physical Activity or Energy Expenditure .............113

3.4.5. Oral GlcNAc Reprograms Fasting Metabolism ...................................................114

3.4.6. Oral GlcNAc Increases Complex N-Glycan Branching in Liver Glycoproteins 116

3.4.7. GlcNAc Increases Nutrient Uptake and Lipid Accumulation in Cultured Cells .117

3.4.8. Oral GlcNAc Partially Restores Anabolic Metabolism in Mgat5-/- Mice ............119

3.5. Discussion ........................................................................................................................120

Chapter 4. Discussion and Future Directions..........................................................................143

4.1. GlcNAc and N-Glycan Branching in Mgat5 Null Mice ................................................144

4.2. HBP and N-Glycan Branching Reprogram Metabolism to Promote Fat Accumulation 145

4.3. Consequences of Long-Term Daily Oral GlcNAc Intake ...............................................147

4.4. GlcNAc Salvage into HBP..............................................................................................148

4.5. GlcNAc Supply and Increased N-Glycan Branching Promote Fat Accumulation .........149

4.6. Increased HBP Activity Promotes Fat Accumulation ....................................................151

4.7. HBP and the Thrifty Genotype/Phenotype Hypothesis ..................................................154

4.8. N-Glycan Branching and Nutrient Uptake .....................................................................155

4.9. HBP Interacts with Gene Polymorphisms in N-Glycan Branching Pathway .................156

4.10. GlcNAc Supplementation and O-GlcNAcylation ...........................................................160

4.11. GlcNAc Supplementation and Gut Microbiota...............................................................162

References ...................................................................................................................................165

x

List of Figures

Figure 1.1 Mgats and Their N-Glycan Branches ..........................................................8

Figure 1.2 Mgat5 in N-Glycan Branching .....................................................................8

Figure 1.3 Glycan-Galectin Lattice Dynamics ............................................................10

Figure 1.4 N-Glycan Branching Pathway ...................................................................11

Figure 1.5 Hexosamine Pathway for Biosynthesis of UDP-GlcNAc ..........................23

Figure 1.6 Glucose Production in Liver During Fasting .............................................38

Figure 2.1 Branching pathway and inducible expression of branching enzymes .......81

Figure 2.2 N-glycan profiles of transgenic Flp-In-TREx HeLa cells ..........................82

Figure 2.3 N-glycan profiles of transgenic Flp-In-TREx Hek293 cells by LC-ESI MS

....................................................................................................................84

Figure 2.4 Metabolite levels are sensitive to tet-induced branching and HBP

stimulation..................................................................................................86

Figure 2.5 Growth of Mgat5 Flp-In-TREx Hek293 cells in defined Glc and Gln

conditions ...................................................................................................88

Figure 2.6 Amino acid levels increase with Mgat5 expression under Gln-deprived

conditions ...................................................................................................89

Figure 2.7 Tet-induced Mgat5 increases TCA cycle intermediates ............................91

Figure 2.8 Tet-induced Mgat5 increases HBP and glycolysis metabolites under

Gln/Glc limiting conditions .......................................................................92

Figure 2.9 Summary of metabolite changes with tet-induced Mgat5 in Hek293 cells

under low Gln/Glc conditions ....................................................................93

Figure 2.10 Tet-induced Mgat5 branching stimulates Gln uptake ................................94

Figure 3.1 Oral GlcNAc is rapidly absorbed by gut to enter bloodstream and be taken

up by tissue from circulation ...................................................................128

xi

Figure 3.2 Oral GlcNAc increases UDP-GlcNAc level and promotes weight-gain in

mice ..........................................................................................................129

Figure 3.3 Oral GlcNAc promotes weight-gain and lipid accumulation ..................131

Figure 3.4 Oral GlcNAc promotes fatty acid oxidation without affecting activity or

energy expenditure ...................................................................................133

Figure 3.5 Oral GlcNAc alters fasting liver metabolism ...........................................134

Figure 3.6 Oral GlcNAc increases tri-antennary N-glycan structures on glycosite Asn

89 of CEACAM1 hepatic transmembrane glycoprotein ..........................135

Figure 3.7 GlcNAc increases UDP-GlcNAc, β-1,6-GlcNAc branched N-glycans and

lipid accumulation ....................................................................................137

Figure 3.8 HBP and N-glycan dependent regulation of cellular metabolism ...........139

Figure 3.9 Oral GlcNAc promotes lipid storage in male and female Mgat5 wild-type

and null mice ............................................................................................141

List of Tables

Table 3.1 Phenotypic differences in serum biochemistry between GlcNAc treated

and untreated mice on 9% fat diet, under fasted and fed conditions .......126

Table 3.2 Global analysis of relative GlcNAc content in liver N-glycans ..............127

xii

List of Abbreviations

3&2-PG 3- and 2-Phosphoglycerate

ACC Acetyl-CoA Carboxylase

A-CoA Acetyl-CoA

Akt Protein Kinase B

ALT Alanine Aminotransferase

AMPK AMP-Activated Protein Kinase

ANOVA Analysis of Variance

Asn Asparagine

ATP Adenosine Triphosphate

AUC Area Under the Curve

BMI Body-Mass Index

CDG Congenital Disorders of Glycosylation

CEACAM1 Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1

ConA Concanavalin A

DEXA Dual-Energy X-Ray Absorptiometry

DHAP Dihydroxyacetone Phosphate

DNA Deoxyribonucleic Acid

EE Energy Expenditure

EGFR Epidermal Growth Factor Receptor

ER Endoplasmic Reticulum

FA Fatty Acid

FASN Fatty Acid Synthase

FFA Free Fatty Acid

Fru-1,6BP Fructose-1,6-Bisphosphate

Fru-6P Fructose-6-Phosphate

Fuc Fucose

GAG Glycosaminoglycans

Gal Galactose

GALE UDP-Galactose-4-Epimerase

xiii

GalNAc N-acetylgalactosamine

Gcgr Glucagon Receptor

GFAT Glutamine-Fructose 6-Phosphate Amidotransferase

Glc Glucose

Glc-6P Glucose-6-Phosphate

GlcNAc N-acetylglucosamine

GlcNAc-P N-acetylglucosamine-Phosphate

Gln Glutamine

Glu Glutamate

Glut Glucose Transporter

GNE UDP-GlcNAc-2-Epimerase/N-Acetylmannosamine Kinase

GNPNAT1 Glucosamine-6-Phosphate Acetyltransferase

GS Glycogen Synthase

GSH Glutathione (reduced)

GSSG Glutathione (oxidized)

HBP Hexosamine Biosynthetic Pathway

I/G Insulin to Glucagon Ratio

Insr Insulin Receptor

IL-3 Interleukin 3

Iso/Leu Isoleucine/Leucine

LC-MS/MS Liquid Chromatography–Tandem Mass Spectrometry

L-PHA Lectin Phaseolus Vulgaris Leukoagglutinatin

m/z Mass to Charge Ratio

Man Mannose

ManNAc N-Acetylmannosamine

MFI Mean Fluorescent Intensity

Mgat Mannosyl α-1,6-Glycoprotein N-acetylglucosaminyltransferase

MMTV Murine Mammary Tumor Virus

MRI Magnetic Resonance Imaging

MS Mass Spectrometry

mTORC1 Mammalian target of rapamycin complex 1

xiv


NAD+ Nicotinamide Adenine Dinucleotide (oxidized)

NADH Nicotinamide Adenine Dinucleotide (reduced)

NADP+ Nicotinamide Adenine Dinucleotide Phosphate (oxidized)

NAGK N-Acetylglucosamine Kinase

NeuAc N-Acetylneuraminic Acid

N-X-S/T Asparagine-X-Serine/Threonine sequon

OAA Oxaloacetate

p Phosphate

PAGE Polyacrylamide Gel Electrophoresis

PC Principle Component

PCA Principal Component Analysis

PEP Phosphophenolpyruvic Acid

PGM3 Phosphoacetyl Glucosamine Mutase

Phe Phenylalanine

PI3K Phosphoinositide-3 Kinase

Pro Proline

Pten Phosphatase and Tensin Homolog

PyMT Polyoma Virus Middle T Oncoprotein

Ras Rat sarcoma oncogene

RER Respiratory Exchange Ratio

rER Rough Endoplasmic Reticulum

S6 Ribosomal Protein S6

SEM Standard Error of the Mean

Ser Serine

Sia Sialic Acid

Slc Solute Carrier Protein

SNP Single Nucleotide Polymorphism

SREBP Sterol regulatory element-binding protein

SW Swainsonine

TCA cycle Tricarboxylic Acid Cycle

xv

Tet Tetracycline

TG Triglycerides

TGF-β Transforming Growth Factor Beta

Thr Threonine

Trp Tryptophan

Tyr Tyrosine

UAP1 UDP-N-Acetylglucosamine Pyrophosphorylase

UDP Uridine-diphosphate

UDP-GalNAc Uridine-Diphosphate N-Acetylgalactosamine

UDP-Glc Uridine-Diphosphate Glucose

UDP-GlcNAc Uridine-Diphosphate N-Acetylglucosamine

UMP Uridine Monophosphate

UTP Uridine Triphosphate

VCO2 Volume of Carbon Dioxide

VO2 Volume of Oxygen

wt Wild-Type

xvi



By


A thesis submitted in conformity with the requirements

for the Degree of Doctor of Philosophy

Graduate Department of Molecular Genetics


© Copyright by Michael Ryczko 2015




Doctor of Philosophy

Department of Molecular Genetics


2015

Abstract

Most mammalian growth factor receptors and solute transporters are co-translationally N-

glycosylated. N-glycans are branched and elongated in the Golgi, and their interaction with

lectins regulates cell surface residency and activity of transmembrane glycoproteins. The Golgi

branching N-acetylglucosaminyltransferases, Mgat1, 2, 4, 5 and 6, require a common donor

substrate uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) generated de novo by the

hexosamine biosynthetic pathway (HBP) from glucose, glutamine and acetyl-CoA, as well as

from GlcNAc salvage pathway. In this thesis I describe evidence for cell-autonomous regulation

of cellular metabolism by the Golgi N-glycan branching pathway. Induced expression of Mgat1,

Mgat5 or Mgat6, and GlcNAc supplementation to the HBP, increased central metabolites in an

additive manner. I show that UDP-GlcNAc levels are also sensitive to dietary GlcNAc

supplementation in vivo, increasing nutrient uptake and promoting anabolic metabolism via the

Golgi N-glycan branching pathway. Chronic oral GlcNAc supplementation in C57BL/6 mice

ii

increased hepatic UDP-GlcNAc and N-glycan branching on liver glycoproteins. Furthermore,

GlcNAc supplementation altered levels of numerous hepatic metabolites, insulin and glucagon

balance, and promoted excess lipid storage, as well as body-weight increase, without affecting

food intake or energy expenditure. In cultured cells, GlcNAc enhanced uptake of glucose,

glutamine and fatty acids, and elevated fatty acid synthesis and storage in an N-glycan-dependent

manner. Mgat5-/- mice exhibit a lean phenotype, and oral GlcNAc rescued fat accumulation,

consistent with functional redundancy of N-glycan branches. However, fat accumulation in this

context was rescued at the expense of lean mass, suggesting that Mgat5 plays a role in lean to fat

body composition. These results suggest that GlcNAc reprograms cellular metabolism by

enhancing nutrient uptake and lipid storage through the HBP and UDP-GlcNAc supply to the

Golgi N-glycan branching pathway.

iii

Acknowledgments

First I would like to thank my supervisor Jim Dennis for his support and motivation

during completion of this thesis. Jim’s dedication and enthusiasm for science is remarkable.

I would like to thank all members of the Dennis and Swallow labs, both past and present,

who have helped me during my graduate odyssey. I would like to particularly acknowledge Judy

Pawling for her scientific contributions and moral support. Special thanks also goes to Ryan

Williams, Anita Johswich, Wendy Johnston, Carol Swallow, and Roland Xu for their friendship,

moral support and advice during my graduate tenure.

Since science is a collaborative endeavor, I also want to thank my scientific collaborators:

Judy Pawling, Anas Abdel Rahman, Rui Chan (from Daniel Figeys’ lab), Miyako Nakano (from

Naoyuki Taniguchi’s lab), Kevin Yau, Anita Johswich, Tania Rodrigues, Aldis Krizus and

Cunjie Zhang, who contributed to work presented in chapters 2 and 3.

I also want to thank Sean Egan and Anne-Claude Gingras for serving as my supervisory

committee members. I am grateful for your comments, input and guidance throughout my Ph.D.

Last but not least, I would like to thank my beloved wife, Rubeena Khan, my dearest

mom, Wandziunia Ryczko, Goldie little potlu, and the rest of my family for unconditional love

and understanding, unwavering support, and personal sacrifice. Thank you for always being

there for me. I am grateful for having you in my life and I would not have been able to reach this

stage without you.

iv

Table of Contents

Abstract ............................................................................................................................... ii

Acknowledgments.............................................................................................................. iv

Table of Contents .................................................................................................................v

List of Figures ......................................................................................................................x

List of Tables ..................................................................................................................... xi

List of Abbreviations ........................................................................................................ xii

Chapter 1. Introduction.................................................................................................................1

1.1. Glycobiology............................................................................................................2

1.1.1. Glycans ........................................................................................................2

1.1.2. Glycans Synthesis and Function ..................................................................3

1.1.3. N-Glycosylation ...........................................................................................5

1.1.3.1. N-Glycan Branching and Glycan-Galectin Lattice .....................................7

1.1.3.2. Golgi N-Glycan Branching Pathway ..........................................................9

1.1.3.2.1. UDP-GlcNAc ...............................................................................................9

1.1.3.2.2. Mgat Branching Enzymes .........................................................................12




1.1.3.2.2.4. Mgat5 and β1,6-linked GlcNAc Branching ..............................................17


1.1.3.2.2.6. Functional Redundancy of N-Glycan Branches ........................................20

v

1.1.4. HBP and UDP-GlcNAc Formation ...........................................................21

1.1.4.1. de novo UDP-GlcNAc Biosynthesis .........................................................21

1.1.4.2. Fate of UDP-GlcNAc ................................................................................22

1.1.4.3. Precursor Metabolites for UDP-GlcNAc Biosynthesis ..............................24

1.1.4.4. HBP and UDP-GlcNAc in Insulin Resistance and Diabetes .....................25

1.1.4.5. UDP-GlcNAc from Salvage Pathway ........................................................26

1.1.4.5.1. GlcNAc Supplementation in vitro and in vivo ...........................................26

1.1.4.5.1.1. GlcNAc Increases UDP-GlcNAc and β1,6-Branched N-Glycans .............27

1.1.4.5.1.2. GlcNAc Supplementation for N-Glycan Compensation ............................29

1.1.4.5.1.3. GlcNAc Safety and Efficacy ......................................................................30

1.1.4.5.2. Dietary Monosaccharide Supplementation as Therapy .............................31

1.2. Metabolism ........................................................................................................................32

1.2.1. Glycolysis and the Tricarboxylic Acid (TCA) Cycle ...........................................33

1.2.2. Insulin, Glucagon and Liver Function in Glucose Homeostasis ...........................34

1.2.2.1. Liver Glycogen .....................................................................................................35

1.2.3. Feeding and Fasting ..............................................................................................36

1.2.4. Glycoprotein Receptors for Glucagon and Insulin, and Glucose Transporters ....39

1.2.4.1. Glucagon Receptor (Gcgr) ....................................................................................39

1.2.4.2. Insulin Receptor (Insr) ..........................................................................................40

1.2.4.3. Glucose Transporters (Gluts)................................................................................40

1.2.5. Nutrient Sensing and Signaling Pathways ............................................................41

1.2.5.1. mTOR ...................................................................................................................41

1.2.5.2. AMPK ...................................................................................................................43

1.2.6. Fatty Acid Synthesis .............................................................................................44

vi

1.2.7. Lipid Storage and Breakdown ..............................................................................44

1.3. Body Weight and Obesity ..................................................................................................46

1.3.1. Body Weight Regulation .......................................................................................47

1.3.1.1. Leptin ....................................................................................................................48

1.3.2. Determinants of Body Weight and Obesity ..........................................................49

1.3.2.1. Energy Balance .....................................................................................................50

1.3.2.2. The Carbohydrate Hypothesis of Obesity ............................................................50

1.4. Rationale, Objectives and Summary ..................................................................................53

Chapter 2. Golgi N-Glycan Branching N-Acetylglucosaminyltransferases I, V and VI

Promote Nutrient Uptake and Metabolism ...............................................................................55

2.1. Summary ............................................................................................................................56

2.2. Introduction ........................................................................................................................57

2.3. Materials and Methods .......................................................................................................60

2.3.1. Materials and Chemicals ........................................................................................60

2.3.2. Cell Culture ............................................................................................................61

2.3.3. Western Blotting ....................................................................................................61

2.3.4. Enzyme Assays ......................................................................................................62

2.3.5. Quantitative Lectin Fluorescence Imaging ............................................................63

2.3.6. Cell Viability Assay ...............................................................................................63

2.3.7. Cell Membrane Preparation for N-Glycan Profiling .............................................64

2.3.8. Enzymatic Release and Purification of N-Glycans ................................................65

2.3.9. LC-ESI MS for Analysis of N-Glycan Alditols .....................................................66

2.3.10. Metabolite Analysis by LC-MS/MS ......................................................................67

2.3.11. LC-MS/MS Measurement of 15N15N-Glutamine Uptake and Metabolism ............70

2.4. Results ................................................................................................................................70

vii

2.4.1. Tet-Inducible Expression of Mgat1, Mgat5 and Mgat6 in Human Cells ..............70

2.4.2. Tet-Inducible Mgat1, Mgat5 and Mgat6 N-Glycan Branching ............................72

2.4.3. Regulation of Cellular Metabolite Levels by Mgat1, Mgat5, Mgat6 and HBP .....73

2.4.4. Tet-Inducible Mgat5 Enhances Amino Acid Uptake and Growth in Nutrient-Poor

Conditions ..............................................................................................................75

2.5. Discussion ..........................................................................................................................77

2.5.1. Mgat6 Enhances Functionality of N-Glycan Branching in Mammalian cells .......78

2.5.2. Mgat5 Enhances Metabolism Under Glutamine-Deprived Conditions .................79

Chapter 3. Metabolic Reprogramming by the Hexosamine Biosynthetic Pathway and Golgi

N-Glycan Branching ....................................................................................................................95

3.1. Summary ............................................................................................................................96

3.2. Introduction ........................................................................................................................97

3.3. Materials and Methods .....................................................................................................100

3.3.1. Chemicals and Materials ......................................................................................100

3.3.2. Mice .....................................................................................................................101

3.3.3. Phenotyping in vivo..............................................................................................102

3.3.4. Targeted Metabolomics .......................................................................................103

3.3.5. Biochemical Studies and Histology .....................................................................104

3.3.6. Site-Specific Characterization of Hepatic N-Glycosylation ................................105

3.3.7. Cell Culture ..........................................................................................................106

3.3.8. Statistical Analysis ...............................................................................................108

3.4. Results ..............................................................................................................................109

3.4.1. Oral GlcNAc Supplementation Alters Liver Metabolism ...................................110

3.4.2. Oral GlcNAc Increases Body-Weight Without Increasing Food Consumption ..111

3.4.3. Oral GlcNAc Increases Lipid Accumulation and Catabolism .............................112

viii

3.4.4. Oral GlcNAc Does Not Alter Physical Activity or Energy Expenditure .............113

3.4.5. Oral GlcNAc Reprograms Fasting Metabolism ...................................................114

3.4.6. Oral GlcNAc Increases Complex N-Glycan Branching in Liver Glycoproteins 116

3.4.7. GlcNAc Increases Nutrient Uptake and Lipid Accumulation in Cultured Cells .117

3.4.8. Oral GlcNAc Partially Restores Anabolic Metabolism in Mgat5-/- Mice ............119

3.5. Discussion ........................................................................................................................120

Chapter 4. Discussion and Future Directions..........................................................................143

4.1. GlcNAc and N-Glycan Branching in Mgat5 Null Mice ................................................144

4.2. HBP and N-Glycan Branching Reprogram Metabolism to Promote Fat Accumulation 145

4.3. Consequences of Long-Term Daily Oral GlcNAc Intake ...............................................147

4.4. GlcNAc Salvage into HBP..............................................................................................148

4.5. GlcNAc Supply and Increased N-Glycan Branching Promote Fat Accumulation .........149

4.6. Increased HBP Activity Promotes Fat Accumulation ....................................................151

4.7. HBP and the Thrifty Genotype/Phenotype Hypothesis ..................................................154

4.8. N-Glycan Branching and Nutrient Uptake .....................................................................155

4.9. HBP Interacts with Gene Polymorphisms in N-Glycan Branching Pathway .................156

4.10. GlcNAc Supplementation and O-GlcNAcylation ...........................................................160

4.11. GlcNAc Supplementation and Gut Microbiota...............................................................162

References ...................................................................................................................................165

ix

List of Figures

Figure 1.1 Mgats and Their N-Glycan Branches ..........................................................8

Figure 1.2 Mgat5 in N-Glycan Branching .....................................................................8

Figure 1.3 Glycan-Galectin Lattice Dynamics ............................................................10

Figure 1.4 N-Glycan Branching Pathway ...................................................................11

Figure 1.5 Hexosamine Pathway for Biosynthesis of UDP-GlcNAc ..........................23

Figure 1.6 Glucose Production in Liver During Fasting .............................................38

Figure 2.1 Branching pathway and inducible expression of branching enzymes .......81

Figure 2.2 N-glycan profiles of transgenic Flp-In-TREx HeLa cells ..........................82

Figure 2.3 N-glycan profiles of transgenic Flp-In-TREx Hek293 cells by LC-ESI MS

....................................................................................................................84

Figure 2.4 Metabolite levels are sensitive to tet-induced branching and HBP

stimulation..................................................................................................86

Figure 2.5 Growth of Mgat5 Flp-In-TREx Hek293 cells in defined Glc and Gln

conditions ...................................................................................................88

Figure 2.6 Amino acid levels increase with Mgat5 expression under Gln-deprived

conditions ...................................................................................................89

Figure 2.7 Tet-induced Mgat5 increases TCA cycle intermediates ............................91

Figure 2.8 Tet-induced Mgat5 increases HBP and glycolysis metabolites under

Gln/Glc limiting conditions .......................................................................92

Figure 2.9 Summary of metabolite changes with tet-induced Mgat5 in Hek293 cells

under low Gln/Glc conditions ....................................................................93

Figure 2.10 Tet-induced Mgat5 branching stimulates Gln uptake ................................94

Figure 3.1 Oral GlcNAc is rapidly absorbed by gut to enter bloodstream and be taken

up by tissue from circulation ...................................................................128

x

Figure 3.2 Oral GlcNAc increases UDP-GlcNAc level and promotes weight-gain in

mice ..........................................................................................................129

Figure 3.3 Oral GlcNAc promotes weight-gain and lipid accumulation ..................131

Figure 3.4 Oral GlcNAc promotes fatty acid oxidation without affecting activity or

energy expenditure ...................................................................................133

Figure 3.5 Oral GlcNAc alters fasting liver metabolism ...........................................134

Figure 3.6 Oral GlcNAc increases tri-antennary N-glycan structures on glycosite Asn

89 of CEACAM1 hepatic transmembrane glycoprotein ..........................135

Figure 3.7 GlcNAc increases UDP-GlcNAc, β-1,6-GlcNAc branched N-glycans and

lipid accumulation ....................................................................................137

Figure 3.8 HBP and N-glycan dependent regulation of cellular metabolism ...........139

Figure 3.9 Oral GlcNAc promotes lipid storage in male and female Mgat5 wild-type

and null mice ............................................................................................141

List of Tables

Table 3.1 Phenotypic differences in serum biochemistry between GlcNAc treated

and untreated mice on 9% fat diet, under fasted and fed conditions .......126

Table 3.2 Global analysis of relative GlcNAc content in liver N-glycans ..............127

xi

List of Abbreviations

3&2-PG 3- and 2-Phosphoglycerate

ACC Acetyl-CoA Carboxylase

A-CoA Acetyl-CoA

Akt Protein Kinase B

ALT Alanine Aminotransferase

AMPK AMP-Activated Protein Kinase

ANOVA Analysis of Variance

Asn Asparagine

ATP Adenosine Triphosphate

AUC Area Under the Curve

BMI Body-Mass Index

CDG Congenital Disorders of Glycosylation

CEACAM1 Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1

ConA Concanavalin A

DEXA Dual-Energy X-Ray Absorptiometry

DHAP Dihydroxyacetone Phosphate

DNA Deoxyribonucleic Acid

EE Energy Expenditure

EGFR Epidermal Growth Factor Receptor

ER Endoplasmic Reticulum

FA Fatty Acid

FASN Fatty Acid Synthase

FFA Free Fatty Acid

Fru-1,6BP Fructose-1,6-Bisphosphate

Fru-6P Fructose-6-Phosphate

Fuc Fucose

GAG Glycosaminoglycans

Gal Galactose

GALE UDP-Galactose-4-Epimerase

xii

GalNAc N-acetylgalactosamine

Gcgr Glucagon Receptor

GFAT Glutamine-Fructose 6-Phosphate Amidotransferase

Glc Glucose

Glc-6P Glucose-6-Phosphate

GlcNAc N-acetylglucosamine

GlcNAc-P N-acetylglucosamine-Phosphate

Gln Glutamine

Glu Glutamate

Glut Glucose Transporter

GNE UDP-GlcNAc-2-Epimerase/N-Acetylmannosamine Kinase

GNPNAT1 Glucosamine-6-Phosphate Acetyltransferase

GS Glycogen Synthase

GSH Glutathione (reduced)

GSSG Glutathione (oxidized)

HBP Hexosamine Biosynthetic Pathway

I/G Insulin to Glucagon Ratio

Insr Insulin Receptor

IL-3 Interleukin 3

Iso/Leu Isoleucine/Leucine

LC-MS/MS Liquid Chromatography–Tandem Mass Spectrometry

L-PHA Lectin Phaseolus Vulgaris Leukoagglutinatin

m/z Mass to Charge Ratio

Man Mannose

ManNAc N-Acetylmannosamine

MFI Mean Fluorescent Intensity

Mgat Mannosyl α-1,6-Glycoprotein N-acetylglucosaminyltransferase

MMTV Murine Mammary Tumor Virus

MRI Magnetic Resonance Imaging

MS Mass Spectrometry


xiii


NAD+ Nicotinamide Adenine Dinucleotide (oxidized)

NADH Nicotinamide Adenine Dinucleotide (reduced)

NADP+ Nicotinamide Adenine Dinucleotide Phosphate (oxidized)

NAGK N-Acetylglucosamine Kinase

NeuAc N-Acetylneuraminic Acid

N-X-S/T Asparagine-X-Serine/Threonine sequon

OAA Oxaloacetate

p Phosphate

PAGE Polyacrylamide Gel Electrophoresis

PC Principle Component

PCA Principal Component Analysis

PEP Phosphophenolpyruvic Acid

PGM3 Phosphoacetyl Glucosamine Mutase

Phe Phenylalanine

PI3K Phosphoinositide-3 Kinase

Pro Proline

Pten Phosphatase and Tensin Homolog

PyMT Polyoma Virus Middle T Oncoprotein

Ras Rat sarcoma oncogene

RER Respiratory Exchange Ratio

rER Rough Endoplasmic Reticulum

S6 Ribosomal Protein S6

SEM Standard Error of the Mean

Ser Serine

Sia Sialic Acid

Slc Solute Carrier Protein

SNP Single Nucleotide Polymorphism

SREBP Sterol regulatory element-binding protein

SW Swainsonine

TCA cycle Tricarboxylic Acid Cycle

xiv

Tet Tetracycline

TG Triglycerides

TGF-β Transforming Growth Factor Beta

Thr Threonine

Trp Tryptophan

Tyr Tyrosine

UAP1 UDP-N-Acetylglucosamine Pyrophosphorylase

UDP Uridine-diphosphate

UDP-GalNAc Uridine-Diphosphate N-Acetylgalactosamine

UDP-Glc Uridine-Diphosphate Glucose

UDP-GlcNAc Uridine-Diphosphate N-Acetylglucosamine

UMP Uridine Monophosphate

UTP Uridine Triphosphate

VCO2 Volume of Carbon Dioxide

VO2 Volume of Oxygen

wt Wild-Type

xv

Chapter 1.

Introduction

1

1.1 Glycobiology

1.1.1 Glycans

Along with nucleic acids, proteins and lipids, glycans are one of the four major

macromolecules in all living organism (Marth, 2008). Biological systems and their complexity

emerge from the layered functional interaction of the genome, proteome, lipidome, glycome, and

their biosynthesis from small-molecule metabolites. Glycans are oligosaccharides or

polysaccharides consisting of a large number of different monosaccharides ligated via a glycosidic

bond, and comprising the carbohydrate portion of glycoconjugates such as glycoproteins,

glycolipids and proteoglycans (Varki, 1993). The potential structural diversity of glycans in

mammalian cells is very large, due to the possible combination of monosaccharide constituents,

anomeric bond configurations, different linkages formed, and extent of branching (Ohtsubo &

Marth, 2006). Approximately 5% of the genome encodes proteins involved in glycan biosynthesis,

degradation and recognition (2009). Glycosylation is the enzyme-catalyzed covalent attachment

of glycans to a polypeptide or other organic compound, typically catalyzed by glycosyltransferases

utilizing specific activated sugar-nucleotide donor substrates.

The vast majority of cell surface proteins are glycosylated. Indeed, the surface of

eukaryotic cells is covered with a dense coating of a myriad sugar chains or glycans covalently

attached to either membrane proteins or lipids, giving rise to the glycocalyx, a complex network

of glycoconjugates (Varki, 1993). Glycan synthesis depends on monosaccharides converted from

other types of sugars inside the cells, salvaged from degraded glycoconjugates, or transported into

the cell from extracellular environment (Du et al, 2009). The most abundant monosaccharides

found in glycans are glucose (Glc), galactose (Gal), mannose (Man), N-acetylglucosamine

(GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia), and fucose (Fuc) (Ohtsubo &

2

Marth, 2006). The extent of glycan biosynthesis is controlled by enzyme concentrations, as well

as the availability of precursor metabolites and activated donor substrates (Du et al, 2009).

Consequently, glycan biosynthesis is very sensitive to the nutrient status of a cell.

1.1.2 Glycan Synthesis and Function

Unlike the biosynthesis of nucleic acids or proteins, glycan formation is not a deterministic

template-dependent process, but rather a stochastic event resulting from the cooperative and

competitive interaction of enzymes, transporters and substrates available (Lauc et al, 2010). Since

glycans are generated through combinatorial action of different glycosidases and

glycosyltransferases in the endoplasmic reticulum (ER) and the Golgi apparatus, their final

structure depends on the level of enzyme expression and activity, acceptor accessibility, and

substrate availability (Schachter, 1986). Thus, glycan biosynthesis is dynamically and reciprocally

linked to both an organism’s genome and its environment, which affect directly the enzymatic

processes, or indirectly the induction of epigenetic changes that modify gene expression. This

makes glycan biosynthesis sensitive to environmental conditions. Moreover, unlike the linearity

of DNA and protein, glycan branching, length and diversity of secondary modifications of

monosaccharides confers on them an extra degree of variation and unsurpassed structural

complexity (Ohtsubo & Marth, 2006). The broad repertoire of biological functions of glycans can

be categorized as having intramolecular and intermolecular functions. Intermolecular functions

include regulation of processes dependent on carbohydrate binding proteins, regulation of cell-to-

cell contact and cell-matrix adhesion, and even interaction with pathogenic molecules (Ohtsubo &

Marth, 2006). Intramolecular functions can influence the properties of glycoproteins such as their

activity, biological half-life, thermal stability, and protease sensitivity (Ohtsubo & Marth, 2006).

3

Additional intramolecular functions of glycans include regulation and monitoring of folding in the

ER, and maintenance of three-dimensional conformation (Ohtsubo & Marth, 2006).

Glycan complexity increases with vertebrate phylogeny, suggesting involvement in

multicellular development and morphogenesis, i.e. the flow and exchange of information required

to transform a collection of cells into a coherent society of different interacting components

(Ohtsubo & Marth, 2006). A large-scale N-glycoproteomic study revealed that more than 10% of

the mouse proteome is N-glycosylated (Zielinska et al, 2010). Intracellularly, glycans within the

secretory pathway regulate protein maturation, quality control, turnover, and trafficking of

molecules to organelles (Ohtsubo & Marth, 2006). The abundance, diversity and ubiquity of N-

glycan structures at the cell surface suggest a significant role for encoded information that is

distinct from the genome. Cell surface N-glycans on glycoproteins serve as ligands for a number

of evolutionarily conserved carbohydrate-binding protein families, such as C-type lectins,

galectins, and siglecs, whose function is to decipher biological information conveyed by the vast

array of N-glycans (Dennis et al, 2009). Functional interactions of galectins with cell surface

glycoconjugates can modulate cellular functions such as cell signaling and cell adhesion (Dennis

et al, 2009). Located at the interface of the inside and outside of the cell, N-glycans are perfectly

positioned to interact with the extracellular environment of the cell, and to mediate a variety of

recognition events and biological processes in health and disease through protein-glycan

interactions (Dennis et al, 2009). Changes in patters of cell surface N-glycans often accompany

development of cancer and metastasis. In fact, altered glycosylation, either increase or decrease of

specific N-glycans, or the appearance of glycans normally restricted to embryonic expression, is

frequently observed in tumours (Dennis et al, 2009). These structural alterations are often the result

of changes in expression and activity of glycosyltransferases, and their substrate availability.

4

1.1.3 N-Glycosylation

N-linked glycosylation occurs through co- and post-translational modification of

membrane and secreted glycoproteins in eukaryotic cells. N-glycans are attached to the nitrogen

atom of asparagine (Asn) residues in the peptide consensus sequon Asn-X-Ser/Thr, where X

corresponds to any amino acid except Pro (Dennis et al, 2009). The presence of a sequon is

necessary but not sufficient for N-glycosylation to occur, as some sequons are not glycosylated

(Zielinska et al, 2010). Thus, when Asn-X-Ser/Thr motifs are present in the amino acid sequence

of a protein they are not identified categorically as N-glycan sites, but rather are referred to as

potential N-glycan sites. The enzyme oligosaccharyltransferase initiates protein N-glycosylation

by transferring a pre-assembled sugar oligosaccharide from dolichol to a nascent protein. Factors

influencing oligosaccharyltransferase catalysis include availability of precursors, enzyme activity,

the number of sequons in a glycopeptide, and their conformational accessibility (Schachter, 1986).

N-glycosylation is catalyzed by a series of enzymes functioning in a sequential and

competitive manner in the rough ER (rER) and Golgi apparatus, where it assumes an assembly-

line style of manufacturing (Schachter, 2010). Different glycosyltransferases reside in different

compartments of the Golgi, where they act in a specific order during glycoprotein transit through

the secretory pathway (Dennis et al, 2009). Moreover, many glycosyltransferases are expressed in

a tissue and time specific manner (Ohtsubo & Marth, 2006). Glycosyltransferases are single-pass

type II integral membrane proteins with a short cytoplasmic amino terminal domain, a

transmembrane anchor domain, a Proline-rich neck or stem region, and carboxyl terminal catalytic

domain (Schachter, 2010). Glycosyltransferases use activated high-energy sugar-nucleotides as

donor substrates, attaching them to polypeptides or to the growing glycan chain (Ohtsubo & Marth,

5

2006). N-linked glycosylation is required for proper function of numerous glycoproteins. For

instance, cell surface delivery and retention of receptors and nutrient transporters depends on their

N-glycan branching, which is both under genetic and metabolic control (Dennis et al, 2009). The

extent of N-glycan branching is dependent on the expression and kinetics of medial Golgi enzymes

- the mannosidases and acetylglucosaminyltransferases, the metabolic flux through the

hexosamine biosynthetic pathway (HBP) to generate the high-energy sugar-nucleotide donor

uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc), as well as protein synthesis rate and

availability of glycoprotein acceptors (Dennis et al, 2009).

N-glycosylation of proteins starts with an en block transfer of pre-assembled lipid-carrier-

linked Glc3Man9GlcNAc2 donor to Asn residue in receptive Asn-X-Ser/Thr sequons of nascent

proteins by oligosaccharyltransferase in the lumen of rER (Schachter, 2010). The transferred pre-

assembled Asn-linked glycan is then extensively remodeled by removal and addition of various

monosaccharides during transition through the rER and Golgi apparatus en route to the cell surface

(Dennis et al, 2009). Remodeling, or lack thereof, results in formation of three main types of N-

linked glycans on mature glycoproteins: high-mannose, hybrid, and complex (Schachter, 2010).

All N-glycans contain a single conserved core structure consisting of two GlcNAc residues

(chitobiose core), and three Man residues (tri-mannosyl core), forming a common penta-saccharide

(Man3GlcNAc2) core structure (Schachter, 2010). This core is further linked to other sugars to

form a variety of different branched N-glycans. UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-

phosphate transferase (GNPTA/DPAGT) is the enzyme in the first committed step of the dolichol-

linked oligosaccharide pathway for N-glycan biosynthesis to utilize UDP-GlcNAc as a substrate

(Schachter, 2010). This initial step can be blocked by the drug tunicamycin, an analog of UDP-

GlcNAc that inhibits GNPTA (Schachter, 2010).

6

In metazoans, trimming is performed by glucosidases and mannosidases, while extension

is carried out by Golgi N-acetylglucosaminyltransferases (Mgats) (Dennis et al, 2009). The high-

mannose structures are formed from the oligosaccharide precursor by α-mannosidase-mediated

cleavage of Man residues, without addition of any other monosaccharides (Schachter, 2010).

Complex-type N-glycans are formed by successive removal and addition of monosaccharides, and

are characterized according to the number of GlcNAc branches attached to terminal Man residues

on the core penta-saccharide structure (Taniguchi & Korekane, 2011). Depending on the number

of branches attached, complex-type N-glycans are subdivided into bi-, tri-, tetra-, and penta-

antennary structures. The hybrid-type N-glycans share structural features found in both high-

mannose and complex-type N-glycans (Schachter, 2010).

1.1.3.1 N-Glycan Branching and Glycan-Galectin Lattice

Biosynthesis and branching of complex N-glycans proceeds via linkage of GlcNAc by

mannosyl glycoprotein N-acetylglucosaminyltransferase (Mgat) enzymes, also known as GlcNAc-

transferases, to the conserved core Man residues (Dennis et al, 2009). Each Mgat transfers GlcNAc

in a specific linkage (Taniguchi & Korekane, 2011) (Figure 1.1). GlcNAc branches in turn are

further extended through sequential addition of Gal and other monosaccharides to complete

elongation of glycan antennas that act as ligands for galectins (Dennis et al, 2009) (Figure 1.2).

Galectins are a family of secreted proteins. Most of them are bivalent or multivalent with regard

to their carbohydrate-binding activities (Dennis et al, 2009). Membrane glycoproteins carrying N-

glycan branches offer binding sites for galectins to facilitate transmembrane glycoprotein cross-

linking, thus forming a glycan-galectin lattice that regulates cell surface residency and activity of

numerous receptors, cell adhesion proteins, and solute transporters (Dennis et al, 2009)

7

Figure 1.1 Mgats and Their N-Glycan Branches

An idealized N-glycan showing different possible branches, with specific linkages and the glycosyltransferase enzymes (Mgat1, Mgat2, Mgat3, Mgat4, Mgat5 and Mgat6) responsible for their formation. Each GlcNAc branch may be elongated with galactose, poly-N-acetyllactosamine, sialic acid and fucose (Taniguchi & Korekane, 2011).

Figure 1.2 Mgat5 in N-Glycan Branching

The branched N-glycans attached at N-X-S/T sequons in mammalian glycoproteins. Mono, bi, tri, and tetra refer to the number of branches in an N-glycan. The glycosyltransferase Mgat5 catalyses the addition of a β1,6-linked GlcNAc branch (green arrows) to form tri- or tetra-antennary N-glycans, the most complex types of branched N-glycans in mammals. In general, the more GlcNAc branches per N-glycan, the more Gal residues are added and elongated to form poly-N-lactosamine [Galβ1,4GlcNAc]n. Galectins bind Gal and form specific cross-linked lattices with glycoproteins, which increases their cell surface expression (Stanley, 2007).

8

(Figure 1.3). Galectins are N-acetyllactosamine (Gal and GlcNAc) binding proteins whose major

ligands are Golgi-remodeled N-glycans common to cell surface glycoproteins (Dennis et al, 2009).

Poly-N-acetyllactosamine glycan structures serve as high affinity ligands for galectins, which bind

to N-glycans of glycoproteins with affinities proportional to GlcNAc content (Dennis et al, 2009).

Glycoproteins with N-acetyllactosamine glycans interact with galectins in a cross-linking manner,

which leads to their retention at the cell surface by slowing later mobility, and delaying loss by

constitutive endocytosis (Dennis et al, 2009). Increased cell surface residency in turn leads to

greater sensitivity to extracellular cues and promotes receptor mediated signaling (Johswich et al,

2014; Lau et al, 2007; Mendelsohn et al, 2007). Sustained surface exposure and clustering of

signaling receptors creates a platform to multiply ligand-induced signal intensity.

1.1.3.2 Golgi N-Glycan Branching Pathway

1.1.3.2.1 UDP-GlcNAc

UDP-GlcNAc is an essential common donor substrate required by all Mgat enzymes. UDP-

GlcNAc is synthesized in the cytosol by the HBP and transported through sugar-nucleotide

transporters into the medial Golgi apparatus (Dennis et al, 2009) (Figure 1.4). The Golgi N-glycan

branching pathway is characterized by multistep ultrasensitivity to UDP-GlcNAc for branching

enzymes Mgat1, Mgat2, Mgat4, and Mgat5 (Dennis et al, 2009). The relative affinity of branching

enzymes for UDP-GlcNAc declines sequentially by ~300 fold (0.04 to 10 mM) moving down the

N-glycan branching pathway from Mgat1 to Mgat5, while this trend is reversed for their respective

glycoprotein N-glycan acceptors (Dennis et al, 2009). Thus, activities of enzymes Mgat1 and

Mgat2 are limited by low affinity for acceptor glycoproteins, while activities of enzymes Mgat4

9

Figure 1.3 Glycan-Galectin Lattice Dynamics

The glycocalyx is the thick carbohydrate layer surrounding the cell. Glycan structures generated in the Golgi differ in affinities for galectins. Galectins cross-link glycoprotein receptors and oppose (1) loss of EGFR to Caveolin 1-positive microdomains, (2) coated-pit endocytosis, (3) precocious clustering of receptors, and (4) F-actin-mediated entry of T-cell receptor into and exit of CD45 from ganglioside GM1-positive microdomains (blue). (5) Nutrient supply and growth signaling increase membrane remodeling, regulate metabolite flux through the hexosamine biosynthetic pathway to UDP-GlcNAc and Golgi N-glycan branching on receptors and transporters to promote surface retention by the glycan-galectin lattice (Dennis et al, 2009).

10

Figure 1.4 N-Glycan Branching Pathway

Oligosaccharyltransterase (OST) utilizes the preassembled donor Glc3Man9GlcNAc2-pp-dolichol to transfer the glycan to N-X-S/T sequons on glycoproteins in the ER. In the secretory pathway, glycoproteins transit from the ER to cis, medial, and trans Golgi apparatus, en route to the cell surface. The N-acetylglucosaminyltransferases enzymes, designated by their gene names (Mgat1, Mgat2, Mgat4, and Mgat5) generate branched N-glycans that display a range of affinities for galectins. The Km values for Mgat1, Mgat2, Mgat4, and Mgat5 are indicated as measured in vitro for UDP-GlcNAc and acceptor glycoproteins. The Golgi UDP-GlcNAc antiporter exchanges uridine monophosphate (UMP) for UDP-GlcNAc and establishes the steady state amounts of UDP-GlcNAc inside the Golgi (Dennis et al, 2009).

11

and Mgat5 are limited by UDP-GlcNAc concentrations generated by the HBP. This implies that

initial branching by Mgat1 and 2 depends on the rate of protein synthesis, while N-glycan

branching by Mgat4 and 5 is determined mostly by UDP-GlcNAc availability. Indeed,

supplementation of extracellular GlcNAc has been shown to increase intracellular UDP-GlcNAc

levels, Mgat5-mediated N-glycan branching, and glycoprotein retention at the cells surface, with

increased sensitivity of cells to growth factors and cytokines (Johswich et al, 2014; Lau et al, 2007;

Mendelsohn et al, 2007).

1.1.3.2.2 Mgat Branching Enzymes

The Man5GlcNAc2 glycan is substrate for the first N-acetylglucosaminyltransferase, or

GlcNAc-transferase enzyme Mgat1 (GlcNAc-TI) (Schachter, 2010). Through transfer of GlcNAc

from UDP-GlcNAc in the medial Golgi, Mgat1 modifies the high-mannose structure to a hybrid

N-glycan. This is an essential step required for synthesis of either hybrid or complex-type glycans,

and cells deficient in Mgat1 can only generate high-mannose type structures (Schachter, 2010).

The product of Mgat1 can then be further stripped of remaining Man residues by α-mannosidase

II, rendering it a substrate for Mgat2 (GlcNAc-TII) (Schachter, 2010). The conversion of a mono-

antennary to a complex bi-antennary structure requires addition of GlcNAc by Mgat2. Specific

Mgat activity requires prior action of a distinct Mgat for catalysis to occur. One exception to this

is Mgat3, which catalyzes the transfer of GlcNAc residue to the core to form a bisected N-glycan.

However, due to the steric hindrance resulting from the presence of a bisecting GlcNAc, this

structure cannot be used as an acceptor by other Mgats, thus preventing further branching reactions

and formation of tri- and tetra-antennary N-glycans (Taniguchi & Korekane, 2011). Since Mgat3

12

inhibits further N-glycan branch formation it has been suggested to play a regulatory role in

biosynthesis of complex and hybrid-type N-glycans (Taniguchi & Korekane, 2011).

In instances where Mgat3 is not involved, additional N-glycan structures can be generated

by Mgat5 (GlcNAc-V) and Mgat4a/b/c (GlcNAc-IVa/b/c) isoenzymes (Taniguchi & Korekane,

2011). Therefore, the number of antennas formed on an N-glycan depends on expression and

dynamic action of different GlcNAc-transferases, the concentration of common donor UDP-

GlcNAc, and acceptor glycoprotein with its immature N-glycan moving through the medial-Golgi.

The abundance of these factors and execution of these processes dictates the ultimate N-glycan

structure produced. Indeed, this varies among species, tissues, cells, glycoproteins, and even varies

with respect to different glycosylation sites on the same glycoprotein. For instance, when protein

synthesis slows down UDP-GlcNAc is spared, providing greater opportunity for the late branching

enzymes Mgat4 and Mgat5 to act and increase branching. Indeed, low glucose media conditions

increase surface β1,6-GlcNAc-branched N-glycans in mouse embryonic fibroblasts (Cheung et al.

2007).

N-glycan products of Mgat1, 2, 4 and 5 can be extended further through sequential addition

of Gal, and terminal capping with Fuc and Sia (Dennis et al, 2009). N-linked glycans represent the

most complex and functionally diverse covalent modification characterized (Ohtsubo & Marth,

2006). N-glycosylation is an inherently noisy and variable process, with potential N-glycan sites

not always being occupied with glycans, and even those that are occupied often varying in their

structure. The structural variability of N-glycans has been described in terms of

microheterogeneity and macroheterogeneity. Microheterogeneity refers to the site-specific

composition, chain-length, and branching pattern variability that occurs at a specific glycosite

amongst different molecules of the same glycoprotein (Schachter, 1986). On the other hand,

13

macroheterogeneity results from variable Asn-X-Ser/Thr sequon usage, suggesting that local

three-dimensional conformation of the polypeptide, and the immediate microenvironment in the

vicinity of the potential glycosite, influences its accessibility to oligosaccharyltransferase

(Schachter, 1986).

1.1.3.2.2.1 Mgat1 in N-Glycan Branching

Although N-glycosylation is dispensable for survival of isolated cells in vitro, it is crucial

for proper functioning in vivo (Schachter, 2010). To elaborate on the N-glycan branching pathway

and impairments caused when its synthesis is perturbed at different points, I will review

phenotypes associated with mutations in genes coding for Mgat enzymes. Mgat1 is the first

branching enzyme to act in the Golgi N-glycan branching pathway, which then allows additional

branches to be added through action of other Mgats. The gene Mgat1 encodes for the enzyme

which starts the branching process by adding GlcNAc to the trimmed core producing the hybrid

N-glycan required for recognition by α-mannosidase II in the medial Golgi (Dennis et al, 2009).

Mgat1 activity is essential for the synthesis of complex-type N-glycans. Genetic ablation studies

in mice have proved informative concerning the structure-function relationship of Mgats.

Mgat1-dependent N-glycans are required for normal mammalian development, as

evidenced by systemic Mgat1 null mouse embryos dying in utero at around embryonic day 9.5,

and presenting underdevelopment including fewer somites, a tube-like heart, defective

vascularisation, and an open neural tube (Schachter, 2010). The phenotype would have been more

severe, and Mgat1 null mice would most likely die much earlier if it was not for maternally derived

Mgat1 gene transcripts, which rescue early embryos (Shi et al, 2004). This suggests that hybrid

14

and/or complex N-glycans might be required for blastocyst formation or implantation (Shi et al,

2004). Lack of Mgat1 affects N-glycan structures at the cell surface, with all hybrid and complex

N-glycans being replaced by Man5GlcNAc2 (Schachter, 2010). Since Mgat1-dependent N-

glycosylated glycoproteins on the cell surface are required for normal cell to cell interaction, as

well as cell surface residency and activity of growth factor receptors and nutrient transporters,

combined disturbance in these fundamental biological processes are most likely responsible for

the lethality of Mgat1 null mice.

Mgat1 loss of function mutation in rice Oryza sativa causes severe developmental defect

with early lethality due to reduced sensitivity to the plant growth hormones cytokinins (Fanata et

al, 2013). Deletion of Mgat1 in Drosophila melanogaster results in viable flies exhibiting defects

in locomotion, brain abnormalities, and severely shortened lifespan (Sarkar et al, 2010). This

phenotype is rescued by neuronal Mgat1 expression, which also increases lifespan in wild-type

flies (Sarkar et al, 2010). These results imply that neuronal glycoproteins dependent on Mgat1

modification play a role in control of fly lifespan by affecting global metabolic changes (Sarkar et

al, 2010). Knockdown of Mgat1 in prostate cancer cells reduces tumor progression both in terms

of tumor size and metastasis (Beheshti Zavareh et al, 2012). This is interesting, as a link between

cancer and metabolism has been rediscovered in recent years (Vander Heiden et al, 2009).


Mgat2 is required for synthesis of complex N-glycans, and is widely expressed in

mammalian cells and tissues (Wang et al, 2001). Mgat2 encodes the enzyme that transfers GlcNAc

onto the tri-mannosyl core. Homozygous deletion of Mgat2, which abolishes complex-type N-

15

glycans but retains hybrid branched structures, results in prenatal and perinatal death with few null

mice surviving to adulthood (Wang et al, 2001). These mice displayed postnatal phenotype similar

to that observed in human patients with congenital disorders of glycosylation IIa (CDG-IIa),

including failure to thrive, dysmorphic facial features, and poor psychomotor development (Wang

et al, 2001). Furthermore, Mgat2 null mice were runted in comparison to wild-type littermates,

had decreased blood glucose, showed gastrointestinal abnormalities, and exhibited reduced body-

weight at all developmental stages (Wang et al, 2001).


Bi-antennary N-glycans can be further modified through addition of GlcNAc onto the tri-

mannosyl core by Mgat4 isoenzymes (Taniguchi & Korekane, 2011). Interestingly, Mgat4b is

upregulated in the liver of fast-growing chickens, and is thought to promote fat deposition in this

context (Claire D'Andre et al, 2013). Mgat4a is expressed in most mouse and human tissues, but

its levels are much higher in pancreas and small intestine (Ohtsubo et al, 2011; Ohtsubo et al,

2005). In pancreatic β-cells, Mgat4a-dependent N-glycosylation is necessary for generating multi-

antennary N-glycans on the glucose transporter 2 (Glut2), which enables galectin-glycan binding

to maintain cell surface residency of Glut2 for proper glucose transport and sensing (Ohtsubo et

al, 2005). Indeed, Mgat4a null mice are hyperglycemic, displaying elevated free fatty acids and

triglycerides, with reduced insulin levels and impaired glucose-stimulated insulin secretion

(Ohtsubo et al, 2005). With aging, glucose intolerance in these mice progressed to metabolic

dysfunction, insulin resistance, and liver steatosis (Ohtsubo et al, 2005).

16

Furthermore, a high-fat diet strongly attenuated Mgat4a expression in pancreatic β-cells of

wild-type mice. This resulted in reduced branching of Glut2 N-glycans required for proper cell

surface retention, which in turn lead to impairment of insulin secretion, and eventually type 2

diabetes and hepatic steatosis (Ohtsubo et al, 2005). In pancreatic β-cells, Mgat4a expression is

transcriptionally regulated by FoxA2 and Hnf1-α, whose intracellular distribution is regulated by

cellular redox balance that might be affected by high-fat diet induced oxidative stress (Ohtsubo et

al, 2011). Protection from high-fat diet-induced metabolic disease was conferred by ectopic

Mgat4a constitutive expression in β-cell of transgenic mice, in which Glut2 glycosylation and its

cell surface residence was maintained (Ohtsubo et al, 2011). Mgat4a was also reduced in

enterocytes from human obese subjects, who exhibited endosomal Glut2 accumulation, most likely

resulting from altered N-glycan branching on Glut2 (Ait-Omar et al, 2011).

1.1.3.2.2.4 Mgat5 and β1,6-linked GlcNAc Branching

Mgat5 encodes for a medial-Golgi N-glycan branching enzyme responsible for catalyzing

addition of β1,6-linked GlcNAc to Man residue, thereby forming complex-type tri- or tetra-

antennary N-glycan structures on glycoproteins (Dennis et al, 2009). The red kidney bean

(Phaseolus vulgaris) lectin L-phytohemagglutinin (L-PHA) exhibits specific and selective

reactivity toward β1-6GlcNAc-branched N-glycans (Grigorian et al, 2009). Since Mgat5 has a

high Km value for UDP-GlcNAc, in comparison to enzymes Mgat1 or Mgat2, the intracellular

concentration of this substrate determines the amount of complex tri- and tetra-antennary N-

glycans found on glycoproteins (Dennis et al, 2009; Lau et al, 2007). Mgat5 products are the

preferred substrate for elongation with N-acetyllactosamine and poly-N-acetyllactosamine, which

is comprised of repeating units of GlcNAc and Gal of variable length, allowing for further

17

modification of the N-glycan chain by fucosylation and sialylation (Dennis et al, 2009; Grigorian

et al, 2009).

Mice deficient in Mgat5 lack tri- and tetra-antennary N-glycans. This results in altered

distribution or clustering of glycoproteins at the cell surface, due to reduced affinity for galectins,

and diminished signal transduction intensities from certain growth factor receptors and adhesion

proteins (Dennis et al, 2009; Grigorian et al, 2009). Mgat5 null mice are viable and appear similar

to wild-type littermates at birth. However, later on they display metabolic phenotypes such as

leaner body composition and smaller size, resistance to weight-gain on high-fat diet,

hypoglycemia, sensitivity to fasting in terms of exaggerated glycogen depletion and lipid

mobilization, as well as increased oxidative respiration by reliance on fatty acid oxidation (Cheung

et al, 2007). Glucose uptake is impaired in mouse embryonic fibroblasts derived from Mgat5 null

mice, especially under conditions of low-glucose or serum-free medium, suggesting that Mgat5-

modified N-glycans promote glucose uptake and anabolic metabolism (Cheung et al, 2007). Mgat5

null mice also display adult phenotypes that may be linked in part through metabolism, including

delayed oncogene-induced tumorigenesis, sensitivity to autoimmune disease, loss of adult stem

cells, and premature aging with accelerated loss of bone and muscle mass (Cheung et al, 2007;

Dennis et al, 2009; Grigorian et al, 2009).

The levels of β1,6-branch on N-linked glycans regulates the cell surface residency of

glycoproteins to affect their function (Dennis et al, 2009). Biological processes regulated by

Mgat5-dependent N-glycan modification include metabolic homeostasis, cell adhesion, motility

and migration, cytokine and growth factor signaling, and cellular proliferation and differentiation

(Dennis et al, 2009). Also, alterations in Mgat5 activity or function, leading to changes in N-glycan

branching pattern on glycoproteins, have been linked to cancer progression and invasive

18

metastastic dissemination. In different types of cancer, Mgat5 transcript levels and enzymatic

activity are upregulated, which in turn leads to increased β1,6-branched N-linked glycan structures

and poly-N-acetyllactosamine content on membrane glycoproteins (Dennis et al, 2009). Mgat5

transcript levels are regulated by a Ras-Raf-Mek-Erk-Ets oncogenic signaling pathway, and

typically show a three to five fold increase when this pathway is activated through transformation

(Taniguchi & Korekane, 2011). The phenotypic effects of overexpression of Mgat5 are decreased

cell–cell and cell–matrix adhesion, and promotion of motility and invasiveness (Taniguchi &

Korekane, 2011). Transgenic mice expressing the polyoma virus middle T (PyMT) oncoprotein

under control of the murine mammary tumor virus (MMTV) long terminal repeat develop

multifocal mammary epithelium carcinomas that metastasize to lungs. Mgat5 null mice

intercrossed with transgenic mice expressing PyMT show a considerable delay in mammary tumor

development, and a reduction in tumor growth and lung metastasis, compared to those observed

in MMTV-PyMT;Mgat5+/- or MMTV-PyMT;Mgat5+/+ mice (Dennis et al, 2009). Mgat5-/- adult

mice also showed suppression of tumor progression in HER2/neu-induced mammary oncogenesis

model, and Pten+/- lymphoma and carcinoma tumor model (Cheung & Dennis, 2007; Granovsky

et al, 2000; Guo et al, 2010). In these models, the effects of Mgat5 deletion were traced to

alterations in PI3K and ERK signaling pathways.


Mgat6 (GlcNAc-VI) catalyzes the formation of the most highly branched penta-antennary

complex-type N-glycan, the β1,4-linked branch (Sakamoto et al, 2000; Watanabe et al, 2006).

Prior action of Mgat5 is a prerequisite for Mgat6 activity (Brockhausen et al, 1989). Mgat6

enzymatic activity was found in avian tissues, such as in the hen oviduct, chicken liver, duck colon

19

and turkey intestine (Brockhausen et al, 1989). This activity has also been seen in fish ovaries

(Brockhausen et al, 1989). The gene coding for Mgat6 is expressed in various chicken (Gallus

gallus) tissues (Sakamoto et al, 2000). However, Mgat6 gene and its enzymatic activity have not

been detected in mammalian tissues (Brockhausen et al, 1989; Sakamoto et al, 2000).

1.1.3.2.2.6 Functional Redundancy of N-Glycan Branches

Genetic studies with Mgat knockout mice revealed that loss of an Mgat enzyme is more

severe the earlier that enzyme acts in the N-glycan branching pathway. In later stages of the

branching pathway a degree of redundancy between N-glycan structures generated by Mgat4 and

Mgat5 may offer compensation for functional defects (Dennis & Brewer, 2013). This is contingent

on substrate availability and spatiotemporal expression of genes coding for each enzyme (Dennis

& Brewer, 2013). Experiments with Mgat4a and Mgat4b single and double knockout mice show

induced glycomic biosynthetic compensation (Dennis & Brewer, 2013). This occurs through

compensatory induction of other Mgats to generate similar N-glycan epitopes, as means of

maintaining overall expression of N-glycan ligands on glycoproteins for cross-linking galectins at

the cell surface (Dennis & Brewer, 2013). Furthermore, functional redundancy or compensation

between N-acetyllactosamine branches in structurally related N-glycans can be promoted by UDP-

GlcNAc, the rate-limiting substrate in their biosynthesis in the Golgi N-glycan branching pathway

(Dennis & Brewer, 2013; Lau et al, 2007). The N-acetyllactosamine units, although repositioned

within N-glycans with GlcNAc treatment, have been shown to substitute and compensate

functionally by rescuing affinity for galectins' association with N-glycan branches at the cell

surface (Dennis & Brewer, 2013; Lau et al, 2007). In fact, GlcNAc supplementation in Mgat5-/-

cells doubled the tri-antennary N-glycan levels, and rescued levels of EGF and TGF-β cell surface

20

receptors and their signaling, suggesting that less-branched N-glycans in larger quantities are

sufficient to restore galectin binding (Dennis & Brewer, 2013; Lau et al, 2007).

1.1.4 HBP and UDP-GlcNAc Formation

The general route for incorporating monosaccharides into a glycan starts with their

conversion into activated sugar-nucleotides, which serve as donors for glycosylation reactions

(Ohtsubo & Marth, 2006). Activated sugar-nucleotides are synthesized by covalently linking a

monosaccharide to a nucleotide, typically nucleoside diphosphate, and in many cases one sugar-

nucleotide can be converted into another (Du et al, 2009). The de novo biosynthetic steps to

generate UDP-GlcNAc follow a variant of the Leloir pathway, elucidated biochemically in the

1950s (Marshall, 2006). However HBP, also called the hexosamine signaling pathway, was only

well delineated and formally described based on genetic and biochemical evidence in the early

1990s (Marshall, 2006). UDP-GlcNAc is the major product of the HBP, and is the primary

substrate in virtually all glycoprotein processing pathways, including Golgi N-glycan branching

pathway.

1.1.4.1 de novo UDP-GlcNAc Biosynthesis

HBP is one of several pathways that divert minor amounts of glucose from glycolysis or

glycogen storage (Buse, 2006; Hardiville & Hart, 2014). Fructose 6-phosphate (Fru-6P), the

obligatory intermediate in glycolysis, enters the HBP through conversion to glutamine-6-

phosphate (GlcN-6P) (Figure 1.5). The rate-limiting enzyme in HBP responsible for catalysis of

this reaction is glutamine-fructose 6-phosphate amidotransferase (GFAT or GFPT), which

21

transfers ammonia from glutamine to Fru-6P, and isomerizes the resulting fructosimine-6P to

GlcN-6P (Buse, 2006; Hardiville & Hart, 2014). UDP-GlcNAc inhibits GFAT allosterically,

thereby regulating its own synthesis through competitive feedback inhibition, and controlling the

amount of glucose entering HBP (Du et al, 2009; Love & Hanover, 2005). The next reaction in

UDP-GlcNAc synthesis involves transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA)

by GlcN-6P acetyltransferase (GNPNAT1 or GNA1), to obtain GlcNAc-6P (Hardiville & Hart,

2014). Isomerization of GlcNAc-6P to GlcNAc-1P is catalyzed by GlcNAc phosphomutase

(PGM3 or AGM1), which is autophosphorylated and dephosphorylated during the reaction cycle.

Finally, activation of monosaccharides as sugar-nucleotides is an essential step in the biosynthetic

pathway. Hence, uridylation of GlcNAc-1P by UDP-GlcNAc pyrophosphorylase (UAP1 or

AGX1) results in formation of the end-product UDP-GlcNAc, which is then transported into the

rER and Golgi apparatus and used as a donor for N-glycosylation by a number of GlcNAc-

transferases (Dennis et al, 2009).

1.1.4.2 Fate of UDP-GlcNAc

In the secretory pathway, UDP-GlcNAc serves as a basic building block for synthesis of

glycans on secreted and membrane-bound glycoproteins. UDP-GlcNAc is also used to make

glycolipids, cell surface O-glycans, glycosaminoglycan (GAG) chains used in proteoglycans of

the extracellular matrix, and hyaluronic acid (2009). In addition, some UDP-GlcNAc is epimerized

to generate UDP-N-acetylgalactosamine (UDP-GalNAc), by the enzyme UDP-galactose-4-

epimerase (GALE), or cleaved into UDP and ManNAc by UDP-GlcNAc-2-epimerase/N-

acetylmannosamine kinase (GNE), the bi-functional and rate-limiting enzyme of sialic acid

biosynthesis (Galeano et al, 2007). The same pool of UDP-GlcNAc generated by HBP also

22

Figure 1.5 Hexosamine Pathway for Biosynthesis of UDP-GlcNAc

An offshoot of the glycolytic pathway, the HBP integrates the metabolism of carbohydrates (glucose), amino acids (glutamine), fats (acetyl-CoA), and nucleotides (uridine-diphosphate) in the synthesis of UDP-GlcNAc. The enzymes and metabolites involved in the synthesis of UDP-GlcNAc are presented, with different precursors color-coded to denote their contribution to UDP-GlcNAc synthesis.

23

functions as the obligatory substrate for intracellular O-GlcNAcylation (Love & Hanover, 2005).

O-GlcNAcylation is a dynamic and reversible posttranslational modification where a single

GlcNAc moiety is attached to the hydroxyl group of serine or threonine residue in cytoplasmic and

nuclear proteins (Hardiville & Hart, 2014). Enzyme O-GlcNAc transferase (OGT) catalyzes the

addition, while O-GlcNAcase the removal of these single GlcNAc moieties (Hardiville & Hart,

2014).

1.1.4.3 Precursor Metabolites for UDP-GlcNAc Biosynthesis

The precursors for UDP-GlcNAc synthesis are nutrient derived. UDP-GlcNAc has been

suggested as an ideal sensor for the metabolic state of a cell, since its levels are sensitive to the

availability and fluctuations in concentrations of various nutrients and metabolites from different

metabolic pathways: glucose (glycolysis, gluconeogenesis, and glycogenolysis), acetyl-coA

(pyruvate or fatty acid oxidation), glutamine (nitrogen metabolism), and uridine-triphosphate

(energy charge and nucleotide metabolism) (Hardiville & Hart, 2014; Love & Hanover, 2005;

Marshall, 2006). Therefore, through its dependence on HBP, N-glycosylation is also dependent on

intermediary metabolism. Since metabolic pathways are deeply interconnected, their

interdependency explains why a perturbation or imbalance in one pathway often affects the

function in another, such as the HBP. For instance, in a calorie-matched experiment rats fed a high-

glucose diet for a week had higher UDP-GlcNAc liver content than rats fed a high-lard diet

(Tepperman et al, 1981). Sustained or chronic hyperglycemia promotes increased HBP flux and

ultimately results in elevated formation of UDP-GlcNAc (Buse, 2006). In cell culture, intracellular

UDP-GlcNAc levels and cell surface β1,6GlcNAc-branched N-glycans were found to be sensitive

24

to the external supply of metabolites Glc, Gln, ammonia, acetoacetate and uridine, as well as to

GlcNAc supplementation (Abdel Rahman et al, 2013; Grigorian et al, 2007; Wellen et al, 2010).

1.1.4.4 HBP and UDP-GlcNAc in Insulin Resistance and Diabetes

Several laboratories have proposed that flux through the HBP plays a role in development

of insulin resistance and other pathologies and complications associated with diabetes, primarily

through O-GlcNAcylation (Love & Hanover, 2005; McClain, 2002). Hyperglycemia, commonly

seen in diabetes mellitus, increases glucose flux through the HBP resulting in increased

biosynthesis of UDP-GlcNAc (Buse, 2006). Mice injected with a low-dose of streptozotocin and

fed a high-fat diet develop hyperglycemia, hyperinsulinemia and obesity, and are used as a model

for type 2 diabetes mellitus (Fricovsky et al, 2012). As the disease progresses, these mice show

increased GFAT levels and elevated concentration of UDP-GlcNAc in the diabetic heart tissue

(Fricovsky et al, 2012). Transgenic mouse models suggest that increased flux through the HBP

plays a role in development of insulin resistance and type 2 diabetes (Love & Hanover, 2005).

Overexpression of GFAT in muscle and fat leads to whole body insulin resistance, as does

overexpression of GFAT solely in pancreatic β-islet cells or in the liver (Love & Hanover, 2005).

The liver-targeted GFAT overexpression in transgenic mice results in obese animals displaying

glucose intolerance, hyperinsulinemia and hyperglycemia as the mice age (McClain, 2002). GFAT

activity and UDP-GlcNAc concentrations were also increased in insulin-resistant genetically obese

mice, while UDP-GlcNAc concentrations were reduced in rats with chronic calorie restriction and

enhanced insulin sensitivity (Buse, 2006).

25

1.1.4.5 UDP-GlcNAc from Salvage Pathway

In addition to de novo HBP synthesis of UDP-GlcNAc from nutritional sources and

precursors, the salvage amino sugar metabolism pathway is also capable of generating UDP-

GlcNAc from intracellularly recycled constituents (Dennis et al, 2009). Free cellular GlcNAc

becomes available either as a result of endogenous lysosomal degradation and catabolism of

glycoconjugates, by direct uptake, or through degradation of dietary glycoconjugates by

glycosidases (Dennis et al, 2009). Thus, UDP-GlcNAc can be obtained or derived from catabolic

and anabolic pathways. The cytosolic enzyme N-acetylglucosamine kinase (NAGK) catalyzes

phosphorylation of free cytoplasmic GlcNAc to GlcNAc-6P, which is then salvaged into HBP to

generate UDP-GlcNAc (Dennis et al, 2009). In cultured mammalian cells, increased intracellular

UDP-GlcNAc, via extracellular GlcNAc supplementation, enhances N-glycan biosynthesis and

branching, and improves association of glycoprotein receptors and transporters with galectins,

thereby increasing their cell surface retention, and sensitivity to extracellular cues (Dennis et al,

2009).

1.1.4.5.1 GlcNAc Supplementation in vitro and in vivo

One of the oldest and most abundant organic compounds on the planet, GlcNAc is a

monosaccharide primarily found in chitin of arthropods, insects and fungi, as well as bacterial

peptidoglycan (Chen et al, 2010; Koropatkin et al, 2012). GlcNAc is also a constituent of

heterogeneous polysaccharides such as hyaluronic acid and GAG, as well as being a major and

essential building block for various forms of glycosylation. GlcNAc is a commercially available

dietary or nutritional supplement used by people and pets all over the world, with numerous uses

26

and abuses attributed to it in modern pharmacopoeia (Chen et al, 2010). Unregulated and readily

available in health and wellness stores, GlcNAc is recommended by doctors, veterinarians, as well

as complementary and alternative medical practitioners. These recommendations, for numerous

conditions and claiming various health benefits, span the whole range from theoretically sound

and encouraging but clinically unproven, to those based on preliminary animal studies, to well-

intentioned but unfounded and speculative. GlcNAc supplements have also been promulgated by

unscrupulous charlatans in guise of glyconutrients (Schnaar & Freeze, 2008). Because of the latter,

research into GlcNAc and related monosaccharides is at times tainted and viewed with skepticism,

especially if the studies are sponsored by a company profiting from positive results associated with

the compound examined (Schnaar & Freeze, 2008). Nevertheless, numerous independent basic

science and clinical research studies utilizing GlcNAc have been conducted, yielding legitimate,

verifiable and interesting results.

1.1.4.5.1.1 GlcNAc Increases UDP-GlcNAc and β1,6-Branched N-Glycans

Defects arising from mutations in the early de novo HBP enzyme GNPNAT1 (GNA1) can

be partially reversed by restoring UDP-GlcNAc accumulation through GlcNAc or UDP-GlcNAc

supplementation in biological systems as diverse as mouse embryonic fibroblasts, Arabidopsis

thaliana and Caenorhabditis elegans (Boehmelt et al, 2000; Johnston et al, 2006; Nozaki et al,

2012). Numerous lines of evidence demonstrate that metabolic flux through HBP and availability

of UDP-GlcNAc regulate Golgi N-glycan branching enzyme activities to control cell surface level

and retention of transmembrane glycoproteins (Dennis et al, 2009; Grigorian et al, 2009; Johswich

et al, 2014; Lau et al, 2007; Mendelsohn et al, 2007; Sasai et al, 2002). In different mammalian

immortalized cells, as well as in primary T-cells and hepatocytes, increased flux through the HBP

27

via extracellular GlcNAc supplementation has been shown to increase intracellular UDP-GlcNAc

levels, glycoprotein β1,6-GlcNAc-branched N-glycans, and transmembrane glycoprotein cell

surface residency, by enhancing association with galectins to increase sensitivity to extracellular

factors (Dennis et al, 2009; Grigorian et al, 2009; Johswich et al, 2014; Lau et al, 2007;

Mendelsohn et al, 2007; Sasai et al, 2002). In addition, by increasing the degree of N-glycan

branching, GlcNAc supplementation also increased cell surface expression level of voltage-gated

potassium channels involved in modulating membrane excitability and action potential (Zhu et al,

2012). Furthermore, GlcNAc was reported to modulate the neuronal ionotropic glutamate receptor

function and ensuing electrophysiological activity by enhancing the receptor's N-glycan-galectin

interaction to alter neuronal excitability (Copits et al, 2014).

GlcNAc has been reported to enter the mammalian cell through passive diffusion or

pinocytosis, without deacetylation (Tesoriere et al, 1972). In cultured mammalian cells salvaged

GlcNAc does not appear to be converted to glucose and used as a fuel source for energy production

in glycolysis, but instead contributes almost exclusively to the UDP-GlcNAc pool for

glycoconjugate formation (Abdel Rahman et al, 2013; Chertov et al, 2011; Wellen et al, 2010).

Treatment of glucose-starved hematopoietic cells with GlcNAc, to maintain HBP flux, promoted

IL-3 surface expression and signaling in a manner dependent on N-glycan branching, which in

turn allowed glutamine uptake for energy production, lipid biosynthesis, cell growth, and uptake

of the amino acid leucine (Wellen et al, 2010). Like GlcNAc, UDP-GlcNAc was also shown to

promote nutrient uptake in cell culture. Treatment of mouse embryonic fibroblasts with UDP-

GlcNAc resulted in increased uptake of glucose, amino acids and uridine, concomitant with

GlcNAc incorporation into N-glycans of cell surface glycoproteins (Natraj & Datta, 1978).

28

1.1.4.5.1.2 GlcNAc Supplementation for N-Glycan Compensation

Mgat5-/- mice display increased levels of circulating hormone glucagon but are glucagon

receptor deficient (Cheung et al, 2007; Johswich et al, 2014). This is linked to a lack of Mgat5-

mediated N-glycan branching on the glucagon receptor, which affects its cell surface residency

and downstream signaling (Johswich et al, 2014). Mgat5-branched N-glycans on hepatic glucagon

receptor increase affinity for galectin binding to counteract cell surface lateral mobility, and

increase responsiveness to glucagon (Johswich et al, 2014). GlcNAc supplementation rescued

glucagon receptor sensitivity, signaling and dynamics in Mgat5-/- primary hepatocytes, and

improved glucagon tolerance response in Mgat5-/- mice (Johswich et al, 2014). This is consistent

with the notion of N-glycan redundancy, where increasing UDP-GlcNAc levels drives

compensating increases in N-glycan branching by other Mgat enzymes (Dennis & Brewer, 2013;

Lau et al, 2007).

Adult Mgat5-/- mice are also hypersensitive to autoimmune disease, and a number of T-cell

glycoproteins are known to suppress autoimmunity in this context (Grigorian et al, 2009). Multiple

sclerosis has been linked to genetic and environmental dysregulation of the Golgi N-glycan

branching pathway, and GlcNAc has been shown to suppress its progression in a mouse model of

this disease (Grigorian et al, 2011). Oral GlcNAc in drinking water increased Mgat5-modified

β1,6-GlcNAc-branched N-glycans on cell surface glycoproteins of T-cells, suppressing

inflammatory T-cell response and clinical course of experimental autoimmune encephalomyelitis

in mice (Grigorian et al, 2011; Grigorian et al, 2007; Grigorian et al, 2009). This suppression is

seen even when oral GlcNAc administration is initiated after disease onset (Grigorian et al, 2011).

These results suggests that oral GlcNAc supplementation in mice increases UDP-GlcNAc supply

to the Golgi N-glycan branching pathway. Furthermore, oral GlcNAc in drinking water suppressed

29

spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice when initiated prior to

disease onset (Grigorian et al, 2007). Collectively, these data suggest that UDP-GlcNAc-

dependent biosynthesis regulates T-cell-mediated autoimmunity by altering GlcNAc branching in

N-glycans (Grigorian et al, 2009).

1.1.4.5.1.3 GlcNAc Safety and Efficacy

Importantly, orally administered GlcNAc showed no significant adverse effects or obvious

indications of toxicity or carcinogenesis in experiments with rats at concentration of ~2500

mg/kg/day (Lee et al, 2004; Takahashi et al, 2009). Also, infusion of GlcNAc into the third

cerebroventricle of rats did not affect feeding, drinking or ambulation, suggesting that the primary

site of action of GlcNAc may be in peripheral cells and organs. (Sakata & Kurokawa, 1992).

Studies with orally administered radioactive GlcNAc in rats found that GlcNAc is not deacetylated

within the intestinal lumen, and that absorbed GlcNAc was incorporated into tissues such as the

liver, stomach and intestine, as well as in serum proteins (Capps et al, 1966). Furthermore, GlcNAc

appeared to be specifically incorporated into glycan components of glycoproteins and GAGs

(Capps et al, 1966). In a mouse model of rheumatoid arthritis, oral GlcNAc decreased the severity

of arthritis symptoms, such as joint swelling, and histopathological joint indicators associated with

disease (Azuma et al, 2012). In normal human subjects acute intravenous administration of high

amount of GlcNAc did not appear to affect blood sugar homeostasis or insulin secretion (Gaulden

& Keating, 1964). Furthermore, unlike for glucose, insulin had no obvious effect on disappearance

of circulating GlcNAc administered intravenously in human subjects (Levin et al, 1961). In

humans, single oral dose of 1 g of GlcNAc per day, the equivalent to ~15 mg/kg/day, resulted in

its absorption from the intestinal track and elevation of serum GlcNAc (Talent & Gracy, 1996).

30

Finally, a pilot study involving treatment of children with severe treatment-resistant inflammatory

bowel disease with oral GlcNAc as adjunct therapy at 3-6 g/day, or ~60–120 mg/kg/day, resulted

in improvement of clinical symptoms in some cases (Salvatore et al, 2000).

1.1.4.5.2 Dietary Monosaccharide Supplementation as Therapy

There are many examples of monosaccharide supplementation as therapy in humans. For

some genetic metabolic diseases, where de novo synthesis of specific monosaccharides is

defective, oral monosaccharide supplementation to salvage pathways can rescue sugar-nucleotide

levels and impaired glycoprotein production, with therapeutic benefit at biochemical and clinical

levels. For example, the congenital disorder of glycosylation Ib (CDG-Ib), a deficiency in

phosphomannose isomerase decreases the supply of guanosine diphosphate mannose (GDP-Man)

for dolichol-pp-oligosaccharide synthesis (Niehues et al, 1998). Oral mannose (Man)

supplementation was found to alleviate chronic postnatal CDG-Ib phenotypes of failure to thrive,

coagulopathies, protein-losing enteropathy, and liver fibrosis (Niehues et al, 1998). In this case,

oral Man administration elevated serum Man concentration increasing the import of Man into cell,

where Man salvage pathway bypassed the defective step - conversion of Fru-6P to mannose-6-

phosphate (Man-6P), to increase Man-6P and GDP-Man (Niehues et al, 1998). Another CDG, the

leukocyte adhesion deficiency type II (CDG-IId) is caused by a hypomorphic mutation in the Golgi

GDP-fucose transporter, with patients suffering recurrent infections due to a deficiency in selectin

ligands (Jaeken & Matthijs, 2007). The fucose (Fuc) salvage pathway can generate higher than

normal concentrations of GDP-Fuc, sufficient to rescue CDG-IId patients' deficit in GDP-Fuc

transport (Marquardt et al, 1999). Similarly, Fuc fed to mice with a deficiency in GDP-keto-4-

31

keto-6-deoxymannose 3,5-epimerase-4-reductase (FX) restored Fuc content in glycoproteins and

rescued associated deficits in postnatal physiology (Smith et al, 2002).

Mutations in the UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) gene, which codes for

a key enzyme in sialic acid biosynthesis, results in a rare autosomal recessive neuromuscular

disorder of adult-onset in humans, and early postnatal death in mice (Hinderlich et al, 1997).

Supplementation of pregnant mice with N-acetylmannosamine (ManNAc) or mannosamine

extended survival of Gne mutant pups by enhancing sialic acid production, restoring renal

glycoprotein sialylation, and improving renal physiology (Galeano et al, 2007). Peracetylated

ManNAc and sialic acid are effective prophylactic therapeutic treatments that prevent development

of biochemical defects and myopathic phenotype in Gne mutant mice expressing the GNE

mutation associated with human disease (Malicdan et al, 2010; Malicdan et al, 2012). In humans

this mutation leads to hereditary inclusion body myopathy. Furthermore, ManNAc and sialic acid

administration are effective in rats and mice, respectively, in significantly decreasing albuminuria

associated with nephrotic syndrome by increasing sialylation of glomerular glycoproteins

(Clement et al, 2011; Ito et al, 2012). Sialic acid enriched diet also proved effective at influencing

neuronal function in old rats by rejuvenating salivation and restoring brain ganglioside bound sialic

acid levels, both of which decline with age (Sprenger et al, 2009). Finally, further supporting the

effectiveness of monosaccharide treatment as metabolic intervention, galactose supplementation

of fibroblasts derived from patients with markedly diminished activity of phosphoglucomutase 1,

as well as dietary supplementation in patients themselves, resulted in restoration of protein

glycosylation and clinical improvement (Tegtmeyer et al, 2014).

1.2 Metabolism

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1.2.1 Glycolysis and Tricarboxylic Acid (TCA) Cycle

Glycolysis is a catabolic pathway that through a series of intricate consecutive metabolic

steps converts hexose sugars, such as glucose, to pyruvate. In animal cells, pyruvate can then be

converted to lactate, or in the presence of oxygen be oxidized in the TCA cycle, also known as the

citric acid cycle or Krebs cycle. Glycolysis takes place in almost all living cells, where it is the key

metabolic pathway for adenosine 5'-triphosphate (ATP) production, generation of reducing

equivalents, and conversion of carbohydrates into compounds which can undergo terminal

oxidation, or be used for biosynthesis (Vander Heiden et al, 2009). Glycolysis occurs in the

cytoplasm and starts with glucokinase or hexokinase-mediated phosphorylation of glucose to

glucose-6-phoshate (Glc-6P). Glycolysis can function under aerobic or anaerobic conditions.

Under anaerobic conditions, or in a situation without sufficient reducing equivalents due either to

the lack of oxygen or high cellular metabolism, pyruvate is converted to lactate (Vander Heiden et

al, 2009). Lactate produced under anaerobic conditions can diffuse from cells into the bloodstream

(Postic et al, 2004). Under aerobic conditions, pyruvate formed during glycolysis is transported

into the mitochondrial matrix, where it is oxidatively decarboxylated by the pyruvate

dehydrogenase complex to generate acetyl-CoA (Vander Heiden et al, 2009). The acetyl residues

formed are subsequently oxidized to carbon dioxide and water in the TCA cycle (Vander Heiden

et al, 2009). Another source of acetyl-CoA supplied to the TCA is derived from β-oxidation of

fatty acids (Postic et al, 2004). Complete oxidation in the TCA cycle is accompanied by release of

relatively large amounts of energy, much of which is salvaged by reducing equivalents through

oxidative phosphorylation to aerobically synthesize ATP (Vander Heiden et al, 2009).

The TCA cycle is a multi-enzyme cyclic metabolic pathway in the mitochondrial matrix

involving eight discrete steps. Each step is catalyzed by a different enzyme, and while some

33

reactions are reversible, others are unidirectional. TCA has numerous functions, both anabolic and

catabolic, and consequently is often described as the hub of intermediary metabolism (Vander

Heiden et al, 2009). The catabolic aspect of TCA initiates terminal oxidation of energy substrates,

since many catabolic pathways generate intermediates of the TCA cycle or supply metabolites

such as pyruvate and acetyl-CoA that can enter the cycle. The TCA cycle also supplies important

precursors for anabolic pathways such as oxaloacetate and malate for gluconeogenesis,

oxaloacetate and 2-oxogluterate for synthesis of amino acids such as glutamate, and citrate for

fatty acids biosynthesis (Vander Heiden et al, 2009). Thus, the TCA not only takes up acetyl-CoA

from fatty acid degradation, but also supplies material for its synthesis by condensing the acetyl

residue with oxaloacetate to form citrate and exporting it into the cytoplasm. In the cytosol, citrate

is cleaved by ATP-dependent citrate lyase into acetyl-CoA and oxaloacetate (Vander Heiden et al,

2009). The rate-determining enzyme acetyl-CoA carboxylase (ACC) carboxylates acetyl-CoA to

malonyl-CoA, which can then be used as a substrate in lipogenesis, through the action of fatty acid

synthase (FASN) complex (Postic et al, 2004).

1.2.2 Insulin, Glucagon and Liver Function in Glucose Homeostasis

Liver is a vital organ responsible for maintaining whole-body nutrient homeostasis,

including maintenance of a relatively stable blood glucose concentration. The pancreatic hormones

insulin and glucagon are involved in coordinating this process of organismal metabolism

(Schwartz et al, 2013). Plasma glucose must remain within a narrow concentration range during

periods of feeding and fasting to avoid hyperglycemia and hypoglycemia, respectively. This tight

control is exerted through coordinated regulation of glucose absorption from the intestine, uptake

and metabolism by peripheral tissues, as well as storage, release and production by the liver. Insulin

34

serves as the primary regulator of blood glucose concentration by promoting glucose uptake in

muscle and adipose tissue, as well as glycogen formation and gluconeogenesis inhibition in the

liver (Schwartz et al, 2013).

Following a meal, increasing concentrations of glucose in the blood stimulate pancreatic

β-cells to secrete insulin. The fundamental role of insulin is to coordinate use of metabolic fuels

in the body, by partitioning them for oxidation or storage. Insulin accelerates glycolysis, stimulates

glucose conversion to glycogen, and lipid biosynthesis in the liver, muscle, and adipose tissue

(Schwartz et al, 2013). To this end, insulin regulates gluconeogenic and lipogenic enzyme

accumulation and function through regulation of gene expression, translation and through post-

translational modifications of select cytosolic proteins (Lin & Accili, 2011). Glucagon is a counter-

regulatory hormone that decreases glycolysis and raises plasma glucose levels in response to

insulin-induced hypoglycemia (Lin & Accili, 2011). High glucose uptake favours glucose storage

as glycogen, as well as glycolysis to generate pyruvate, which in liver and adipose tissue is then

converted to fatty acid and triglyceride (Postic et al, 2004). Glucose is also metabolized by the

pentose phosphate pathway to generate ribose 5-phosphate, a precursor in nucleotide biosynthesis,

and NADPH, which is required for biosynthesis of fatty acids (Postic et al, 2004).

1.2.2.1 Liver Glycogen

Once absorbed, carbohydrates are carried to the liver where they are converted to glucose.

Glycogen, a branched polymer of glucose consisting of up to 50,000 residues, is the primary form

of stored carbohydrate in animal tissue. Liver is the central organ involved in glycogen formation

(glycogenesis), storage and breakdown (glycogenolysis) (Lin & Accili, 2011). These processes are

35

coordinated to maintain normal glucose levels in the blood during glycemic fluctuations associated

with states of feeding and starving. Due to its hydrated state, glycogen requires ample storage

space, and as such liver can only store a limited amount of glucose in this form. Liver glycogen

represents a short-term reserve of glucose for peripheral organs and tissues. Indeed, glycogen-

metabolizing enzymes have the property that enables the liver to act as a sensor of blood glucose

and to store or mobilize glycogen according to peripheral needs. Glycogen synthase (GS) is the

rate-limiting enzyme for glycogen synthesis and deposition, responsible for transfer of glucose

from UDP-glucose, its nucleotide-sugar donor form (Postic et al, 2004). GS phosphorylation on

Ser641 leads to inactivation, while glycogenesis is stimulated by activating GS through

dephosphorylation by protein phosphatase 1, which is controlled by insulin (Postic et al, 2004).

1.2.3 Feeding and Fasting

All cells and organs in the body have a constant requirement for nutrients and metabolites,

which serve as energy substrates. Since nutrient supply in nature is irregular and variable in amount

and type, organs such as the liver and adipose tissue act as temporary reservoirs storing energy

bearing metabolites and nutrients that can be readily deployed. In metabolism, a distinction is often

made between the fed state, also known as the absorptive state, immediately following a meal, and

the fasting or starving state, which develops later if food is not consumed. The two phases operate

on different metabolic programs, which are dictated by plasma levels of various metabolites and

the hormonal signaling cascades they trigger (Schwartz et al, 2013). During the absorptive state,

the insulin to glucagon ratio increases and the availability of substrates trigger an anabolic phase,

whereby liver forms increased amounts of glycogen and fats, muscle synthesizes proteins from

amino acids and stores glycogen, and adipose tissue synthesizes triglycerides and stores them in

36

lipid droplets (Lin & Accili, 2011). Without food intake the post-absorptive state gradually evolves

into the fasting state (Figure 1.6).

During fasting, blood glucose and insulin levels decrease, and glycogen synthesis is

inhibited, as is fatty acid synthesis (Schwartz et al, 2013). In response to lowered blood glucose

concentration, pancreatic α-cells release increased amounts of glucagon, which act on the liver to

stimulate glucose release by glycogenolysis to maintain blood glucose homeostasis and provide

other tissues with an energy source (Lin & Accili, 2011). Glucagon also plays an important role in

initiating and maintaining hyperglycemic conditions in diabetes (Lin & Accili, 2011). In fact,

insulin resistance is manifested by hyperinsulinemia, increased hepatic glucose production, and

decreased glucose disposal. The reduced insulin to glucagon ratio results in a switch to different

fuel metabolism, as the body engages in using its energy reserves by breaking down glycogen, fats

and proteins, and distributing these energy supplying metabolites between organs (Lin & Accili,

2011). Increased level of glucagon relative to insulin also stimulates the mobilization of fatty acids

from adipose tissue. Glucagon release also activates hormone-sensitive lipases, which catalyze

triglyceride hydrolysis in adipocytes to release glycerol and free fatty acids into the circulation

(Lin & Accili, 2011).

Liver is a major site for fatty acid oxidation to acetyl-CoA and fat-derived ketone body

formation (Lin & Accili, 2011). Free fatty acids and ketone bodies released into the blood serve as

important energy sources during hunger. After prolonged glucose starvation, muscles and other

tissues begin to degrade protein to amino acids, which are then oxidized or secreted and used in

gluconeogenesis by the liver. Gluconeogenesis is the formation of glucose from non-carbohydrate

sources, primarily glycerol produced through degradation of fats, and lactate, alanine, glutamine

and other glucogenic amino acids derived from muscles (Lin & Accili, 2011). In animals

37

Figure 1.6 Glucose Production in Liver During Fasting

During fasting, when plasma glucose levels drop due to lack of new supplies or rapid use, the pancreatic islet cells increase glucagon secretion, and decrease insulin release. To maintain plasma glucose homeostasis, glucagon stimulates liver glycogenolysis and increases the enzymatic activity required for gluconeogenesis, utilizing glycerol, lactate and amino acids released by adipocytes and muscle cells into the bloodstream.

38

gluconeogenesis takes place primarily in the liver, and its main function is to generate sufficient

glucose supply for organs such as brain and muscles during fasting or starvation (Lin & Accili,

2011). Typically this system is activated once hepatic glycogen has been depleted. Most of the

reaction steps involved in gluconeogenesis represent a reversal of glycolysis reactions, catalyzed

by the same enzymes that are used in glycolysis (Postic et al, 2004). Other enzymes are specific to

gluconeogenesis and are only synthesized under the influence of glucagon when needed (Lin &

Accili, 2011).

1.2.4 Glycoprotein Receptors for Glucagon and Insulin, and Glucose Transporters

Transmembrane receptors for the hormones glucagon and insulin are N-glycosylated, as

are glucose transporters. All of these are specialized glycoproteins residing at the cell surface,

where they respond to the extracellular environment and trigger changes in function of the cell.

1.2.4.1 Glucagon Receptor (Gcgr)

The glucagon receptor (Gcgr) is a G-protein coupled receptor with four and five N-glycan

sequons in man and mice, respectively. Glucagon binding to Gcgr results in a conformational

change that activates adenylate cyclase, forming the secondary messenger cyclic AMP (cAMP),

which in turn activates protein kinase A (PKA) (Johswich et al, 2014). PKA then phosphorylates

and inactivates GS, thereby terminating the synthesis of glycogen (Lin & Accili, 2011). Recently,

the Dennis laboratory has shown in primary hepatocytes that Mgat5-branched N-glycans on Gcgr

increase receptor binding to galectin-9, which slows its membrane mobility and enhances

39

sensitivity to glucagon (Johswich et al, 2014). This study suggests that N-glycan branching on

Gcgr acts as a positive regulator of glucagon responsiveness.

1.2.4.2 Insulin Receptor (Insr)

The insulin receptor (Insr) belongs to a family of related tyrosine kinase receptors.

Interestingly, human and murine Insr both have eighteen N-glycan sequon motifs. A natural variant

of human Insr has a substitution of Lysine for Asparagine at a position immediately preceding the

first N-glycan sequon (Kadowaki et al, 1990). This was found to decrease the affinity of insulin

binding to the receptor (Kadowaki et al, 1990). Furthermore, site-directed mutagenesis of the first

and second Insr N-glycan sequons significantly reduced insulin binding to the receptor on the cell

surface (Caro et al, 1994). Insulin binding to Insr leads to tyrosine phosphorylation of several

insulin receptor substrates (IRSs), which then act as docking proteins for other signaling proteins

(Lin & Accili, 2011). Phosphorylation of IRSs proteins activates the phosphatidylinositol-3-kinase

(PI3K) and Akt signaling pathway, which is responsible for most of the metabolic actions of insulin

(Lin & Accili, 2011). One of these is to increase translocation of glucose transporters (Gluts) from

cytoplasmic vesicles to the cell surface, where they can mediate glucose import from the blood via

facilitated diffusion (Lin & Accili, 2011).

1.2.4.3 Glucose Transporters (Gluts)

Most Glut proteins, encoded by Slc2a genes, typically have a single N-glycan sequon.

Dependence of Gluts on N-glycan branching for increased residency and activity at the cell surface

has been shown for Glut1, Glut2 and Glut4 (Haga et al, 2011; Kitagawa et al, 1995; Lau et al,

40

2007; Ohtsubo et al, 2005; Zhu et al, 2012). In both mouse and human pancreatic β-cells, cell

surface expression of Glut2 depends on binding of its Mgat4-mediated N-glycan structure to

galectin-9, which slows mobility at the cell surface and loss to endocytosis, thereby enhancing

glucose transport (Ohtsubo et al, 2011; Ohtsubo et al, 2005). There is also pharmacological

evidence for N-glycan requirement on nutrient transporters at the cell surface. Indeed, membrane

transport of three major classes of nutrients, namely glucose, amino acids and uridine, was

inhibited by tunicamycin, an inhibitor of protein N-glycosylation in the ER (Olden et al, 1979). A

similar result was obtained using a different tool to perturb N-glycan function, suggesting that

interfering with N-glycans on glycoproteins alters plasma membrane transport systems to impair

nutrient uptake. In this case, wheat germ agglutinin, a lectin that binds GlcNAc within N-glycans

on cell surface glycoproteins, has been shown to have an inhibitory effect on transport of amino

acids through the plasma membrane (Li & Kronfeld, 1977).

1.2.5 Nutrient Sensing and Signaling Pathways

Cells have evolved several sensory systems and homeostatic regulatory mechanisms to

sense nutrient levels, cellular energy and metabolic status, as well as to respond to fluctuations by

adjusting flux through metabolic pathways. Pathways that detect differences in extracellular and

intracellular levels of nutrients and metabolites are often integrated and coordinated. The

mammalian target of rapamycin (mTOR) and the AMP-activated protein kinase (AMPK)

pathways are two well characterized nutrient sensing and signaling pathways.

1.2.5.1 mTOR

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mTOR controls cell growth and metabolism in response to nutrients, growth factors and

cellular energy, by positively and negatively regulating several anabolic and catabolic processes,

respectively, to collectively determine mass accumulation (Cornu et al, 2013; Zoncu et al, 2011).

mTOR is a Ser/Thr protein kinase that interacts with several distinct proteins to form two

complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Cornu et al,

2013). mTORC1 activates p70 S6 kinase, which in turn phosphorylates ribosomal protein S6, thus

activating translation and transcriptional up-regulation of anabolic pathways (Cornu et al, 2013).

The abundance of phosphorylated ribosomal protein S6 is directly related to mTORC1 activity,

and inversely related to level of cellular autophagy (Zoncu et al, 2011). Autophagy is involved in

regulating intracellular lipid metabolism in the liver through macrolipophagy (Singh et al, 2009).

Macrolipophagy promotes breakdown of intracellular lipids stored in lipid droplets to

supply fatty acids for β-oxidation or other uses (Singh et al, 2009). Hepatic mTORC1 activity

negatively regulates autophagy and production of ketone bodies for peripheral tissues to use as

energy source in response to fasting (Zoncu et al, 2011). Hence, fasting response typically

suppresses mTORC1 activity, however its chronic activation by overabundance of nutrients and/or

hyperinsulinemia can drive ectopic accumulation of lipids in the liver (Zoncu et al, 2011). Indeed,

mTORC1 activity is significantly elevated in liver, muscle, and adipose tissue of both genetically

induced and high-fat diet induced obese mice, suggesting its involvement in the pathogenesis of

obesity and obesity-associated metabolic disorders (Rui, 2007). In addition to its key role in lipid

metabolism within the liver, hepatic mTORC1 signaling also affects systemic glucose and insulin

homeostasis, most likely due to its effects on Akt and hepatic glucose uptake (Albert & Hall, 2014).

Hyper-activation of mTORC1 signaling upon fasting results in metabolic stress due to systemic

and hepatic glutamine depletion, and thereby an inability of glutaminolysis to sustain the TCA

42

cycle (Albert & Hall, 2014). This is consistent with the finding that mTORC1 inhibition by

rapamycin increases intracellular glutamine levels (Albert & Hall, 2014). In certain settings, such

as low-nutrient conditions, AMPK acts as a negative regulator of mTORC1 signaling (Hardie,

2014).

1.2.5.2 AMPK

AMPK is a master regulator of energy homeostasis, as well as cellular and organismal

metabolism (Hardie, 2014). It senses low ATP levels and restores cellular energy by inhibiting

energy consuming anabolic while stimulating catabolic pathways such as autophagy and fatty acid

oxidation (Hardie, 2014). To maintain energy homeostasis, a delicate balance between fat storage

and breakdown is crucial. The adipose tissue hormone adiponectin, whose actions are mediated by

AMPK, inhibits fatty acid synthesis, stimulates fatty acid uptake and oxidation, and sensitizes liver

and muscle tissues to insulin (Unger et al, 2010). AMPK activity can be measured through its

phosphorylation at Thr172, as well as by phosphorylation of its downstream target acetyl-CoA

carboxylase (ACC) at Ser79 (Hardie, 2014). ACC catalyzes the pivotal step in fatty acid synthesis,

the formation of malonyl-CoA, by attaching a bicarbonate ion to acetyl-CoA (Jump, 2011).

Malonyl-CoA inhibits transport of acyl-CoA into the mitochondrial matrix and its subsequent

breakdown (Jump, 2011). Thus, de novo fatty acid synthesis is in part negatively regulated by

AMPK. Dephosphorylated ACC is enzymatically active, while phosphorylation inactivates its

enzymatic activity and promotes fatty acid oxidation instead (Hardie, 2012). The inactivating

phosphorylation of ACC is catalyzed by AMPK, which in turn is regulated by an activating

phosphorylation by cAMP dependent PKA, while the reactivating dephosphorylation of ACC is

catalyzed by protein phosphatase 2A (Hardie, 2014).

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1.2.6 Fatty Acid Synthesis

The rate of fatty acid synthesis can be influenced by diet. In well-fed state with an ample

supply of glucose, insulin is secreted, which causes a low degree of ACC phosphorylation, and

increased formation of malonyl-CoA (Jump, 2011). The biosynthesis of fatty acids, or de novo

lipogenesis, occurs in many tissues, however post-absorptively this occurs primarily in the liver

and fatty tissues. Fatty acid synthase (FASN) is a key biosynthetic multi-enzyme protein complex

responsible for de novo lipogenesis (Jump, 2011). By catalyzing the synthesis of long-chain fatty

acids from acetyl-CoA and malonyl-CoA, in the presence of NADPH as a reducing agent, FASN

produces long-chain saturated fatty acids for storage in the liver or export to other tissues (Jump,

2011). FASN uses acetyl-CoA as a starter molecule, and in a cyclic reaction, acetyl residue is

elongated by two carbon units at a time for seven cycles, until formation of sixteen carbon palmitic

acid as the major product (Jump, 2011). The liver is the most important site for synthesis of fatty

acids and triglycerides, most of which are subsequently released into the blood. The cytosolic

buildup of acetyl-CoA encourages de novo hepatic lipogenesis, which over time can lead to

triglyceride accumulation and even development of fatty liver disease, or hepatic steatosis (Jump,

2011). FASN is transcriptionally regulated by sterol regulatory element binding protein-1c

(SREBP-1c) in response to feeding and insulin (Jump, 2011). Long-term adaptive control of

lipogenesis occurs though changes in the rate of synthesis at the transcriptional level, and

degradation of the participating enzymes (Jump, 2011).

1.2.7 Lipid Storage and Breakdown

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Due to the limited storage capacity of glycogen, all carbohydrates consumed beyond

energy needs are converted to and stored as fat. Fats are the most important energy reserves in an

animal. This reserve pool is practically almost unlimited. Triglycerides are neutral lipids, each

composed of three fatty acids in ester linkage with a single glycerol (Walther & Farese, 2012). In

animals, triglycerides are highly concentrated stores of metabolic fuel, and yield more energy per

weight upon oxidation than either proteins or carbohydrates (Speakman & O'Rahilly, 2012).

Although most tissues are capable of producing triglycerides, their primary site of accumulation

is the adipose tissue. In vertebrates, lipid droplets are the major cytoplasmic storage organelles for

triglycerides (Walther & Farese, 2012). They are characterized as ubiquitous components of

different cell types, even those that, unlike adipocytes, only have a limited capacity for triglyceride

storage (Walther & Farese, 2012). Lipid droplets play a crucial role in regulating cellular lipid

levels through hydrolysis and trafficking, and provide energy and substrates for synthesis and

repair of cell membranes (Walther & Farese, 2012).

During fasting or starvation the body’s strategy is to minimise the use of carbohydrate and

protein, and to obtain as much energy as possible from fat stores. Hence, lipogenesis is inhibited,

and fatty acids activated in a process requiring ATP to form acyl-CoA, which can then be

transported into the mitochondrial matrix for β-oxidation and acetyl-CoA generation (Walther &

Farese, 2012). Resulting acetyl residues can be oxidized to carbon-dioxide in the TCA cycle,

producing reduced coenzyme and ATP derived through oxidative phosphorylation (Vander Heiden

et al, 2009). Fatty acid breakdown in the liver is increased when levels of free fatty acids are

elevated, such as after consumption of high-fat foods, and increased lipolysis during fasting or

starvation (Jump, 2011). Short chain fatty acids are dissolved in plasma, while longer chain fatty

acids are bound to albumin. Fatty acids are transported across the cell membrane by membrane-

45

associated fatty acid-binding proteins, or fatty acid transporters, which not only facilitate but also

regulate cellular fatty acid uptake (Schwenk et al, 2010). A number of fatty acid transporters have

been identified, including CD36, which is extensively glycosylated. Indeed, mouse CD36 has eight

N-glycan sequons, while human CD36 has ten. These are situated in a region coding for the large

extracellular loop. Insulin treatment stimulates simultaneous recruitment of CD36 and glucose

transporter Glut4 from the recycling endosomal storage compartment to the cell membrane in order

to increase fatty acid and glucose uptake, respectively (Schwenk et al, 2010).

1.3 Body Weight and Obesity

Body weight is a simple and effective way to measure tissue mass and estimate body

composition of an animal. In humans, the body mass index (BMI) provides a more accurate

measurement, since it is independent of height and correlates fairly well with the total body fat

(Speakman & O'Rahilly, 2012). BMI is calculated from body weight in kilograms divided by the

square of height in meters. To specifically measure body composition in terms of fat and lean

muscle mass, various imaging modalities are available. Dual-energy X-ray absorptiometry

(DEXA) passes two photons of varied energy intensity through body tissues, and body fat

percentage is estimated based on the attenuation patterns of these photons. Alternatively, to obtain

even more accurate measures of fat mass, computed tomography (CT) or magnetic resonance

imaging (MRI) can be used. In humans, obesity is clinically defined as BMI of greater than 30

kg/m2, while a number of 25 up to 30 indicates that a person is overweight. Around the globe, in

both industrialized and developing countries, there has been an increase in adiposity among people,

often progressing to obesity. It is estimated that currently about ~2 billion people worldwide are

overweight and ~700 million are obese (Speakman & O'Rahilly, 2012). These are staggering

46

numbers compared to 50 years ago (Speakman & O'Rahilly, 2012). Obesity is a risk factor for

chronic diseases such insulin resistance, diabetes, metabolic syndrome, cardio-vascular disease,

fatty-liver disease, and even some forms of cancer (Speakman & O'Rahilly, 2012; Unger et al,

2010). The organs affected in these conditions, including the liver, pancreatic islets, skeletal

muscle, heart and kidney, display ectopic lipid accumulation (steatosis), which can ultimately lead

to lipotoxicity, apoptosis, and gradual organ failure (Unger et al, 2010). The increased prevalence

of obesity and these chronic diseases related to nutrition and metabolism have prompted

considerable efforts to understand their origin and to identify effective prevention strategies or

treatments.

1.3.1 Body Weight Regulation

The biology of body weight regulation is complex and operates through effects on energy

intake, metabolic activity, energy expenditure, and caloric partitioning into tissues such as fat and

muscle. Since there is a constant flux in the nutritional supply and energy needs of an organism,

strategies that maintain a steady state under varying nutritional conditions are crucial for healthy

functioning of an organism. Although body weights across a population vary considerably,

individuals show a remarkable weight stability, suggesting that body weight is physiologically

regulated (Flier & Maratos-Flier, 2007). Regulation of fat depots involves a coordinated interplay

between central regulators of feeding behavior in the hypothalamus, neuroendocrine signals, and

metabolic regulators of energy expenditure and fat storage (Flier & Maratos-Flier, 2007). Dramatic

alterations in body weight have been shown to result from mechanical or pharmacological lesions

of the hypothalamus (Flier & Maratos-Flier, 2007). Peripheral tissues such as the gut, white and

brown adipose, and skeletal muscles also regulate body weight and its composition (Flier &

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Maratos-Flier, 2007). One of the most dramatic illustration of this comes from animals with a

mutation in myostatin, or animals treated with compounds that block or antagonize the activity of

myostatin such as follistatin, which results in significantly larger skeletal muscle mass and

increased muscle strength (Gangopadhyay, 2013).

1.3.1.1 Leptin

The adipose-derived hormone leptin regulates systemic energy homeostasis by linking an

individual’s fat stores with caloric intake and expenditure (Flier & Maratos-Flier, 2007). The

receptors for leptin are located in the hypothalamus, and have eighteen N-glycan sequons in

humans, and seventeen and sixteen in rat and mouse, respectively. Leptin functions as a satiety

signal released from adipose tissue in proportion to fat stores, and is part of a signaling pathway

that acts to maintain the size of body fat depot (Flier & Maratos-Flier, 2007). Leptin activity

impinges on the hypothalamic regulatory neuro-circuitry to increase energy expenditure and

inhibit feeding (Flier & Maratos-Flier, 2007). Both leptin and insulin have been characterized as

adiposity signals, since their plasma levels positively correlate with body weight and are in direct

proportion to the amount of energy stores in adipose tissue (Schwartz et al, 2013). Leptin also

stimulates fatty acid oxidation in non-adipose tissues, so as to minimize ectopic lipid accumulation

and protect against lipotoxicity (Unger et al, 2010). Humans with a rare congenital loss-of-function

mutations in the leptin gene are clinically obese, due to abnormalities in energy expenditure and

increased food intake (Speakman & O'Rahilly, 2012). These symptoms are reversed by

administration of recombinant human leptin (Speakman & O'Rahilly, 2012). Mice deficient in

leptin or in the leptin receptor exhibit overfeeding and develop obesity and diabetes (Flier &

Maratos-Flier, 2007).

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1.3.2 Determinants of Body Weight and Obesity

Factors affecting weight regulation and obesity have been identified at a number of

different levels of analysis. These span the whole spectrum, from social to economic,

psychological, behavioral, developmental, physiological, biochemical, metabolic, genetic and

molecular, with interaction and feedback across and between factors. Adiposity is a highly

heritable trait, and genetics appears to explain around 65% of weight variation between individuals

(Speakman & O'Rahilly, 2012). Although many polymorphisms associate robustly with obesity,

much of the genetic variation that underpins differences in adiposity across the normal population

remains unexplained (Speakman & O'Rahilly, 2012). It appears that body weight and composition

is a phenotype at the interface of genes and environment, where genes most likely determine

susceptibility to environmental factors. Environmental factors such as gut microbiota, stress,

endocrine disruptors have also been linked to risk of developing obesity (Speakman & O'Rahilly,

2012).

The most widely accepted explanation for the rise in adiposity around the globe involves

increased availability and access to palatable calorie-dense foods, which leads to overeating and

excess calorie intake; combined with decreased energy expenditure through sedentary lifestyle

resulting from modernization and technological advances, which have greatly reduced labour

intensive activities (Speakman & O'Rahilly, 2012; Unger et al, 2010). The idea of a toxic-

environment and exposure to chemical obesogens, such as bisphenol-A and pesticides, has also

been proposed to account for the rise in obesity rates in the last few decades (Grun, 2010). This

theory argues that our environment has become contaminated due to pharmaceutical drugs,

chemical pollutants, as well as hormones and antibiotics used in farming, which disrupt the energy

49

balance, endocrine function, lipid metabolism and fat storage by a variety of mechanisms (Grun,

2010). However, the prevailing explanation for the observed increase in body weight and obesity

is that of energy imbalance, i.e. eating too many calories and not getting enough physical activity

(Speakman & O'Rahilly, 2012).

1.3.2.1 Energy Balance

The guiding principle of body regulation bioenergetics is that of energy balance or energy

homeostasis, which proclaims that to maintain a constant body weight, energy or calorie intake

from food consumed must equal energy or calories burned by the metabolism of the organism

(Flier & Maratos-Flier, 2007). Hence, body weight is maintained in a steady state by a balance

between caloric intake and energy expenditure. Any imbalance between energy intake and

expenditure, which includes total physical activity, metabolic rate and thermogenesis, is reflected

in a change in the amount of stored energy as fat (Flier & Maratos-Flier, 2007). Thus, when

nutritional intake chronically exceeds the energy needs of an organism, the excess energy is stored

in body fat, which in turn leads to weight gain. This dominant theory is firmly rooted in the first

law of thermodynamics, i.e. the law of conservation of energy, which states that energy can neither

be crated nor destroyed, but only converted from one form to another. However, the dogma of

energy imbalance between calories consumed and calories expended being the fundamental cause

of obesity is not universally accepted (Lustig et al, 2012; Taubes, 2013).

1.3.2.2 The Carbohydrate Hypothesis of Obesity

50

The theory of energy balance has been challenged with arguments that physiology is not

physics, and that not all calories are created equal, but rather that nutrient composition affects fat

accumulation (Taubes, 2012). In this theory, sugar has been singled out as the toxic culprit

responsible for increase in obesity, diabetes and the metabolic syndrome around the globe (Lustig

et al, 2012; Taubes, 2013). This alternative hypothesis suggests that adiposity is not caused by

excess calories alone, but rather by the quantity and quality of carbohydrates and starches

consumed. The logic behind this theory is that since insulin regulates carbohydrate metabolism

and stimulates the synthesis and storage of fats in the liver and fat depots, and blood levels of

insulin are effectively determined by carbohydrate intake, then consumption of sweet

carbohydrates with a high-glycemic index will result in more insulin release and more fat

accumulation (Taubes, 2013). With elevated insulin levels (hyperinsulinemia) in the bloodstream

for prolonged periods, adipocytes respond by accumulating more fat, thus promoting more weight

gain (Taubes, 2013).

A variant of this theory suggests that it is specifically excess consumption of fructose,

found in sucrose and high-fructose corn-syrup, that is responsible for the current epidemic of

obesity and chronic metabolic disease (Lustig, 2013). Indeed, fructose consumption has increased

worldwide, as it is often used as a sweetener during food processing, paralleling the increase in

obesity and chronic metabolic diseases over the last few decades (Lustig et al, 2012). Unlike

glucose, fructose is very sweet, and does not generate an insulin response (Lustig, 2013). Its

hepatic metabolism in the post-absorptive state impairs β-oxidation, and promotes de novo

lipogenesis, triglyceride formation and hepatic steatosis (Lustig, 2013). This is often followed by

hepatic insulin resistance, hyperglycemia, as well as formation of reactive oxygen species,

resulting in cellular toxicity and dysfunction (Lustig, 2013). Furthermore, fructose also has

51

dependence-producing properties, which promote changes in the brain’s reward system, leading

to excessive consumption (Lustig, 2013). Consequently, fructose has been deemed toxic and a

danger to individuals and society requiring prompt regulation, since it exerts its detrimental health

effects beyond its calories, and in ways that are similar to alcohol (Lustig et al, 2012).

However, it should also be noted that the idea that carbohydrates are mostly to blame for

making animals fat has many detractors, who instead point to fat as the real culprit. Most nutrients

consumed in excess of energy needs are converted to fat, however since carbohydrates must first

be metabolized for this purpose, their conversion is actually more ineffective than fats, which can

be directly integrated into triglycerides and lipid droplets. Moreover, there is plenty of evidence

from mouse experimental research to support the claim that calorie enriched fatty diet leads to

body weight increase (Cheung et al, 2007). The incompleteness in our understanding of body

weight regulation and what causes obesity is illustrated by the lack of a general consensus on the

issue, or a universally accepted explanation. Indeed, competing diet programs promising success

and results are often polar opposites of one another. Furthermore, the fact that dieting, weight-loss

and fad diets are a billion dollar industry, with most being ineffective, and those that do lose weight

initially not being able to maintain the weigh-loss they have obtained either through hard-work or

deprivation, is a testament to the fact that we do not fully understand the underlying biology of

body weight regulation, and hence lack actionable knowledge for impactful intervention in public

health advice.

52

1.4 Rationale, Objectives and Summary

N-glycosylation and Golgi N-glycan branching are essential modifications of proteins

translated in the secretory pathway. Glycoproteins traffic through the Golgi apparatus, where

exposure to N-glycan branching enzymes is dependent on enzyme expression and UDP-GlcNAc

supply. Glucose transporters Glut1, Glut2 and Glut4 are dependent on N-glycan branching

pathway and galectin binding for optimal cell surface retention and glucose transport (Dennis et

al, 2009). Mutations in genes encoding branching enzymes Mgat4a and Mgat5 in the N-glycan

branching pathway disrupt glucose homeostasis in mice (Cheung et al, 2007; Ohtsubo et al, 2005).

When I began my research project, Mgat4a expression in the pancreas was reported to be regulated

at the level of gene expression, and both Mgat5 and Mgat4a mutant mice displayed aberrant

regulation of body-weight on high-fat diet (Cheung et al, 2007; Ohtsubo et al, 2005). Relatively

little consideration had been given to the contribution of metabolic feedback through the HBP or

GlcNAc salvage to UDP-GlcNAc, the common donor substrate used by Mgat enzymes, until a

publication from the Dennis laboratory reported that elevated UDP-GlcNAc supply to the N-

glycan branching pathway enhanced cell surface residency and activity of growth factor receptors

and Glut4 (Lau et al, 2007).

The main objective of my thesis was to determine whether metabolic input through the

HBP to UDP-GlcNAc contributed to control of N-glycan branching pathway in vivo, and whether

this could be seen to occur cell-autonomous in vitro. Another objective was to examine the effect

of increased expression of N-glycan branching enzymes and/or UDP-GlcNAc supply on cellular

metabolism using targeted mass-spectrometry based metabolomics, and to explore the potential

mechanisms by which HBP-mediated increase in N-glycan branching exerts its effect on

metabolism.

53

Effects of overexpressing Mgat1, Mgat5 or Mgat6 on central metabolites were investigated

in cells growing under normal cell culture media conditions supplemented with GlcNAc. Induced

overexpression of Mgat enzymes increased respective N-glycan branches on cell surface

glycoproteins, and increased central metabolites in different metabolic pathways. Further increases

were observed by using GlcNAc to supplement the HBP and increase UDP-GlcNAc pool for N-

glycan branching enzymes. Importantly, GlcNAc supplementation and Mgat5 overexpression

displayed synergistic increase in Mgat5-mediated N-glycan branching. Moreover, my findings

suggest that N-glycan branching cooperates with the HBP to regulate nutrient import and

metabolism in a cell-autonomous manner.

I also examined whether UDP-GlcNAc levels are sensitive to dietary GlcNAc

supplementation in vivo, and might increase nutrient uptake and promote anabolic metabolism via

Golgi N-glycan branching. Chronic oral GlcNAc supplementation in C57BL/6 mice increased

hepatic UDP-GlcNAc and N-glycan branching on liver glycoproteins. GlcNAc supplementation

altered hepatic metabolism resulting in excess lipid storage and body-weight increase. In cultured

cells, GlcNAc enhanced nutrient uptake and promoted fatty-acid synthesis and storage in an N-

glycan-dependent manner. These results suggest that GlcNAc salvage promotes nutrient uptake

and lipid storage by enhancing UDP-GlcNAc supply to the Golgi N-glycan branching pathway to

modify N-glycan branches on transmembrane glycoproteins. GlcNAc supplementation also

restored fat accumulation in Mgat5 null mice, which exhibit a lean phenotype with reduced fat

depots. In both genders of Mgat5 null mice oral GlcNAc increased fat accumulation, a result

consistent with functional redundancy of N-glycan branches. The rescue of fat accumulation in

Mgat5 null mice occurred at the expense of lean mass, indicating a requirement for Mgat5 in the

normal lean to fat balance.

54

Chapter 2

Golgi N-Glycan Branching N-Acetylglucosaminyltransferases I, V and VI

Promote Nutrient Uptake and Metabolism

A version of this chapter was accepted for publication in Glycobiology 2015 Feb;25(2):225-40

Anas Abdel Rahman*, Michael C. Ryczko*, Miyako Nakano*,

Judy Pawling, Tania Rodrigues, Anita Johswich, Naoyuki Taniguchi, and James W. Dennis

* co-first authors

Attributions:

The LC–ESI-MS chromatogram results for N-glycan analysis displayed in Figures 2.2C-E and

2.3D-F were performed by Miyako Nakano.

The targeted metabolomics mass spectrometry experiments in Figures 2.4A-C, 2.5C, 2.6A and C,

2.7, 2.8, and 2.10 were performed together with Anas Abdel Rahman.

All other experiments were designed and performed by Michael Ryczko.

55

2.1 Summary

Nutrient transporters are critical gate-keepers of extracellular metabolite entry into the cell.

As integral membrane proteins, most transporters are N-glycosylated, and the N-glycans are

remodeled in the Golgi apparatus. The Golgi branching enzymes N-acetylglucosaminyltransferase

I, II, IV, V, and avian VI (encoded by Mgat1, Mgat2, Mgat4a/b/c, Mgat5 and Mgat6), each catalyze

the addition of GlcNAc in N-glycans. Here I asked whether N-glycan branching promotes nutrient

transport and metabolism in immortal human HeLa carcinoma and non-malignant Hek293

embryonic kidney cells. Mgat6 is absent in mammals, but ectopic expression can be expected to

add an additional β1,4 linked branch to N-glycans, and may provide evidence for functional

redundancy of the N-glycan branches. Tetracycline-induced over-expression of Mgat1, Mgat5,

and Mgat6 resulted in increased enzyme activity and increased N-glycan branching concordant

with the known specificities of these enzymes. Tet-induced Mgat1, Mgat5 and Mgat6 combined

with stimulation of the HBP to UDP-GlcNAc, increased cellular metabolite levels, lactate and

oxidative metabolism in an additive manner. I then tested the hypothesis that N-glycan branching

alone might promote nutrient uptake when glucose and glutamine are limiting. In low glutamine

and glucose medium, tet-induced Mgat5 alone increased amino acids uptake, intracellular levels

of glycolytic and TCA intermediates, as well as Hek293 cell growth. More specifically, tet-induced

Mgat5 and HBP elevated the import of Gln, although transport of other metabolites may be

regulated in parallel. Our results suggest that N-glycan branching cooperates with HBP to regulate

metabolite import in a cell autonomous manner, and can enhance cell growth in low-nutrient

environments.

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2.2 Introduction

The medial Golgi N-acetylglucosaminyltransferases I, II, IV, V (Mgat1, Mgat2,

Mgat4a/b/c, Mgat5) form a linear pathway that initiate the GlcNAc branches on newly synthesized

glycoproteins (Figure 2.1A) (Schachter, 1986). In the trans Golgi, the branches are extended with

galactose, fucose and sialic acid to generate sequences recognized by galectins, C-type lectins and

siglecs. The N-glycan branching pathway is required for tumor progression, and molecular

mechanisms have emerged that intersect with basic metabolism (Lau et al, 2007; Shirato et al,

2011). Tumor growth rates are reduced in Mgat5-/- mice with a PyMT transgene or in Pten

heterozygous mice (Cheung & Dennis, 2007; Granovsky et al, 2000). Suppression of branching

by either Mgat1 shRNA (Beheshti Zavareh et al, 2012) or over-expression of N-

acetylglucosaminyltransferase III (Mgat3) (Song et al, 2010; Yoshimura et al, 1995) also inhibit

tumor progression in mice. Furthermore, over-expression of Mgat5 in epithelial cells promotes

transformation (Demetriou et al, 1995), and in human mammary and colon cancer, increased

Mgat5-branched N-glycans are associated with disease progression (Fernandes et al, 1991;

Seelentag et al, 1998).

Previously it was shown that galectins bind branched N-glycans and cross-link

glycoprotein to form a highly dynamic lattice that slows loss of membrane glycoproteins to

endocytosis (Lau & Dennis, 2008). In Mgat5-/- tumor cells, the retention of surface receptors is

reduced along with sensitivity to growth factors and cytokines. The mutant phenotype could be

rescued by either (i) Mgat5 re-expression, (ii) inhibition of constitutive endocytosis, or (iii)

increased UDP-GlcNAc, the common sugar-nucleotide donor for the Mgat enzymes (Lau et al,

2007; Partridge et al, 2004). In the absence of Mgat5, compensating amounts of N-

acetyllactosamine branches are produced by Mgat1, Mgat2 and Mgat4 when cells are

57

supplemented with GlcNAc to increase UDP-GlcNAc (Lau et al, 2007). This suggests redundancy

of the branches and cooperative action of N-glycans in support of growth factor receptors and other

glycoproteins at the cell surface (Dennis & Brewer, 2013). Interestingly, the chicken (Gallus

gallus) GnT-VI (Mgat6) enzyme catalyzes the GlcNAcβ1,4 linkage to

GlcNAcβ1,6Manα1,6Manβ in the trimannosyl core of N-glycans (Figure 2.1A). Mgat6 is

expressed widely in birds (Watanabe et al, 2006), with homologues in frog (Xenopus laevis), duck-

billed platypus (Ornithorhynchus anatinus) and a few species of fish. Mgat6 gene and activity are

absent in mammals. If N-glycan branching is indeed cooperative and regulates metabolism, it is

reasonable to assume that ectopic expression of Mgat6 in mammalian cells may reveal evidence

for both.

Growth factor receptors activate Akt, Ras and mTor/S6K signaling, which leads to

increased Glc and amino acid transporter activity (Frauwirth et al, 2002; Wellen et al, 2010; Wise

et al, 2008; Yun et al, 2009). Many of the ~600 mammalian solute transporters have extracellular

domains with sites of N-glycosylation, and like receptor kinases, may be regulated by N-glycan

branching. Gluts are dependent on N-glycan branching for surface residency of Glut2 in β-cells

(Ohtsubo et al, 2005), Glut1 in tumor cells (Kitagawa et al, 1995), and insulin-stimulated or HBP-

stimulated increases in surface Glut4 (Haga et al, 2011; Lau et al, 2007). The GlcNAc β1,4

branching enzyme GnT-IVa (Mgat4a) is selectively expressed in insulin producing β cells of the

pancreas, and Mgat4a-/- mice develop type 2 diabetes (Ohtsubo et al, 2005; Takamatsu et al, 2010).

The branched N-glycans on Glut2 bind galectin-9 which promotes surface residency of Glut2, and

transport of Glc.

N-glycan branching depends on de novo synthesis of UDP-GlcNAc by the HBP and/or

salvage of GlcNAc. Moreover, growth factor signaling increases UDP-GlcNAc levels, and shRNA

58

inhibition of GFAT in the de novo HBP reduced Ras-driven tumor growth (Ying et al, 2012).

Ras/Mapk and PI3K signaling also stimulates Mgat5 gene expression (Buckhaults et al, 1997;

Dennis et al, 1989; Kang et al, 1996), as well as Mgat4 in human carcinomas (D'Arrigo et al, 2005;

Takamatsu et al, 1999). The affinity of Mgat1, Mgat2, Mgat4a/b/c and Mgat5 enzymes for UDP-

GlcNAc decreases ~300 fold moving from Mgat1 to Mgat5. As such, Mgat1 functions near

saturation and Mgat5 well below its Km value for UDP-GlcNAc (Lau et al, 2007; Sasai et al, 2002).

The action of Mgat1 is required to generate substrates for further remodeling by α-Mannosidase

II, Mgat 2, Mgat4 and Mgat5. Gln supplied to cells above typically used cell culture levels (>4

mM) increases UDP-GlcNAc levels, similar to GlcNAc supplementation in normal culture

conditions, while Glc below physiological concentration (<5 mM) reduced UDP-GlcNAc levels

(Abdel Rahman et al, 2013). Thus both Glc and Gln flux through HBP regulate UDP-GlcNAc

levels, but at the low and high end of their concentration ranges, respectively. These studies offer

the intriguing possibility that cellular regulation of N-glycan branching on solute transporters and

receptors has evolved as a mechanism of adaptation to changing nutrient and environmental cues.

Mgat5-deficient mice are hypoglycemic, hyposensitive to glucagon (Johswich et al, 2014),

and resistant to weight-gain on a high-fat diet, although their food intake is equivalent to wild-type

littermates (Cheung et al, 2007). This suggests that N-glycan branching may promote a “thrifty

phenotype”, i.e. efficiency in uptake and/or utilization of nutrients (Cheung et al, 2007). As an

experimental model, I generated HeLa tumor cells and Hek293 immortalized cells with inducible

transgenes for Mgat1, Mgat5, or Mgat6 over-expression. Here I have characterized the effects of

short-term (~24 h) transgene over-expression on nutrient levels and central metabolism in cells in

culture. I report that Mgat1, Mgat5 or Mgat6 over-expression alone, and with HBP stimulation to

UDP-GlcNAc, increases metabolite levels, suggesting cooperative action of the N-glycan

59

branching enzymes. Mgat5 transgene over-expression alone restored metabolite levels and

Hek293 cell growth to normal in low Gln and Glc culture conditions. The uptake rate of Gln and

the intracellular levels of other amino acids were enhanced by Mgat5 over-expression. This is the

first evidence of cell autonomous regulation of nutrient uptake and metabolism by N-glycan

branching.

2.3 Materials and Methods

2.3.1 Materials and Chemicals

PhosSTOP Phosphatase Inhibitor Cocktail and Complete Protease Cocktail were

purchased from Roche. Alexa Fluor-488 conjugated lectins Concanavalin A (ConA) and L-PHA

were purchased from (Invitrogen). Metabolite standards and reagents were obtained from Sigma

Chemicals (St. Louis, MO) with minimal purity of 98%. Stable-isotope 15N15N-L-Glutamine was

purchased from Cambridge Isotope Laboratories Inc. (Andover, MA). All organic solvents and

water used in sample and LC/MS mobile phase preparation were HPLC grade and obtained from

Fisher Scientific (Fair Lawn, NJ). N-Glycosidase F (PNGase F, EC 3.5.1.52, recombinant cloned

from Flavobacterium meningosepticum and expressed in E. coli) was purchased from Roche

Diagnostics (Mannheim, Germany). PVDF membrane (Immuno-Blot, 0.2 µm, 7.0 x 8.5 cm) was

purchased from Bio-Rad (Hercules, CA). Cation-exchange columns were made of (30 mg) Dowex

50W-X8 200-400 mesh resins (Wako Chemical, Osaka, Japan) packed on top of a 10 μl ZipTip u-

C18 (Millipore, Billerica, MA). The resins were protonated with 1M HCl (50 µL, 3 times), were

rinsed with methanol (50 µl, 3 times), and then equilibrated with water (50 µl, 3 times) before use.

Microtiter plates (96-well flat bottom, MaxiSorp) were purchased from Nunc (Roskilde,

60

Denmark). All other chemicals were purchased from Sigma-Aldrich, Nacalai Tesque (Kyoto,

Japan) or Wako Chemical. Other reagents and solvents were of HPLC or LC/MS grade.

2.3.2 Cell Culture

HeLa Flp-In-TREx cells were a kind gift from Dr. Stephen Taylor (University of

Manchester), while Hek293 Flp-In-TREx cells (Ward et al, 2011) were purchased from Invitrogen.

Both adherent cell lines were grown in an incubator at 37°C and 5% CO2 in a humidified

atmosphere. They were maintained in high-Glc DMEM (Sigma) supplemented with 10% fetal

bovine serum (FBS), 2 mM Gln, penicillin/streptomycin, and 3ug/ml blastocidin and 100 ug/ml

zeocin, unless indicated otherwise. Human Mgat1 cDNA, human Mgat5 cDNA, and chicken

Mgat6 cDNA were FLAG-tagged at the N-terminus, and each construct was cloned individually

into the pcDNA5/FRT/TO expression vector. Each plasmid was integrated into the genome of the

HeLa or Hek293 Flp-In-TREx cells described above, at a pre-integrated FRT recombination site,

by co-transfection with Flp recombinase encoding POG44 plasmid, using Lipofectamine

(Invitrogen) and OptiMEM media lacking FBS or antibiotics. Following selection in 200 ug/ml of

hygromycin, resistant clones were individually picked, expanded and treated with 1 ug/ml

tetracycline for 24h. To verify gene expression, cell lysates were analyzed by Western-blotting

using monoclonal Anti-FLAG M2 antibody (Sigma).

2.3.3 Western Blotting

Cells were rinsed with ice-cold PBS and lysed in ice-cold lysis buffer (50 mM HEPES, 120

mM NaCl, 2 mM EDTA, 1.0% NP-40, one tablet of phosphatase inhibitor cocktail per 10 ml, and

one tablet of EDTA-free protease inhibitors per 10 ml). The soluble fractions of cell lysates were

61

isolated by centrifugation at at 20,000G for 10 min by centrifugation in a microfuge at 4°C.

Proteins were denatured by addition of 5X sample buffer, boiled for 5 min, resolved by 8% or 10%

SDS-PAGE, and analyzed by immunoblotting for the FLAG-tagged proteins.

2.3.4 Enzyme Assays

Enzyme sources were cell lysates obtained from large plates of tetracycline induced and

not induced cells lysed on ice in 0.9% NaCl, 1% Triton X-100, and including protease inhibitors

(Beheshti Zavareh et al, 2012; Korczak et al, 2000). Mgat1 enzyme activity was measured using

synthetic acceptors. The assays contained 10 µl of cell lysate, 2 mM Man(1,3) Man(1,6) Glc-

O(CH2)7CH3 acceptor (Toronto Research Chemicals), 0.5 µCi [6-3H]-UDP-GlcNAc (44,400

dpm/nmol) in 50 mM MES pH 6.5, 2 mM Mn2Cl2, 5mM AMP in total volume of ~20 µl. After

1.5 h of incubation at 37°C, 1 ml of ice-cold water was added to stop further reaction and assays

were either frozen or processed immediately. Mgat5 enzyme activity was determined as transfer

of UDP-[6-3H] GlcNAc to the synthetic acceptors. The reaction contained 10 µl of cell lysate, 1

µCi of [6-3H]-UDP-GlcNAc (44,000 dpm/nmol), 50 mM MES pH 6.5, 0.5 M GlcNAc, 1mM UDP-

GlcNAc, 25mM AMP, in a final volume of 20 µl, with synthetic acceptors 1 mM

βGlcNAc(1,2)αMan(1,6)βGlc-O(CH2)7CH3 for Mgat5. Reactions were incubated at 37°C for 180

min for the Mgat5 assay. Endogenous activity for the enzymes was measured in the absence of

acceptor, and subtracted from values determined in the presence of added acceptor. Reactions

were stopped with 1ml of H2O, and enzyme products were separated from radioactive substrates

by binding them to 50 mg C18 cartridges (Alltech), preconditioned with methanol rinsing and water

washes. Reactions were loaded and the columns washed 3 times with water. Radio labeled

products were eluted with methanol directly into scintillation vials with two separately applied 250

62

µl aliquots of methanol, and the radioactivity determined by liquid scintillation counting. Mgat6

enzyme activity was assayed according to the method of Taguchi et al (Taguchi et al, 2000) using

as acceptor substrate, PA-agalacto [(2,6),(2,4)] tetra-antennary and 100 mM UDP-GlcNAc in

buffer 150 mM HEPES-NaOH, 60 mM MnCl2, 200 mM GlcNAc, 0.5% Triton X-100 and 2 mg/ml

BSA for 4h at 37oC. The product was separated by HPLC on TSKgel ODS-80TM (0.46 x 15 cm,

Tosoh, Tokyo, Japan), eluted with 20 mM ammonium acetate (pH 4.0)/0.02% 1-butanol at flow

rate of 1 ml/min, column temp at 55℃ and detection by fluorescence (excitation/emission

=320/400 nm)

2.3.5 Quantitative Lectin Fluorescence Imaging

Cells were plated in a 96-well plate at 2000 cells/well in high-Glc DMEM supplemented

with 10% FBS, 2 mM Gln, and incubated at 37°C and 5% CO2 with and without tetracycline for

24h to induce gene expression. Cells were then fixed for 15min with 4% paraformaldehyde,

washed with PBS, and incubated for 1h at room temperature in 50 µl PBS, 1/5000 of Hoechst

33342, and either 1/1000 of 2mg/ml Alexa Fluor-488 conjugated lectin ConA or L-PHA

(Invitrogen, Carlsbad, CA). After washing with PBS, cells were imaged and staining per cell

quantified using IN Cell Analyzer 1000 automated fluorescence imaging system.

2.3.6 Cell Viability Assay

Following plating in a 96-well cell culture plate, and treatment with various concentrations

of Glc, Gln and FBS in DMEM for at least 24h, AlamarBlue cell viability reagent (Invitrogen) was

added at 1/10th the volume directly to cells in cell culture media. The plate was then returned to

the incubator at 37°C and 5% CO2 in a humidified atmosphere for an additional incubation of 6h.

63

Cell viability was measured fluorometrically, as relative fluorescence units (RFU), in a microplate

reader (Gemini Fluorescence from Molecular Devices) following the reduction of the non-

fluorescent oxidized dye AlamarBlue by viable and metabolically active cells to its fluorescent

product, using fluorescent excitation and emission wavelengths of 570 and 585 nm respectively.

The number of metabolically active, proliferating, viable cells correlates with the magnitude of

dye reduction and fluorescence emission intensity from each well.

2.3.7 Cell Membrane Preparation for N-Glycan Profiling

For N-glycan analysis by LC-MS, HeLa and Hek293 Flp-In-TREx cells were cultured in

standard culture medium, DMEM plus 10% FBS at 37°C and 5% CO2 in a humidified atmosphere

for 24h, with and without 1ug/ml tetracycline to induce gene expression. ~2x107 cells per treatment

were rinsed with phosphate buffer saline (PBS), trypsinized, collected in pellets, frozen on dry ice,

and stored at -80°C. The frozen pellets were suspended in 2 ml of lysis buffer containing 50 mM

Tris-HCl (pH 7.4), 0.1 M NaCl, 1 mM EDTA and protease inhibitor cocktail (Roche Diagnostics,

Mannheim, Germany) and kept on ice for 20 min and then homogenized using a polytron

homogenizer (Omni TH tissue homogenizer, Omni International, Inc., VA; 15 sec, 7 times on ice

bath). The following approach was taken, according to the procedure reported by Nakano and

colleagues (Nakano et al, 2011). The homogenized cells were centrifuged at 2000g for 20 min at

4°C. The supernatant was diluted with 2 ml of Tris-buffer (50 mM Tris-HCl (pH 7.4), 0.1 M NaCl)

and then were sedimented by ultracentrifugation at 120000g for 80 min at 4°C (swing rotors,

Himac, Hitachi Koki). The supernatant was discarded, and the membrane pellet was suspended in

100 µl Tris-buffer. After adding 400 µl Tris-buffer containing 1% (v/v) Triton X-114, the

suspended mixture was homogenized by pipetting strongly. The homogenate was chilled on ice

64

for 10 min and incubated at 37°C for 20 min and then phase partitioned by centrifugation at 1940g

for 2 min. The upper aqueous phase was removed. The lower detergent phase was further mixed

with 1 ml of ice-cold acetone and kept at -25°C overnight to precipitate proteins and remove any

detergent. After centrifugation at 1940g for 2 min, the precipitated cell membrane proteins were

stored at -25°C if not used immediately.

2.3.8 Enzymatic Release and Purification of N-Glycans

The precipitated membrane proteins were dissolved with 10 µl 8 M Urea. The solubilized

proteins were dotted (2.5 µl x 4 times) onto PVDF membrane pre-wetted with ethanol. After drying

the PVDF membrane with cold blast of a dryer for 10 min, the PVDF membrane was rinsed with

ethanol for 1 min and then rinsed three times for 1 min with water. The protein on the membrane

was stained for 5 min with (800 µl solution A: 0.1% (w/v) Direct Blue 71 (Sigma-Aldrich) in 10

ml solution B: acetic acid : ethanol : water = 1:4:5). After destaining with solution B for 1 min, the

PVDF membrane was dried with cold blast of a dryer for 10 min. Protein stained blue were cut

from the PVDF membrane and placed in separate wells of a 96-well microtiter plate. The spots

were then covered with 100 μL of 1% (w/v) polyvinylpyrrolidone 40000 in 50% (v/v) methanol,

agitated for 20 min, and rinsed with water (100 µl x 5 times). PNGase F (3U in 10 μl of 30 mM

phosphate buffer (pH 7.3)) was added to each well and incubated at 37°C for 15 min. An additional

10 µl of water was added to each well and incubated at 37 °C overnight to release N-glycans.

During the incubation, the sample wells were sealed with amplification tape to prevent

evaporation. To collect the released N-glycans, the samples were sonicated (in the 96-well plate)

for 10 min, and the released N-glycans (20 µl) were transferred to 1.5-ml polypropylene tubes.

The sample well was rinsed with water (50 µl twice), and the washings combined. To completely

65

generate the reducing terminus, ammonium acetate buffer (100 mM, pH 5.0, 20 µl) was added to

the released N-glycans for 1 hour at room temperature. After evaporating to dryness, the glycans

were resolved with 10 µl of 50 mM KOH and then reduced by adding 10 µl of 2 M NaBH4 in 50

mM KOH at 50°C for 3 hours. 1 µL of acetic acid was added to stop the reaction, and the N-glycan

alditols were desalted using a cation-exchange column. The glycan alditols were eluted with water

(50 µl twice), dried, and the remaining borate was removed by the addition of (100 μL x 3)

methanol and drying under vacuum. To remove sialic acids from the glycans 100 µl of 2 M acetic

acid was added to the dried glycan samples and the samples were incubated at 80oC for 2 hours

and then were evaporated to dryness. The desialylated N-glycan alditols were re-suspended in 10

mM NH4HCO3 (20 µL) immediately before glycan analysis by liquid chromatography-

electrospray ionization mass spectrometry (LC-ESI MS).

2.3.9 LC-ESI MS for Analysis of N-Glycan Alditols

N-Glycan alditols were separated using a HyperCarb porous graphitized carbon column (5

μm HyperCarb, 0.32 mm I.D. × 100 mm, Thermo Scientific) under the following gradient

conditions. The separation was performed using a sequence of isocratic and two segmented linear

gradients: 0–8 min, 10 mM NH4HCO3; 8–53 min, 6.75–15.75% (v/v) CH3CN in 10 mM

NH4HCO3; 53–73 min, 15.75–40.5% (v/v) CH3CN in 10 mM NH4HCO3; and increasing to 81%

(v/v) CH3CN in 10 mM NH4HCO3 for 6 min and re-equilibrated with 10 mM NH4HCO3 for 15

min at a flow rate of 5μl/min (LC/MSD Trap XCT Plus Series 1100, Agilent Technologies, USA).

The injection volume of samples was 5 µl. In the MS (Esquire HCT, Bruker Daltonics GmbsH,

Bremen, Germany), the voltage of the capillary outlet was set at 3.5 kV, and the temperature of

the transfer capillary was maintained at 300°C. The flow rate of nitrogen gas for drying was 5

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l/min. The MS spectra were obtained in the negative ion mode over the mass range m/z 150 to m/z

3000. The scan rates were 8100 amu/s for the MS mode and the MS/MS mode.

Monoisotopic masses were assigned with possible monosaccharide compositions using the

GlycoMod tool available on the ExPASy server (http://au.expasy.org/tools/glycomod; mass

tolerance for precursor ions is +/- 0.1 Da) and the proposed oligosaccharide structures were

provided at UnicarbKB database (http://unicarbkb.org/), and further verified through annotation

using a fragmentation mass matching approach based on the MS/MS data. Validation of the

technical reproducibility of the analytical conditions such as retention time and mass number was

carried out using known glycans derived from bovine fetuin before analyzing any experimental

samples. The relative abundance of each glycan structure on the cell membrane glycoproteins was

calculated based on the peak area of the ion chromatogram of the corresponding glycan structure

extracted using the mass of the [M-2H]2- ion, after processing of the peaks (smoothing algorithm;

Gauss, smoothing widths; 1pnts, S/N thresholds; 1, no exclusion mass, using Bruker Daltonics

DataAnalysis software ver. 3.4) (Nakano et al, 2011).

2.3.10 Metabolite Analysis by LC-MS/MS

To determine the relative levels of metabolites, HeLa and Hek293 Flp-In-TREx cells were

cultured in 6-well plates at 37°C and 5% CO2 in a humidified atmosphere for 24h in various

nutrient conditions as described before (Abdel Rahman et al, 2013), with and without tetracycline

to induce gene expression. Media was aspirated and cells rinsed on the plates with warm PBS. The

plates were snap frozen in liquid N2 and moved to -80°C until extraction. The metabolites were

rapidly extracted by addition of 1 ml ice-cold solution of (40% acetonitrile, 40% methanol, and

20% water). After quenching, the cells were scraped and transferred to 1.5 ml tube and shaken for

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http://unicarbkb.org/

1 h at 4°C and 1000 rpm in a Thermomixer (Eppendorf, Germany). The samples were spun down

at 14,000 rpm, for 10 min at 4°C (Eppendorf, Germany), and then the supernatant transferred to

fresh tubes to be evaporated to dryness in a CentreVap concentrator at 40°C (Labconco, MO). The

dry extract samples were stored at -80°C for LC/MS analysis. Cell number for each culture

condition was determined for normalization purposes by trypsinization of parallel replicate wells.

The dry metabolite extracts were reconstituted in 200 µl of water containing internal

standards (500 µg/ml and 300 µg/ml of D7-glucose and 13C915N-Tyrosine, respectively), for the

purpose of calibration, normalization and quantification. The mixture of metabolites was injected

twice through the HPLC (Dionex Corporation, CA) in gradient reversed phase column Inertsil

ODS-3, 4.6 mm internal diameter, 150 mm length, and 3-µM particle size for positive and negative

mode analysis. In positive mode analysis, the mobile phase gradient ramps from 5% to 90% of

acetonitrile in 16 min, then after 1 min at 90%, the composition returns to 5% acetonitrile in 0.1%

acetic acid in two min. In negative mode, the acetonitrile composition ramped from 5 to 90% in

10 min, then after 1 min at 90%, the gradient ramped back to 5% acetonitrile in buffer-A (0.1%

tributylamine, 0.03% acetic acid, 10% methanol). The total runtime in both modes was 20 min,

the samples were stored at 4°C, and the injection volume was 10 µl. An automated washing

procedure was developed before and after each sample to avoid any sample carryover.

The eluted metabolites were analyzed at the optimum polarity in MRM mode on

electrospray ionization (ESI) triple-quadrupole mass spectrometer (ABSciex4000Qtrap, Toronto,

ON, Canada). The mass spectrometric data acquisition time for each run is 20 min, and the dwell

time for each MRM channel is 10 ms. Common mass spectrometric parameters are the same as

tuning conditions described below, except: GS1 and GS2 were 50 psi; CUR was 20 psi, and CAD

was 3 and 7 for positive and negative modes, respectively, and source temperature (TEM) was

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400°C. Signal was normalized to internal standard and cell number. The LC-MS/MS system does

not resolve hexose, hexosamine and sugar-nucleotide stereoisomers, including Glc/Galactose,

GlcNAc/GalNAc, GlcNAc-6P/GlcNAc-1P, and UDP-GlcNAc/UDP-GalNAc. To monitor trends

in metabolic pathways, I referred to these stereoisomers in their Glc forms.

Stock solutions were prepared for each metabolite standard at a concentration of 100 µM

in 40/60 methanol/water (v/v), 0.1%NaOH. A final concentration (10µM) of each metabolite was

obtained for mass spectrometric tuning. A standard mixture of all metabolites was prepared at 200,

500, and 3000 nM as a sensitivity and specificity quality control. Ten additional serially diluted

samples were prepared ranging from 1 nM to 2 µM for linearity assessments. A mixture of all the

standard metabolites was used daily to check the LC-MS/MS system for optimal ionization

polarity, declustering potential (DP), precursor ion (Q1), product ion (Q3), and collision energy

(CE). Ion source potential (ISP) was 4500 V for positive and negative modes. Nebulizer gas (GS1)

and bath gas (GS2) were 10 psi, curtain gas (CUR) was 15 psi, and collision gas (CAD) was 4 psi.

Source temperature (TEM) was set to zero and interface heater was ON. The mass spectrometer

was maintained and calibrated using a special kit designed by the manufacturer (ABSciex,

Canada).

The raw LC-MS/MS data was imported to MultiQuant version 2.0.0 (ABSciex, Toronto,

ON, Canada) and the extracted ion chromatogram (XIC) peaks were integrated. The result table

contains the area, area ratio (area of analyte/area of internal standard), retention time, and

concentration, and is then exported from MultiQuant as text files to MarkerView version 1.2.11

(ABSciex, Toronto, ON, Canada) for normalization with cell number and protein content. Cell

number normalized data was analyzed using the MetaboAnalyst online software for metabolomic

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analysis (http://www.metaboanalyst.ca/MetaboAnalyst/), and the KEGG databases

(http://www.genome.jp/kegg/pathway.html) (Xia et al, 2012).

2.3.11 LC-MS/MS Measurement of 15N15N-Glutamine Uptake and Metabolism

Flp-In-TREx Hek293 Mgat5 cells were treated for ~24h with 1 mg/ml of tetracycline, to

induce Mgat5 gene expression, in DMEM with 2.5 mM glucose and no glutamine plus 10% FBS,

with and without 15 mM GlcNAc. The media was then changed to DMEM with 2.5 mM glucose

and 1 mM 15N15N-glutamine without FBS, for 1, 5 and 10 min. After incubation, cells were

collected and metabolites extracted using the procedure detailed above. The chromatographic

peaks for metabolite interest, and correct transitions for the labelled form of the metabolite were

integrated using MultiQuant (ABSciex, Toronto, ON, Canada) (Soliman et al, 2014).

2.4 Results

2.4.1 Tet-Inducible Expression of Mgat1, Mgat5 and Mgat6 in Human Cells

To generate transgenic cell lines, HeLa and Hek293 cell lines with a single Flp-In-TREx

insertion site were transfected with vectors for insertion of tetracycline (tet)-inducible FLAG-

tagged Mgat1, Mgat5 or Mgat6. The Flp-In-TREx site is designed for controlled expression from

a single insertion, and indeed, tet-inducible expression of FLAG-tagged protein was detected in

multiple independent HeLa cell clones (Figure 2.1B-E). Independent clones displayed similar

levels of tet-inducible expression, with increases in Mgat1 and Mgat5 enzyme activity by 4 and 30

fold, respectively, while Mgat6 introduced a novel activity not present in mammalian cells (Figure

2.1F-H). Changes in N-glycan composition at the cell surface were determined by ConA and L-

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PHA lectin staining. ConA binds high-mannose or mono-antennary N-glycans, while L-PHA

binds Mgat5-modified tri- and tetra-antennary N-glycans. In HeLa cells, tet-induced Mgat1

enhanced ConA but not L-PHA binding. L-PHA binding was enhanced in tet-induced Mgat5,

while ConA was unchanged (Figure 2.2A and B). Tet-induced Mgat6 reduced L-PHA binding

suggesting the addition of the Mgat6 branch to Mgat5-branched N-glycans may interfere with L-

PHA binding (Figure 2.2B).

To further characterize the tet-induced Mgat1, Mgat5 and Mgat6 changes in N-glycan

distributions, structures from the Flp-In-TREx HeLa cell glycoproteins were released by PNGase,

and N-glycan alditols analyzed by LC-ESI MS. We interpret this data in light of the known

specificity of the enzymes (Schachter, 1986; Watanabe et al, 2006) and prior knowledge of the

Golgi pathway in mammalian cells (Schachter, 1986; Watanabe et al, 2006). We limited our

interpretation to structures with the N-glycan core plus 1-4 Hex-HexNAc units, and the cartoon

structures are drawn without branch linkages. The MS features corresponding to the masses of 16

predicted N-glycan structures were quantified. Twenty-three additional structures are indicated by

their m/z values and probable compositions (Figure 2.2C-E). The profile for tet-induced Mgat1

HeLa cells revealed a 4-10 fold increase in the immediate product of Mgat1 (structures 14, 15 and

16) but not the down-stream tri- and tetra- antennary N-glycans (Figure 2.2C). This was consistent

with the lectin binding profile of the cells (Figure 2.2A and B), and suggests a backup in N-glycan

processing due to insufficient α−mannosidase II activity. Tet-induced Mgat5 revealed a small

increase in peaks with m/z of the expected products; 2,2,6 tri- and 4,2,2,6 tetra-antennary N-

glycans with and without fucose (Figure 2.2D, structures 3, 5, 8 and 11).

In tet-induced Mgat6 HeLa cells, novel peaks were observed with m/z predicted to be the

4,2,2,4 tetra- and 4,2,2,4,6 penta-antennary products of Mgat6 with and without fucose (Figure

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2.2E). Structures 10, 12 and 13 are predicted to be tetra- and penta- antennary products of avian

Mgat6, while the native 4,2,2,6 tetra-antennary N-glycans (structures 8 and 11) were reduced. This

is consistent with our earlier studies showing that Mgat5-modified intermediates are the preferred

acceptor for Mgat6 (Watanabe et al, 2006), thus conversion of tri- and tetra- to tetra- and penta-

branched N-glycan (Figure 2.2E). Structures 8 and 11 are preferred ligands for L-PHA binding,

and their conversion to 10, 12 and 13 is associated with reduced L-PHA binding (Figure 2.2B).

A low amount of Mgat6 activity was detected in the absence of tet (Figure 2.1H), as well as a

small amounts of structures 10, 12 and 13, suggesting some background (leaky expression) of the

Mgat6 transgene (Figure 2.2E). HeLa cells had insufficient α-mannosidase II activity to process

the tet-induced Mgat1 product (i.e. HexNAc-Hex5-HexNAc2-Asn), which appeared to be the

limiting factor for tet-induced increases in Mgat5 and Mgat6 branched N-glycans.

2.4.2 Tet-Inducible Mgat1, Mgat5 and Mgat6 N-Glycan Branching

Hek293 is an immortalized human embryonic cell line with epithelial morphology

previously reported to be growth-sensitive to changes in N-glycan branching and HBP (Lau et al,

2008b). Hek293 cell lines with tet-inducible Mgat1, Mgat5 or Mgat6 were made by the Flp-In-

TREx system, and displayed tet-inducible expression of the enzymes, comparable to that seen in

the HeLa cell lines (Figure 2.3A). Flp-In-TREx Hek293 clones also displayed very similar

induction of enzyme activities and glycan branching, as illustrated for tet-inducible Mgat5 (Figure

2.3B). Tet-induced Mgat5 expression alone increased L-PHA binding by ~13%, 30 mM GlcNAc

supplementation to HBP by ~30%, and addition of both by ~83%, clearly displaying a synergistic

interaction (Figure 2.3C).

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To characterize the structural effects of tet-induction, N-glycans were released by PNGase

and the N-glycan alditols were analyzed by LC-ESI MS. Overall, Hek293 cells displayed a greater

fold change in tri- and tetra-antennary N-glycans than HeLa cells (Figure 2.3D-F). The tetra-

antennary structures 8 and 11 were reduced in tet-induced Mgat1 Hek293 cells (Figure 2.3D).

This may seem counter-intuitive, but is consistent with multistep ultrasensitivity to UDP-GlcNAc

that defines the Golgi N-glycan branching pathway in non-transformed epithelial cells (Lau et al,

2007). Briefly, multistep ultrasensitivity is due to declining affinities of Mgat1, Mgat2, Mgat4 and

Mgat5 enzymes for UDP-GlcNAc (Km values increases from 0.04 mM to 10 mM across the

pathway). Mgat1 over-expression has been shown to reduce substrate available to Mgat4 and

Mgat5, but rescue is possible by extracellular GlcNAc supplementation to increase UDP-GlcNAc

pool (Lau et al, 2007). Over-expression of Mgat4 and Mgat5 in malignant cells reduces pathway

dependence on UDP-GlcNAc, and thereby loss of regulatory control by HBP (Lau & Dennis,

2008).

Tet-induced Mgat5 Hek293 cells displayed a ~2 fold increase in tri-antennary (structure 3

and 5), and a ~2 fold increase in tetra-antennary (structure 8 and 11) N-glycans (Figure 2.3E).

Tet-induced Mgat6 Hek293 cells produced the same novel oligosaccharide features observed in

HeLa cells, predicted to be the 2,2,4,6 tetra- (structures 7 and 10), 4,2,2,4 tetra- (structures 9 and

12) and 4,2,2,4,6 penta- (structure 13), while native 4,2,2,6 tetra- (structures 8 and 11) were

reduced (Figure 2.3F).

2.4.3 Regulation of Cellular Metabolite Levels by Mgat1, Mgat5, Mgat6 and HBP

Next, metabolites in HeLa and Hek293 Flp-In-TREx cells were measured by liquid

chromatography-tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring

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(MRM), targeting glycolysis, amino acids, nucleotides, TCA cycle, and HBP metabolites.

Supplementing cells with GlcNAc increases UDP-GlcNAc available to the N-glycan branching

enzymes, and should interact with tet-induced Mgat1, Mgat5 or Mgat6 to reveal more intense

phenotypes (Lau et al, 2007; Sasai et al, 2002). Cells were cultured in standard nutrient-rich

medium, with and without tet, in the presence of 0, 15 or 30 mM GlcNAc for 24h, and cellular

metabolites were extracted and analyzed by LC-MS/MS. In both Flp-In-TREx HeLa and Hek293

cells, GlcNAc supplementation increased intracellular UDP-GlcNAc up to ~5 fold over control.

Unsupervised clustering of metabolite data revealed that GlcNAc and tet-induced Mgat1, Mgat5

or Mgat6 increased the levels of many metabolites in HeLa cells, and Hek293 cells (Figure 2.4A).

In the absence of GlcNAc, tet-induced Mgat1, Mgat5 or Mgat6 was associated with modest

increases in metabolite levels, which was further enhanced by GlcNAc supplementation. For

example, tet-induced Mgat5 and Mgat6 were additive with GlcNAc supplementation for increases

in lactate in Hek293, suggesting increased glycolytic flux (Figure 2.4B).

Increased lactate production is commonly observed in proliferating cells, along with high

rates of oxidative respiration that require mechanisms of stress tolerance (Kaplon et al, 2013;

Levine & Puzio-Kuter, 2010). Reactive oxygen species (ROS) are toxic byproducts of oxidative

respiration that require buffering by glutathione (GSH) in a reaction that produces glutathione

disulfide (GSSG) (Suzuki et al, 2010). The ratio of glutathione (GSH) to glutathione disulfide

(GSSG) declined markedly with GlcNAc supplementation in Hek293 cells, with and without tet-

induction for all three enzymes (Figure 2.4C). Mgat6 Hek293 cells displayed lower GSH/GSSG

ratio without induction, which declined further with tet-induction and GlcNAc treatment, possibly

due to low-level expression of Mgat6 in the absence of tet. Taken together, the results suggest that

N-glycan branching and HBP increase the levels of many metabolites, including glycolysis and

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TCA cycle intermediates, as well as amino acids, with increased disposition of carbon into

oxidative respiration (Figure 2.4D). However, tet-induction and/or GlcNAc supplementation did

not increase cell proliferation in the rich-medium conditions used in these experiments (Abdel

Rahman et al, 2013).

2.4.4 Tet-Inducible Mgat5 Enhances Amino Acid Uptake and Growth in Nutrient-Poor

Conditions

Nutrient-rich culture medium does not reflect typical in vivo conditions, where glucose and

amino acid availability are frequently limiting. Therefore, Glc and Gln were titrated in the medium,

and cell growth compared for non-induced and tet-induced N-glycan branching. HeLa tumor cells

displayed growth autonomy that did not benefit from tet-induced Mgat5 when Glc and/or Gln

supply was limiting. In contrast, tet-induced expression of Mgat5 alone in Hek293 cells increased

cell growth under low Glc/Gln conditions (Figure 2.5A). This is also consistent with the greater

inducibility of Mgat5-branched N-glycans in the Hek293 cell line (Figure 2.2D and 2.3D).

Therefore, Hek293 cells were cultured for 24h in DMEM medium containing four Gln/Glc

concentrations (0/2.5, 0/10, 2/2.5, 2/10 mM), selected to reveal growth-sensitive effect of tet-

induced Mgat5 (Figure 2.5A and B). Cellular metabolites were analyzed by LC-MS/MS, and

principal component analysis (PCA) of data revealed clear separation of experimental groups

based on Glc and Gln supply (Figure 2.5C). Using PCA technique, which reduces the

dimensionality of data but maintains the variation, we observe Mgat5 overexpression having the

greatest effect in low Gln. In 2 mM Gln, separation was observed for high versus low Glc, but no

further separation was detected with tet-induced Mgat5, suggesting Glc uptake is not likely the

primary effect of tet-induced Mgat5. However, in 0 mM Gln, the tet-treated and untreated cells

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were separated, and with greater distance when Glc supply was also low (Figure 2.5C). Note that

in 0 mM Gln without tet, Glc had no effect on separation in the PCA plot. We conclude that in

Gln-depleted conditions, tet-induced Mgat5 enhances intracellular metabolite levels and growth

by increasing amino acid uptake, while Glc supply acts as a modifying factor.

In Gln-depleted conditions, most metabolites that were measured increased with tet-

induced Mgat5, while the effect of tet-induced Mgat5 in richer medium (i.e. 2 mM Gln plus 2.5 or

10 mM Glc) was minimal (Figure 2.6 and 2.8). In Gln-depleted medium, intracellular Gln was

reduced by ~10 fold, and glutamate (Glu) and α-ketoglutarate by ~2-3 fold (Figure 2.6A and 2.7).

Tet-induced Mgat5 increased intracellular amino acid content, while depleting them from the

growth media (Figure 2.6B). Increased consumption from the media by tet-induced Mgat5 cells

was observed for Arg, His, Met, Phe, Ser, Trp, Tyr, Val, Ileu, Leu, and Cys, which includes the

essential amino acids (EAA). The catabolism of amino acids provides NH4+ for Glu and Gln

synthesis, as well as anaplerotic support to the TCA cycle. Indeed, tet-induced Mgat5 increased

Glu and TCA cycle intermediates including succinyl-CoA, succinate, fumarate and malate (Figure

2.7). Glycolysis and HBP intermediates were also increased in low Gln conditions, suggesting a

reduced demand for Glc due to the increased uptake and catabolism of amino acids (Figure 2.8).

Tet-induced Mgat5 increased GlcN-P, GlcNAc-P and UDP-GlcNAc, which contribute positive

feedback to the N-glycan branching enzymes, and downstream effectors. Mgat5 has a low affinity

for UDP-GlcNAc, and is therefore sensitive to Glc and Gln flux through HBP (Abdel Rahman et

al, 2013). Our results suggest that up-regulation of Mgat5 may generate positive feedback to

nutrient uptake by increasing UDP-GlcNAc supply to the Golgi (Figure 2.4D). The effect of tet-

induced Mgat5 on central metabolite levels are summarized in Figure 2.9.

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Increased Gln uptake and catabolism is a critical feature of metabolism in proliferating

cells (Levine & Puzio-Kuter, 2010; Wellen & Thompson, 2012). Branched-chain EAA uptake is

mediated by a bidirectional transporter complex (Slc7a5 and Slc3a2), that exports Gln in exchange

for the import of branched-chain EAA (Nicklin et al, 2009). To measure Gln uptake directly, cells

were pulse-labeled with dual labeled 15N15N-Gln for 1, 5 and 10 min. Tet-induced Mgat5 increased

L-PHA staining and modestly increased uptake of 15N15N-Gln, which was further increased in an

additive manner by 15 mM GlcNAc (Figure 2.10A). 15N15N-Gln conversion to 15N-Glu and re-

amination to single-labeled 15N-Gln showed similar kinetics (Figure 2.10, right). Tet-induced

Mgat1 suppressed L-PHA staining by ~20%, consistent with observed suppression of tetra-

antennary structures 8 and 11 by mass spectrometry (Figure 2.3D). Tet-induced Mgat1 alone had

little effect on 15N15N-Gln uptake (Figure 2.10B). These results demonstrate that Mgat5-modified

N-glycan branching and HBP cooperate to up-regulate Gln import.

2.5 Discussion

In this report, transgenic Hek293 and HeLa cell lines with single site insertion for tet-

inducible over-expression of Mgat1, Mgat5, and avian Mgat6 enzymes were generated, and their

effects on N-glycan profiles and central metabolism characterized by mass spectrometry. N-glycan

profiling revealed unexpected differences between HeLa and Hek293 cell lines following tet-

inducible transgene over-expression. In HeLa cells, tet-induced Mgat1 markedly increased the

expected hybrid N-glycans, but had a much smaller effect on downstream branching, suggesting

the cells have insufficient α-mannosidase II activity to process the additional Mgat1 product. In

Hek293 cells, tet-induced Mgat1 did not increase hybrid N-glycans, but did partially suppress

downstream branching, consistent with previous characterization of the pathway as multistep

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ultrasensitive to UDP-GlcNAc (Lau & Dennis, 2008; Lau et al, 2007). Tet-induced Mgat5

increased tri- and tetra-antennary N-glycans in Hek293 more robustly than in HeLa cells. HBP

supplementation was additive with tet-induced Mgat1, Mgat5 and Mgat6 for increasing levels of

cellular metabolites in nutrient-rich medium. The nutrients were not a stimulus for cell

proliferation, but rather catabolism as indicated by increased lactate and oxidative respiration.

GlcNAc supplementation at >15 mM overcame the UDP-GlcNAc-limiting effects of Mgat1 over-

expression in Hek293T cells (Lau et al, 2007). Similarly, GlcNAc supplementation acts down-

stream of α-mannosidase II and Mgat1, and markedly up-regulated metabolite levels in HeLa cells.

GlcNAc supplementation might mimic nutrient abundance in glucose, nitrogen and/or fatty-acid

metabolism, and such conditions might arise in hyperglycemia, and excess intake or catabolism of

amino acids and/or fat.

2.5.1 Mgat6 Enhances Functionality of N-Glycan Branching in Mammalian Cells

The Mgat5 product is the preferred acceptor for Mgat6, which adds a GlcNAcβ1,4 branch

that is not present in mammals (Watanabe et al, 2006). The mature N-glycan branches function in

an additive manner to regulate galectin binding to growth factor receptors and nutrient transporters

at the cell surface (Lau et al, 2007). The addition of an ectopic branch to N-glycans in mammalian

cells might display an additive and stronger phenotypic effect compared to over-expression of

Mgat5. Tet-induced Mgat6 alone, and with GlcNAc supplementation, in both HeLa and Hek293

cell lines, displayed the strongest enhancement of metabolite levels of the three enzymes tested

(Figure 2.4A). It is possible that over-expression of Mgat6 qualitatively changes the interaction

between mammalian branching enzymes and HBP, or that Mgat6 has substrate preferences that

alter the relationship between nutrient transporters activities. Overall, our results provide

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compelling evidence that functional redundancy of branches and their conditional regulation by

HBP is a critical feature of metabolic regulation (Dennis & Brewer, 2013). The emergence of

Mgat6 in tetrapod dinosaurs and retention of the gene in modern birds may play a critical role in

their metabolic rates and fitness.

2.5.2 Mgat5 Enhances Metabolism Under Glutamine-Deprived Conditions

Instead of using the typical cell culture conditions, which have excess amounts of Glc

(25mM) and Gln (4mM), more limiting condition were used to discern the effect of Mgat5

overexpression on metabolism and cell growth. In low Gln/Glc culture conditions, tet-induced

Mgat5 alone increased proliferation in cultures of Hek293 cells, but not in HeLa cells, consistent

with the relative change in N-glycan branching upon tet-Mgat5 induction in the two cell lines. Tet-

induced Mgat5 in Hek293 cells stimulated the uptake of essential amino acids, and increased the

levels of glycolytic, HBP and TCA pathway intermediates (Figure 2.6 and 2.7). In a more direct

assay of uptake, tet-induced Mgat5 and 15 mM GlcNAc stimulated 15N15N-Gln uptake in a

cooperative manner (Figure 2.8). GlcNAc supplementation to HBP has been shown to stimulate

Gln uptake and catabolism in Bax-/-Bak-/- lymphoma cells in the absence of Glc (Wellen et al,

2010). In these experiments, surface IL-3 receptor was reduced in Glc-starved condition, but could

be rescued by GlcNAc supplementation to UDP-GlcNAc and N-glycan branching (Wellen et al,

2010). Furthermore, IL-3 receptor-dependent signaling stimulated transcription of SLC1A5, a

high-affinity Gln transporter, and SLC6A19, a transporter of Leu and Gln. However, the present

study is the first to show a direct effect of Mgat5-mediated branching on amino acid uptake and

metabolic flux, in low Gln/Glc (i.e. 0 / 2.5 mM) +10% FBS culture conditions.

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Gln is not an essential amino acid, but high growth rates in embryonic and cancer cells

depend on the import of Gln, and anaplerotic conversion to α-ketoglutarate which supports the

TCA cycle (DeBerardinis et al, 2008). SLC7A5/SLC3A2 is a bidirectional transporter that imports

branched-chain essential amino acids (BCAA) (Leu, Ile,Val) in exchange for Gln efflux (Nicklin

et al, 2009). The Gln transporter, SLC1A5 is widely expressed and required to support BCAA

uptake, although in some cells, Gln biosynthesis from α-ketoglutarate and glutamate can support

internal needs and SLC7A5/SLC3A2 activity (Hassanein et al, 2013). Tet-induced Mgat5 alone in

Hek293 cells was sufficient to increase uptake of Gln and EAA in low Gln/Glc culture conditions.

Gln is also a positive regulator of HBP (Abdel Rahman et al, 2013) and may drive reciprocal

positive feedback between N-glycan branching and metabolism (Figure 2.4D and 2.9). Mgat5-

deficient mice are hypoglycemic and display a reduced sensitivity to glucagon (Johswich et al,

2014), and here we present the first evidence of a cell autonomous regulation of metabolism by N-

glycan branching. Up-regulation of Mgat5 in Hek293 cells stimulated amino acid uptake and

increased metabolite levels under low Gln/Glc culture conditions. Further work is required to

identify the various nutrient transporters regulated by N-glycan branching and their contribution

to development, disease and environmental stress.

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Figure 2.1 Branching pathway and inducible expression of branching enzymes.

(A) N-glycan branching pathway modified from Essentials of Glycobiology 2nd edition, Chapter 8 Figure 5 (Varki et al, 2009). GlcNAcT-I (Mgat1), GlcNAcT-V (Mgat5), avian GlcNAcT-VI (Mgat6) are circled. (B-D) Expression of transgene proteins detected by Western blots probed with antibodies to Flag and tubulin. Flag-tagged GlcNAcT-I, GlcNAcT-V (upper band), and GlcNAcT-VI have expected molecular weights of 53kD, 86kD and 54kD, respectively. Flp-In-TREx HeLa clones were cultured in DMEM with 25 mM Glc, 4 mM Gln, 10% FBS (normal culture conditions), with or without 1 ug/ml of tetracycline (tet) for 24h. (E) Tet dose response in clone 8 Mgat5 Flp-In-TREx HeLa cells. (F-H) N-acetylglucosaminyltransferases activities measured in cell lysates from tet-inducible expression of Flag-tagged Mgat1, Mgat5 and Mgat6 in different Flp-In-TREx HeLa cell clones.

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Figure 2.2 N-glycan profiles of transgenic Flp-In-TREx HeLa cells.

(A) ConA and (B) L-PHA lectin binding to Mgat1, Mgat5 and Mgat6 in Flp-In-TREx HeLa clones, with and without 1 ug/ml tet for 24h, measured by fluorescence microscopy, *p<0.05 and **p<0.01 by student t-test. (C-E) LC-ESI MS chromatogram for N-glycans in Flp-In-TREx HeLa cells expressing (C) Mgat1 clone 3, (D) Mgat5 clone 8, and (E) Mgat6 clone 6. The red chromatogram is tet-induced and black is non-induced. Orange circles indicate increases, and blue circles indicate decreases. Cells were grown in DMEM, 25 mM Glc and 4 mM Gln +10% FBS (standard conditions) with and without tet for 24h.

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Figure 2.3 N-glycan profiles of transgenic Flp-In-TREx Hek293 cells by LC-ESI MS.

(A) Expression of transgene proteins detected by western blot probed with anti-Flag and tubulin antibodies in three different Flp-In-TREx Flag-tagged Mgat5 Hek293 clones, and Mgat5 enzyme activity measured in cell lysates from tet-inducible expression of Flag-tagged Mgat5 in clone 4. (B) Tet-induced Mgat5 increases complex-type N-glycan branching in Hek293 Mgat5 clones 4 and 8, quantified by Alexa-488 conjugated L-PHA fluorescence imaging. For quantification, mean ±SD, one-way ANOVA with Dunnett's multiple comparison test. (C) L-PHA binding of tri- and tetra-antennary N-glycans displays a synergistic effect for GlcNAc supplementation with tet-induced Mgat5 in clone 4. *p<0.05 and **p<0.01 by student t-test compared to -tet control. (D-F) LC-ESI MS chromatogram of N-glycans in Flp-In-TREx Hek293 cells expressing (D) Mgat1, (E) clone 4 Mgat5, and (F) Mgat6. Experimental conditions as described in Figure 2.

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Figure 2.4 Metabolite levels are sensitive to tet-induced branching and HBP stimulation.

(A) Mgat1, Mgat5 and Mgat6 Flp-In-TREx HeLa and Hek293 cells were grown in standard culture conditions with and without tet (T) and 0, 15 or 30 mM GlcNAc supplementation for 24h. Each row represents a biological replicate (n=4-5). Metabolites in cell lysates were measured by targeted LC-MS/MS and normalized to cell number. Data for 129 metabolites was analyzed by unsupervised clustering and presented as heat maps. (B) Lactate and (C) GSH / GSSG ratios in Flp-In-TREx Hek293 cells with and without tet-induced Mgat1, Mgat5 and Mgat6 induction for 24h. Additive effects of tet and GlcNAc, *p<0.05 by student t-test. (D) Scheme for increased nutrient uptake and flow to catabolism with tet-induced increased N-glycan branching and GlcNAc supplementation. Green and yellow arrows indicate putative positive feedback from central metabolism through de novo HBP to N-glycan branching, which in turns promotes cell surface residency of transporters and more nutrient uptake.

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Figure 2.5 Growth of Mgat5 Flp-In-TREx Hek293 cells in defined Glc and Gln conditions.

Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured with and without tet in medium modified for Glc/Gln content as indicated + 10% FCS for 48h. (A) Cell growth as a function of Glc and Gln concentrations, with and without tet-induced Mgat5 (cell count ± SD, n=3). (B) Growth conditions (grey bars) corresponding to (C) metabolite profiling by LC-MS/MS, and analyzed by principal component analysis (PCA).

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Figure 2.6 Amino acid levels increase with Mgat5 expression under Gln-deprived conditions.

(A) Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions indicated, and data was normalized to 2.5/10 Gln/Glc no-tet (second bar from the right, red line) and plotted as fold change (mean ±SD, n=5, *p<0.05). The axis labels are shown for Ala at the top. The pathway scheme is from Chapter 20 (Amino Acid Degradation and Synthesis) Lippincott's Illustrated Reviews: Biochemistry. (B) Heat map of amino acid levels in cells and medium showing data for each of 4-5 replicates. In limiting conditions, tet-induced Mgat5 increased cellular amino acids content, and the depleted growth medium of the same, indicating increased amino acids uptake. Red is high, green is low. White boxed area highlights contrasting effect of +tet on cells and medium.

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Figure 2.7 Tet-induced Mgat5 increases TCA cycle intermediates.

Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions indicated. The data was normalized to 2.5/10 mM Gln/Glc no-tet conditions (second bar from the right, red line). *p<0.05 by student t-test for tet-induced change at 0/2.5 mM Gln/Glc condition.

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Figure 2.8 Tet-induced Mgat5 increases HBP and glycolysis metabolites under Gln/Glc limiting conditions.

Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions, and normalized data graphed as fold change. The data was normalized to 2.5/10 mM Gln/Glc no-tet conditions (second bar from the right, red line). *p<0.05 by student t-test for tet-induced change at 0/2.5 mM Gln/Glc condition.

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Figure 2.9 Summary of metabolite changes with tet-induced Mgat5 in Hek293 cells under low Gln/Glc conditions.

Metabolites in bold were measured by LC-MS/MS, and the EAA are marked in blue. * indicates 2 fold or more decrease as a result of growth in low Gln/Glc 0/2.5 mM conditions. Green arrows mark metabolites increased by tet-induced Mgat5.

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Figure 2.10 Tet-induced Mgat5 branching stimulates Gln uptake.

Flp-In-TREx Hek293 (A) Mgat5 clone 4 and (B) Mgat1 clone 7 cells, were grown with and without tet and 15 mM GlcNAc in standard culture conditions. Gln was removed for 16 h and cells were pulsed with 1 mM 15N15N Gln for the indicated times. 15N15N Gln and 15N Gln levels in cell lysates were measured by LC-MS/MS. *p<0.05 and **p<0.01 by student t-test comparing nil to tet or GlcNAc to GlcNAc+tet, with n=5-6 samples. The bar graphs on the left compare branching by L-PHA staining of Mgat1 and Mgat5, with and without tet-induction.

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Chapter 3

Metabolic Reprogramming by the Hexosamine Biosynthetic Pathway and

Golgi N-Glycan Branching

A version of this chapter is in revision at the Journal of Biological Chemistry

Michael C. Ryczko, Judy Pawling, Rui Chen, Anas M. Abdel Rahman, Kevin Yau,

Daniel Figeys, and James W. Dennis

Attributions:

Mouse-work and targeted metabolomics mass-spectrometry experiments in Figures 3.1A-F, 3.2A-

G, 3.3A,D-F,I, 3.5E-G, 3.7A,G,I, 3.8A-C,E, and 3.9A-D,G-I were performed together with Judy

Pawling.

Site-specific characterization of N-glycan structures on liver glycoproteins summarized in Table

3.2 and displayed in Figure 3.6 was performed by Rui Chen using LC-MS/MS.

All other experiments were designed, performed and analyzed by Michael Ryczko.

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3.1 Summary

UDP-GlcNAc is an essential substrate for protein N-glycosylation and Golgi remodeling

of N-glycans, a crucial modification to cell surface receptors and nutrient transporters. de novo

synthesis requires glucose, glutamine, acetyl-CoA and uridine-triphosphate, and cellular levels of

UDP-GlcNAc are sensitive to the supply of these central metabolites. GlcNAc is salvaged from

dietary and glycoconjugate turnover into the HBP to generate UDP-GlcNAc. Herein I examined

the effects of GlcNAc supplementation and salvage on metabolism in C57BL/6 mice. Fat and

body-weight increased without affecting calorie-intake, activity, or energy-expenditure. Chronic

oral GlcNAc increased hepatic UDP-GlcNAc, and GlcNAc content in N-glycans on hepatic

glycoproteins, indicating increased Golgi N-glycan branching. Furthermore, glucose homeostasis,

hepatic glycogen and lipid metabolism were altered with GlcNAc treatment, especially during

fasting. In cultured cells, GlcNAc enhanced the uptake of glucose, glutamine and fatty acids, and

increased synthesis and lipid accumulation, while inhibition of N-glycan branching blocked

GlcNAc-dependent lipid accumulation. The N-acetylglucosaminyltransferase (Mgat1,2,4,5)

enzymes of the N-glycan branching pathway display multistep ultrasensitivity to UDP-GlcNAc,

due to declining affinity for the common substrate, with Mgat5 operating below its Km, thus

making it sensitive to increased UDP-GlcNAc. Mgat5-/- mice exhibit a lean phenotype, and oral

GlcNAc rescued fat accumulation, consistent with functional redundancy of N-glycan branches.

However, fat accumulation was rescued at the expense of lean mass, suggesting Mgat5 plays a role

in lean to fat body composition. Our results suggest that GlcNAc reprograms cellular metabolism

by enhancing nutrient uptake and lipid storage through UDP-GlcNAc supply to N-glycan

branching pathway.

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3.2 Introduction

GlcNAc is found widely in glycoconjugates, many with ancient origins, notably the

GlcNAc polymer chitin found in arthropods, molluscs, insects and fungi, as well as the N-glycans

that modify glycoproteins produced in the secretory pathway (Banerjee et al, 2007; Ruiz-Herrera

et al, 2002). Biosynthesis of glycoconjugates requires high-energy sugar-nucleotide donors. de

novo UDP-GlcNAc biosynthesis by the HBP requires Glc, Gln, Ac-CoA, and UTP, metabolites

that are central to carbon, nitrogen, fatty acid, nucleotide, and energy metabolism. In cell culture,

starvation levels of Glc suppress UDP-GlcNAc levels, whereas elevated Gln increases UDP-

GlcNAc (Abdel Rahman et al, 2013). The rate limiting step in HBP is the conversion of Fru-6P

and Gln to GlcN-6P and glutamate by GFAT (Broschat et al, 2002). Overexpression of GFAT

increases UDP-GlcNAc, and transgenic mice overexpressing GFAT in the liver displayed obesity,

enhanced glycogen storage, impaired glucose tolerance and insulin resistance at 8 months of age

(Veerababu et al, 2000). GFAT overexpression in skeletal muscle, adipose tissue, or pancreatic β-

cells also resulted in insulin resistance (Cooksey & McClain, 2002).

Glucosamine-6P N-acetyltransferase (GNPNAT1/GNA1) converts GlcN-6P and Ac-CoA

to GlcNAc-6P (Oikawa et al, 1986), which flows primarily into UDP-GlcNAc in mammalian cells

(Wellen et al, 2010). GNA1 is essential for de novo UDP-GlcNAc synthesis, but GNA1-deficient

mouse embryo fibroblasts expressed glycoproteins containing GlcNAc, indicating that salvage of

GlcNAc from autophagy and exogenous glycoconjugates is a significant source of UDP-GlcNAc

(Boehmelt et al, 2000). No mammalian plasma membrane transporter for GlcNAc has been

identified, and uptake of extracellular GlcNAc is thought to occur by bulk- or fluid-phase

endocytosis (Dennis et al, 2009). Intracellular GlcNAc is converted to GlcNAc-6P by N-

acetylglucosamine kinase (NAGK), and enters HBP as substrate for phosphoglucomutase (PGM3)

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and UDP-N-acetylglucosamine pyrophosphorylase (UAP1). GlcNAc-6P N-deacetylase has been

reported (AMDHD2) (Bergfeld et al, 2012), but its activity is very low in mammalian cells, and

therefore salvaged GlcNAc contributes almost exclusively to the UDP-GlcNAc pool and protein

glycosylation (Wellen et al, 2010).

The glycosylation pathways utilizing UDP-GlcNAc include N-glycosylation of membrane

glycoproteins and O-GlcNAcylation of cytosolic proteins. Cytoplasmic, nuclear and mitochondrial

O-GlcNAc modified proteins have been identified as regulators of cell signaling and gene

transcription (Hardiville & Hart, 2014). O-GlcNAcylation of proteins in insulin-Akt signaling

pathway, and transcription factors and cofactors that regulate glycogen synthesis, gluconeogenesis

and lipogenesis have been shown to play a role in metabolic homeostasis (Dentin et al, 2008; Ruan

et al, 2013; Yang et al, 2008). OGT catalyzes the addition of GlcNAc, while O-GlcNAcase

removes it. Interestingly, increasing O-GlcNAcylation globally using a selective inhibitor of O-

GlcNAcase did not result in insulin resistance or perturb glucose homeostasis in rodents or 3T3-

L1 adipocytes (Macauley et al, 2010a; Macauley et al, 2010b). On the other hand, mutations in

genes encoding Golgi N-glycan branching enzymes have been shown to sense glucose and regulate

insulin release by pancreatic β-cells, as well as control hepatic glucagon receptor sensitivity

(Johswich et al, 2014; Ohtsubo et al, 2005). Enzymes Mgat1, Mgat2, Mgat4a/b/c and Mgat5 each

catalyze the addition of GlcNAc in a specific β-linkage to the trimannosyl core of N-glycans

(Schachter, 1986), and the branching pathway is characterized by multistep ultrasensitivity to

UDP-GlcNAc. Multistep ultrasensitivity arises from a ~300 fold decline in affinity for the common

donor substrate UDP-GlcNAc moving down the pathway from Mgat1 to Mgat5 (Lau et al, 2007).

Therefore, Mgat5 and the synthesis of tri- and tetra-antennary N-glycans are most sensitive to

UDP-GlcNAc levels, and metabolite flux through HBP.

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The GlcNAc branches are further extended with galactose, fucose and sialic acid,

generating glycan structures with affinity for galectins, C-type lectins and siglecs at the cell surface

(Dennis et al, 2009). Galectins bind N-acetyllactosamine (Galβ1-4GlcNAcβ) branches on N-

glycans, and their affinities for membrane glycoproteins are proportional to both the extent of

branching and the N-glycan number (NXS/T consensus site) encoded in the protein sequence

(Hirabayashi et al, 2002; Lau et al, 2007). For example, Glc transporter Glut4 has a single N-

glycan chain and is retained at the cell surface in a UDP-GlcNAc dependent manner (Lau et al,

2007). Galectin-9 binds to the single multi-antennary N-glycan chain on pancreatic Glut2 and

prevents its loss to endocytosis, thus allowing glucose import and glucose-stimulated insulin

secretion (Ohtsubo et al, 2013; Ohtsubo et al, 2005). Mgat4a-/- mice fail to retain Glut2 on the

surface of pancreatic β-cell cells, resulting in reduced secretion of insulin and hyperglycemia

(Ohtsubo et al, 2005). N-glycan branches are partially redundant and act cooperatively to support

glycoproteins at the cell surface (Dennis & Brewer, 2013). Inducible expression of Mgat5 in

Hek293 human embryonic kidney cells growing in low Glc and Gln medium increased N-glycan

branching, intracellular metabolites, uptake of amino acids, and cell growth (Abdel Rahman et al,

2014). With a Km value of ~10 mM for UDP-GlcNAc, Mgat5 is very sensitive to HBP activity and

output, which may vary with feeding conditions in animals.

Here I asked whether GlcNAc supplemented to drinking water of mice can be salvaged in

the HBP and interact through N-glycan branching to increase anabolic metabolism and weight-

gain. In wild-type C57BL/6 mice GlcNAc supplementation increased liver UDP-GlcNAc levels,

and enhanced fat accumulation and weight-gain. Chronic GlcNAc intake altered hepatic

metabolite levels, decreased respiratory exchange ratio (RER), and increased lipid accumulation

and body-weight, without affecting food intake, physical activity or energy expenditure.

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Furthermore, GlcNAc supplementation globally increased the relative GlcNAc content of N-

glycans found on hepatic glycoproteins, by increasing Golgi N-glycan branching. Mgat5-deficient

mice display adult phenotypes that may be linked in part through metabolism, including delayed

oncogene-induced tumor progression (Granovsky et al, 2000), autoimmune sensitivity (Demetriou

et al, 2001), depression-like behavior (Soleimani et al, 2008), resistance to weight-gain on a high

fat diet, and loss of adult stem cells and early aging (Cheung et al, 2007). GlcNAc supplementation

partially restored anabolic metabolism in Mgat5-/- mice and primary hepatocytes, as indicated by

increased lipid accumulation. In cultured cells, GlcNAc enhanced uptake of Glc, Gln and fatty

acid, and promoted lipid accumulation in an N-glycan-dependent manner. My results indicate that

GlcNAc supplementation increases nutrient uptake and lipid storage by enhancing UDP-GlcNAc

supply to the Golgi N-glycan branching pathway.

3.3 Materials and Methods

3.3.1 Chemicals and Materials

GlcNAc was obtained as “Ultimate Glucosamine®” (Wellesley Therapeutics, Toronto,

Ontario, Canada), GlcN was obtained from Sigma Chemicals (St. Louis, MO), and both were

dissolved in water. Metabolite standards and reagents were obtained from Sigma Chemicals (St.

Louis, MO) with minimal purity of 98%. Stable-isotope 15N2-L-Glutamine was purchased from

Cambridge Isotope Laboratories Inc. (Andover, MA). All organic solvents and water used in

sample and LC/MS mobile phase preparation were LC/MS grade and obtained from Fisher

Scientific (Fair Lawn, NJ). Antibodies to phospho-Thr172 AMPK-α, phospho-Ser79 ACC,

phospho-Ser235/236 S6, phospho-Ser473 Akt, phospho-Ser641 GS were obtained from Cell

Signaling Technology. Antibodies to Tubulin were purchased from Sigma-Aldrich, fatty acid

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synthase from BD Scientific, and mAb CTD 110.6 from Covance. PhosSTOP Phosphatase

Inhibitor Cocktail and Complete Protease Cocktail were purchased from Roche. PVDF membrane

(Immuno-Blot, 0.2 µm, 7.0 x 8.5 cm) was purchased from Bio-Rad (Hercules, CA). Alexa Fluor-

488 conjugated lectins ConA and L-PHA, BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-

4-Bora-3a,4a-Diaza-s-Indacene), and 2-NBD-Glucose (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-

yl)Amino)-2-Deoxyglucose) were purchased from Invitrogen (Carlsbad, CA). QBT Fatty Acid

Uptake Assay Kit containing BODIPY-dodecanoic acid fluorescent fatty acid analog was

purchased from Molecular Devices.

3.3.2 Mice

Weight and age-matched young C57BL/6 male mice were used in GlcNAc

supplementation experiments. For experiments involving Mgat5 wild-type and null mice, age and

sex-matched littermates on the C57BL/6 background were used as described previously (Cheung

et al, 2007; Johswich et al, 2014). All mice were maintained in cages of up to 5 mice per cage, in

a normal 12 h light/12 h dark cycle on either 4%, 9% or 22% fat food diet (Tekland rodent diet),

with or without GlcNAc (0.5, 5.0 or 15 mg/ml) or GlcN (0.5 mg/ml) in the drinking water, as

indicated, for the specified duration of time. Bottles with drinking water containing GlcNAc or

GlcN were changed twice weekly. Mice were euthanized using CO2 inhalation, and dissections

carried out rapidly to remove, weigh, and freeze liver tissue samples on dry-ice, and store in tubes

at -80°C until further biochemical and molecular analysis. All experiments using mice were

conducted according to protocols and guidelines approved by the Toronto Centre for

Phenogenomics animal care committee.

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3.3.3 Phenotyping in vivo

Body-weight of mice was measured on a weekly basis. Daily food consumed was

determined every 24 h over 10 days, and expressed as calorie intake per body-weight, at 21 weeks

following start of GlcNAc supplementation. Body composition, in terms of lean and fat tissue

mass, was determined by dual energy X-ray absorptiometry (DEXA) (PIXImus) or magnetic

resonance imaging (MRI) (Echo Medical Systems). Whole-body O2 consumption and CO2

production rates were recorded for 20 h with the use of an open-circuit indirect calorimeter

(Oxymax Lab Animal Monitoring System, Columbus Instruments). Respiratory exchange ratios

(RER) was calculated as the molar ratio of VCO2 to VO2 for 5 mice per group, averaging the

measurements for the light and dark cycle. The activity of mice in three spatial dimensions plus

time was continuously measured during the same 20 h using infrared photocells attached to the

metabolic cage during dark and light cycles. Total activity included ambulatory movement

(locomotion), and body movements (grooming and rearing on hind legs). Energy Expenditure (EE)

per mouse was calculated as EE = (3.815 + 1.232 × VCO2/VO2) × VO2. Water and food were

available ad libitum in the metabolic chamber and determined for individual mouse. To minimize

the potential influence of circadian rhythms on experimental outcomes, standardized periods of

fasting or experimental analyses were utilized. For intraperitoneal glucose tolerance test, mice

were fasted for 18 h before intraperitoneal injection of 0.01 ml/g of body-weight of a glucose

solution containing 150 mg/ml. For the glucagon tolerance test, mice were fasted for 5 h and

injected intraperitoneally with a glucagon solution of 1.6 g/ml (0.01 ml/g of body-weight). For

both tolerance tests blood samples were drawn from the tail vein at different time intervals over

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the course of 2 h, and blood glucose levels measured using a Glucometer Elite blood glucose meter

(Bayer, Toronto, Canada).

3.3.4 Targeted Metabolomics

Frozen liver tissue (~100 mg per sample) was pulverized using the CellCrusherTM

cryogenic tissue pulverizer under liquid nitrogen, which reduces most tissues to a fine, easily

recoverable powder. The soluble polar metabolites were extracted by addition of 1 ml of ice-cold

extraction solvent consisting of 40% acetonitrile, 40% methanol and 20% water, followed by

vortexing for 30 sec, and shaking at 1,000 rpm for 1 h at 4°C in a ThermoMixer (Eppendorf,

Germany). For cells grown in cell culture plates, following a PBS wash and flash freezing in liquid

nitrogen, metabolites were extracted by adding 1 ml of same ice-cold extraction solvent to the

wells of the plate, scraping the cells, collecting in 1.5-ml tubes, and vortexed and shaken as

described above. Following extraction, samples were spun down at 20,000G for 10 min at 4°C,

and the supernatant transferred to fresh tubes to be evaporated to dryness in a CentriVap

concentrator at 40°C (Labconco, MO). The dry extract samples were stored at -80°C for later LC–

MS/MS analysis. Samples were separated twice on a reversed phase HPLC column Inertsil ODS-

3 of 4.6-mm internal diameter, 150-mm length, and 3 µM particle size (Dionex Corporation,

Sunnyvale, CA) for MS analysis in positive and negative modes. The eluted metabolites were

analyzed at the optimum polarity in MRM mode on electrospray ionization triple-quadrupole mass

spectrometer (4000 QTRAP; ABSciex, Toronto, Canada) as previously described (Abdel Rahman

et al, 2013). The LC-MS/MS system does not resolve isomers of hexose or n-acetyl-hexosamine,

including glucose/galactose/mannose, GlcNAc/GalNAc/ManNAc, or their phosphorylated or

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sugar-nucleotide forms. In the metabolomic data and throughout the manuscript I refer to the Glc

form of these isomers. UDP-GlcNAc/UDP-GalNAc isomerase is widely present in mammalian

cells and may maintain homeostatic balance of these sugar-nucleotides.

3.3.5 Biochemical Studies and Histology

Western blot analysis was performed on frozen liver tissue and cells. Protease inhibitor and

phosphatase inhibitors were added to all buffers before experiments. Protein were resolved by 8%

or 10% SDS-PAGE, analyzed by immunoblotting with primary antibodies incubated overnight at

4°C, followed by incubation with secondary infrared fluorescence antibodies, and visualization of

specific proteins using the Odyssey infrared imaging system (LI-COR). To measure relative

protein abundance, band intensities were quantified using ImageJ software. Intensity values were

normalized to Tubulin. Blood samples at sacrifice were collected via cardiac puncture. For plasma

preparation, blood samples were supplemented with trasylol, EDTA, and diprotin and centrifuged

at 4,000G at 4°C for 5 min. Basic plasma clinical chemistry analyses were performed. Plasma

leptin, insulin and glucagon hormones were measured using blood samples obtained at sacrifice

with a mouse endocrine LINCOplex kit (Linco Research) following the manufacturer’s protocol.

Adiponectin and leptin were measured using ELISA kits (EMD Millipore) according to

manufacturer’s instructions. To measure glycogen content in the liver, 20–50 mg of tissue was

acid-hydrolyzed in 2 M HCl at 95°C for 2 h and neutralized using 2 M NaOH. The liberated

glucose was assayed spectrophotometrically using the glucose assay reagent (hexokinase method)

(Amresco) following the manufacturer’s protocol.

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Liver lipids were extracted using the Folch-Lees method. The extracts were filtered, and

lipids recovered in the chloroform phase. Individual lipid classes were separated by thin layer

chromatography using Silica Gel 60 A plates developed in petroleum ether, ethyl ether, acetic acid

(80:20:1) and visualized by rhodamine 6G. Phospholipids, diglycerides, triglycerides and

cholesteryl esters were scraped from the plates and methylated using BF3/methanol. The

methylated fatty acids were extracted and analyzed by gas chromatography. Gas chromatographic

analyses were carried out on an Agilent 7890A gas chromatograph equipped with flame ionization

detectors, a capillary column (SP2380, 0.25 mm x 30 m, 0.25 µm film, Supelco). Helium was used

as a carrier gas. The oven temperature was programmed from 160°C to 230°C at 4°C/min. Fatty

acid methyl esters were identified by comparing the retention times to those of known standards.

Inclusion of lipid standards with odd chain fatty acids permitted quantitation of the amount of lipid

in the sample. For histology, frozen liver sections were stained with oil red O, while formalin-

fixed liver sections were stained with periodic acid-Schiff, and mounted on glass slides for

microscopy imaging.

3.3.6 Site-Specific Characterization of Hepatic N-Glycosylation

Frozen mouse liver tissue was cut into pieces (diameter less than 2 mm) and rinsed with

cold PBS to remove any residual blood. Liver tissue was homogenized in 2% SDS and 100 mM

Tris-HCI lysing buffer (pH=7.4), with protease inhibitor, by blade homogenizer. The homogenate

was sonicated to shear the DNA content, and the lysate was then centrifuged at 20,000G for 15

min to remove the insoluble content. A 10 fold volume of cold acetone was added to the

supernatant, and the protein was precipitated at -20oC overnight. Protein precipitant was collected

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by centrifugation at 20,000G for 15 min, and rinsed with cold ethanol/acetone (50%:50%, v/v)

twice to remove trace SDS. Dried protein precipitant was dissolved with 8M urea in PBS, and

protein concentration was measured by DC Protein Assay (Bio-Rad). Liver lysates from mice in

the same group were pooled, and 500 µg of protein from pooled lysate was used as the starting

material for the proteomics study. Protein was denatured, reduced by adding 10 mM DTT, and

incubated at 56oC for 45 min. Reduced protein was alkylated by adding 20 mM IAA and incubated

at room temperature in the dark for 30 min. The sample solution was diluted 5 times with 100 mM

TEAB, and trypsin was added at 1:50 ratio. The protein was digested by incubation at 37°C

overnight. After digestion, peptides from control and GlcNAc treated liver samples were labelled

with “light” and “heavy” reagents respectively, as described elsewhere (Boersema et al, 2009).

Labelled peptides were mixed and desalted with a C18 cartridge. 10% of the elution was dried by

Speed Vac for total proteome analysis, and the rest was used for glycopeptide enrichment by

hydrophilic interaction chromatography (HILIC SPE). The elutions from HILIC SPE were divided

into two aliquots: one aliquot was dried by Speed Vac, and the other one was incubated with

peptide-N-glycosidase F (PNGase F) for deglycosylation. Both deglycosylated peptides and intact

glycopeptides were analyzed by LC-MS/MS with an Orbitrap-Elite mass spectrometer.

Quantification of total proteome and N-glycosylated sites were achieved with Maxquant, and

results were processed with Perseus (Boersema et al, 2013). Intact glycopeptides were identified

and quantified by matching the Y1 ion from the MS/MS spectrum to the deglycosylated peptides

identified with Mascot, and extracting the peak of the peptides precursor (Chen et al, 2014). Glycan

compositions of intact glycopeptides were validated manually after software analysis.

3.3.7 Cell Culture

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AML12 immortal hepatocytes were purchased from ATCC and grown in 1:1 mixture of

Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F12 medium with 0.005 mg/ml insulin,

0.005 mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone, and 10% fetal bovine

serum (FBS). Mouse primary hepatocytes were isolated as previously described (Johswich et al,

2014), seeded in Williams E media supplemented with serum, 2 mM Glutamax, 100 U/ml

penicillin, and 100 mg/ml streptomycin on Primaria plates (BD Biosciences, Mississauga, ON),

and treated overnight with different concentrations of GlcNAc. Cellular lipid accumulation in lipid

droplets was detected using the lipophilic fluorescent probe BODIPY 493/503, while lectin

binding to N-glycans was determined using ConA and L-PHA. For quantitative microscopic

fluorescence imaging, cells were seeded in a 96-well plate (Costar 3595) at 2000 cells/well in

regular media at 37°C and 5% CO2, and treated with GlcNAc for indicated times. Cells were then

fixed for 15 min with 4% paraformaldehyde, washed with PBS, and incubated for 1 h at room

temperature in 50 µl PBS containing Hoechst 33342, and either BODIPY 493/503, or Alexa Fluor-

488-conjugated ConA or L-PHA. After three washes with PBS, cells were imaged and staining

per cell quantified using IN Cell Analyzer 1000 automated fluorescence imaging system and the

accompanying software package.

For the endpoint measure of glucose uptake, AML12 cells were grown under normal

conditions with and without GlcNAc for 20 h. Media was changed to glucose-free media and 100

µM 2-NBD-Glc was added for 1 h at 37°C. The amount of 2-NBD-glucose inside the cells was

measured as mean fluorescence intensity (MFI), with cells gated for similar size, using the

Beckman Coulter Gallios flow cytometer, and analyzed with Kaluza analysis software. For the

LC–MS/MS measurement of 15N2-Gln uptake and metabolism in AML12 and HeLa cells, cells

were grown in their respective standard media for 24 h, followed by a switch to respective media

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without Gln or serum but containing 5.5 mM Glc, with or without GlcNAc for an additional 24 h.

Cells were then treated with 1 mM 15N2-Gln containing media for indicated times. After

incubation, cells were washed in PBS, quenched in liquid nitrogen, and metabolites extracted using

the procedure detailed above to measure the relative intracellular levels of 15N2-Gln, 15N-Glu, and

15N-Glu metabolites by LC–MS/MS.

3T3-L1 fibroblast cells were kindly provided by Dr. Amira Klip (Sick Kids Hospital), and

maintained in 25 mM Glc containing DMEM with 10% FBS. To differentiate 3T3-L1 cells into

adipocytes, cells were grown to confluency, and after 2 days post-confluent cells were stimulated

with the differentiation cocktail containing 5 µg/ml insulin, 1 µM dexamethasone, 0.5 mM

isobutylmethylxanthine, and 5 µM troglitazone (all purchased from Sigma) in DMEM with 10%

FBS. Cells were treated with differentiation cocktail for 2 days, after which cells were grown in

DMEM containing 10% FBS and 5 µg/ml insulin with media replaced every 2 days. 3T3-L1

fibroblasts differentiated into adipocytes, and treated with GlcNAc for 24 h prior to the assay, were

seeded in a 96-well plate for the QBT fatty acid uptake assay performed according to the

manufacturer’s instructions outlined in the kit. The kinetic reading monitoring the uptake of non-

esterified long-chain fatty acid analog, using fluorescent BODIPY-FA, was quantified as Area

Under the Curve (AUC) for Relative Fluorescence Units (RFU).

3.3.8 Statistical Analysis

All data are expressed as mean ± SEM values for experiments, including numbers of mice

as indicated. Statistical significance was determined using unpaired, two-tailed Student’s t-tests to

compare differences between two group means (two samples and equal variance), or one-way

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ANOVA with Dunnett’s multiple comparison test using Microsoft’s Excel or GraphPad Prism

Software). In all experiments, a p-value of 0.05 or less was considered to be statistically significant.

Metaboanalyst (http://www.metaboanalyst.ca/MetaboAnalyst/), a comprehensive online software

suite for metabolomic data analysis was used to generate the PCA scatter plot (Xia & Wishart,

2011).

3.4 Results

3.4.1 Oral GlcNAc Supplementation Alters Liver Metabolism

To identify dietary conditions where a modifying effect of GlcNAc supplementation on

body-weight could be detected, three groups of young C57BL/6 male mice were maintained on

diets containing 4%, 9% and 22% fat content. As expected, dietary fat content correlated with

increased body-weight and with decreased RER (Figure 3.1A-C). Rates of weight-gain differed

significantly in mice on different fat diets, with near cessation of growth on 4% diet, while 9% and

22% showing continued weight-gain. The 4% and 9% fat diets were selected for further

experiments, as both left capacity for GlcNAc-dependent weight-gain, and represent diverse base-

line dietary conditions.

To determine if oral GlcNAc readily enters the bloodstream and accumulates in tissue, an

oral gavage in mice with a bolus administration of heavy-GlcNAc (13C6-GlcNAc) was performed.

Blood samples were collected at different time intervals over a 3 hour period. Heavy-GlcNAc in

blood peaked at 30 minutes and then gradually started to be cleared from the circulation, suggesting

that it was being taken up by tissue (Figure 3.1D). At 180 minutes the blood level of heavy-

GlcNAc has returned to normal, and heavy-GlcNAc was taken up by tissues and assimilated into

UDP-GlcNAc via salvage HBP (Figure 3.1F). Heavy-GlcNAc was also co-administered with

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http://www.metaboanalyst.ca/MetaboAnalyst/

heavy-glucose (Glc-d7). As expected, the uptake of heavy-Glc peaked earlier than heavy-GlcNAc

(Figure 3.1E), but the similarity displayed by the two curves suggests that a specific transporter

might be responsible for GlcNAc uptake from the gut and subsequent release into circulation,

where it is made available to the tissues.

GlcNAc is commercially available as a dietary supplement, and oral GlcNAc in rats has

shown no overt toxicity (Lee et al, 2004; Takahashi et al, 2009). A clinical study in children with

severe treatment-resistant inflammatory bowel disease showed clinical improvement in a majority

of cases following treatment with 3–6 g/day (~60–120 mg/kg/day) of oral GlcNAc as an adjunct

therapy (Salvatore et al, 2000). Oral GlcNAc at 0.25 mg/ml in drinking water, estimated to be ~40

mg/kg/day, suppressed spontaneous autoimmune diabetes in non-obese diabetic mice when

initiated prior to disease onset (Grigorian et al, 2007). Importantly, oral GlcNAc increased UDP-

GlcNAc supply and Mgat5-modified β1,6-GlcNAc-branched N-glycans on cell surface

glycoproteins of T-cells in vivo (Grigorian et al, 2007). To explore dosage, GlcNAc at 0.5, 5.0 and

15 mg/ml (~80-2,500 mg/kg/day) was continuously provided in drinking water to weight and age-

matched male mice on 4% fat diet starting at weaning. Body-weight was measured weekly, and

hepatic metabolites were analyzed by liquid-chromatography tandem mass-spectrometry (LC-

MS/MS) 90 days following the beginning of GlcNAc treatment. Body-weight during this period

of rapid growth increased with 5.0 and 15 mg/ml GlcNAc, on 4% fat diet (Figure 3.2A).

GlcNAc at 0.5 mg/ml or greater in the drinking water increased hepatic GlcNAc-P and

UDP-GlcNAc by 20%, while ATP remained unchanged (Figure 3.2B). Moreover, principal

component analysis (PCA), a visualization technique that reduces the dimensionality of data by

maintaining as much variance as possible, performed with the entire metabolite data set revealed

a directional trend with GlcNAc dosage, moving away from the cluster of untreated controls

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(Figure 3.2C). Intermediates in hepatic glycolysis and gluconeogenesis were increased with oral

GlcNAc: including Glc-6P, Fru-6P, Fru-1,6BP, phosphoglycerate (3- & 2-PG),

phosphophenolpyruvic acid (PEP), lactate, glycerol, and glycerol-3P (Figure 3.2D). Oral GlcNAc

also increased hepatic levels of certain TCA cycle metabolites (Figure 3.2E), as well as the

glucogenic polar amino acid Gln, and decreased essential amino acids Thr, Trp, Phe, and Iso/Leu

(Figure 3.2F). Pyridine nucleotides soluble coenzymes, present as oxidized and reduced

nicotinamide adenine dinucleotides: NAD+, NADH and NADPH were increased; as were the

reduced and oxidized forms of the antioxidant glutathione (GSH and GSSG respectively) (Figure

3.2G). The GSH to GSSG ratio however was unaffected, while the ratio of NADH to NAD+ was

elevated, consistent with catabolism supplying oxidative phosphorylation (Figure 3.2G).

3.4.2 Oral GlcNAc Increases Body-Weight without Increasing Food Consumption

I estimate that 0.5 mg/ml of GlcNAc (translating into ~40-80 mg/kg per day) was

equivalent to less than 0.1% of total weight of daily food intake per mouse. Hence, even if GlcNAc

was catabolized it would be an insignificant source of calories, suggesting that conversion to UDP-

GlcNAc may regulate metabolism through protein glycosylation. A GlcNAc dose of 0.5 mg/ml

was used to test for interaction with fat-enriched diet in mice. Oral GlcNAc was initiated at 14

weeks of age in weight-matched wild-type C57BL/6 male mice maintained on 4% and 9% fat diets

(low fat and high fat, respectively), and continued for 30 weeks. Mice were also treated with

glucosamine (GlcN), an amino-sugar dietary supplement used extensively in cell culture

experiments to increase UDP-GlcNAc levels, primarily in O-GlcNAcylation studies (Hardiville &

Hart, 2014). GlcN is taken up efficiently by cultured cells via Glut2 transporter (Uldry et al, 2002),

and converted to GlcN-6P by hexokinases followed by two possible fates, either N-acetylation by

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GNPNAT1/GNA1 to enter HBP, or deamination by GNPDA1 to Fru-6P and use in glycolysis

(Wolosker et al, 1998). GlcNAc treated mice on 9% fat diet displayed significantly increased

weight-gain compared to 9% fat diet alone, while GlcN had a negligible effect, suggesting that it

did not follow the same path as GlcNAc (Figure 3.3A). After 20 weeks of continuous GlcNAc

treatment, the mice had on average 13% and 19% increase in body-weight on 4% and 9% fat diet

respectively, without any discernible increase in daily calorie intake (Figure 3.3B). In fact, after

adjusting for body-weight, GlcNAc treated mice on 9% fat diet consumed fewer calories than

control mice (Figure 3.3C). This suggests that exogenous GlcNAc promotes more efficient uptake

and/or utilization of nutrients, as measured by conversion to biomass. These results were

confirmed in five independent cohorts of mice and apply to both genders.

3.4.3 Oral GlcNAc Increases Lipid Accumulation and Catabolism

At the time of sacrifice, GlcNAc treated mice weighed 10% more on 4% fat diet, and 16%

more on 9% fat diet, than their control counterparts (Figure 3.3D). GlcNAc treated mice had

similar muscle mass, but displayed increased fat content on both diets, as determined by DEXA

(Figure 3.3E). The epidydymal fat-pads were increased by GlcNAc treatment by 46% and 12%,

on the 4% and 9% fat diets respectively (Figure 3.3F). GlcNAc treatment with 9% fat diet resulted

in hepatomegaly, an overall 42% increase in total liver weight, or a 22% increase after correcting

for body-weight (Figure 3.3F). Thus, GlcNAc dependent weight-gain in C57BL/6 mice at 10

months of age was largely due to excess lipid accumulation in multiple sites. Serum free fatty acids

were increased, whereas the level of triglycerides (TG) was the same in GlcNAc treated mice

(Figure 3.3G-H). Consistent with increased fatty acid turnover, serum levels of glycerol and

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glyceraldehyde were elevated; whereas, pyruvate, glycerol-3-phosphate, phosphoglyceric acid,

propionate, and ketone-body 3-ketobutyrate were decreased (Figure 3.3I).

The hepatomegaly observed in mice on GlcNAc with 9% fat diet suggests metabolic stress

that might be revealed by nutrient-sensing signaling pathways. Notably, mTOR is inhibited in low-

nutrient or stressed conditions, which promotes autophagy, hydrolysis of macromolecules and

salvage of the constituents to promote cell survival (Efeyan et al, 2013; Rabinowitz & White,

2010). Western blot analysis of liver lysates revealed a dramatic reduction in the level of ribosomal

protein p-S6, a marker of mTORC1 pathway activity, in GlcNAc treated mice on 9% fat diet

(Figure 3.3J). Since hepatic mTORC1 activity negatively regulates autophagy and production of

ketone bodies for peripheral tissues to use as an energy source, this observation suggests increased

autophagy and macrolipophagy in these mice (Singh et al, 2009). No significant change in AMP-

activated protein kinase (p-AMPK) or Ac-CoA carboxylase (p-ACC) was observed with GlcNAc,

indicators of energy charge and fatty acid synthesis and oxidation (Hagiwara et al, 2012; Hardie,

2012).

3.4.4 Oral GlcNAc Does Not Alter Physical Activity or Energy Expenditure

Open circuit indirect calorimetry was used to estimate whole-body O2 consumption and

CO2 production, while the activity of mice was measured by infrared photocells (Figure 3.4A-C).

The RER was calculated from the volumes of O2 consumed and CO2 produced, and provides a

measure of nutrients oxidized that ranges from 0.7 for oxidation of pure fats, to 1.0 for oxidation

of pure carbohydrates. GlcNAc treated mice on 9% fat diet displayed increased oxidation of fat

compared to mice on 9% fat diet alone (Figure 3.4D-E). GlcNAc treated and untreated mice on

9% fat diet were indistinguishable in total activity (ambulatory, grooming and rearing on hind

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legs), and energy expenditure per mouse (Figure 3.4A and 3.4F). GlcNAc treated mice also

consumed less food and water, but stored more fat, and relied more on oxidation of fat than their

control counterparts (Figure 3.3C and 3.4D-H).

Oral GlcNAc increased hepatic free fatty acids in mice on the 4% fat diet, either fasted or

fed, and to a lesser degree in mice on 9% fat diet, where free fatty acids in controls were already

high (Figure 3.4I). Hepatic TG levels in fasted and fed mice were increased by GlcNAc, but only

on the 9% fat diet (Figure 3.4J). The livers of GlcNAc treated mice were larger, heavier and

appeared pale, suggesting hepatic steatosis. Histology revealed extensive lipid deposition and

confirmed that TG and neutral lipids were increased by GlcNAc treatment over levels observed

with 9% fat diet alone (Figure 3.4K). However serum alanine aminotransferase (ALT), a non-

specific marker of liver damage was unchanged (Table 3.1). Blood plasma TG and electrolytes on

9% fat diet, in both fasted and fed mice, were not different in GlcNAc and respective control

groups (Table 3.1).

3.4.5 Oral GlcNAc Reprograms Fasting Metabolism

Insulin receptor signaling phosphorylates/activates Akt, which leads to

dephosphorylation/activation of glycogen synthase (GS), resulting in glycogen synthesis (Patel et

al, 2004). Upon fasting, hepatic glycogen content, p-Akt, and fatty acid synthase (FASN) were

increased in GlcNAc treated mice, while phosphorylated/inactivated GS (p-GS) was decreased,

relative to fasted GlcN treated and untreated controls (Figure 5A-D). Collectively, these findings

suggest that GlcNAc supplementation enhanced the efficiency of nutrient uptake, and/or provided

extra support for hepatic anabolic metabolism from muscle and adipose tissue, to delay depletion

of glycogen during fasting. Alternatively, the increased reservoir of lipid and the greater

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dependence of GlcNAc treated mice on fatty acid oxidation may reduce glucose consumption and

depletion of glycogen. Indeed, p-AMPK-α and its downstream target p-ACC were increased in

livers of fasted animals on GlcNAc or GlcN (Figure 3.5C-D). p-S6 levels were also elevated in

GlcNAc and GlcN treated mice compared to controls upon 18 h of fasting, possibly a reflection of

both reduced metabolic stress and a delayed fasting response. In this measure, GlcN has a similar

effect to GlcNAc, consistent with some conversion of GlcN to UDP-GlcNAc. The increased

phosphorylation of Akt and S6, and decreased phosphorylation of GS, along with FASN level,

hormonal profile, and hepatic glycogen content suggest delayed fasting; while increased

phosphorylation of AMPK-α and ACC is consistent with fasting and/or stress. Thus, GlcNAc

treated mice show a delay in signaling that triggers glycogen breakdown and autophagy, along

with enhanced AMPK and ACC signaling for promotion of mitochondrial fatty–acid oxidation.

Normally, blood glucose, insulin and leptin levels decrease during fasting, while glucagon

increases. However, GlcNAc treated mice fasted for 18 h also displayed higher blood glucose and

insulin levels, lower glucagon levels, and elevated hepatic glycogen and p-Akt, comparable to that

of GlcNAc treated fed mice (Table 3.1, Figure 3.5A-D). The ratio of circulating insulin to

glucagon was indistinguishable between fasted and fed mice treated with GlcNAc, while the ratio

changed 12-fold in untreated mice (Table 3.1). Circulating serum leptin was increased during both

fasted and fed states in GlcNAc treated wild-type mice on 9% fat diet (Table 3.1 and Figure 9J),

which implies increased fat depots, satiety and reduced appetite, and is consistent with the lowered

food-intake observed in GlcNAc treated mice discussed above (Figure 3.3C and 3.4G). Serum

leptin and insulin levels positively correlate with body-weight and energy stores in adipose tissue,

while adiponectin inversely correlates with the same (Ye & Scherer, 2013). Adiponectin levels

were lower in GlcNAc treated mice on 9% fat diet in the fed state (Table 3.1).

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Hepatic HBP and Mgat5 activity have recently been shown to regulate the sensitivity of

glucagon receptor to glucagon (Johswich et al, 2014). After 5 months of oral GlcNAc treatment,

sensitivity to an injection of glucagon was increased, as indicated by elevated release and lower

disposal rate of hepatic glucose, while glucose tolerance was not affected (Figure 3.5F-G). The

hypersensitivity of glucagon receptor should place a higher demand on insulin to clear the excess

hepatic glucose. In addition to lactate, amino acids are a major source of carbon for

gluconeogenesis. After 7 months of oral GlcNAc, metabolite analysis in 18 h fasted mice revealed

elevated serum levels of gluconeogenic precursors lactate, Gln, Phe and Ile (Figure 3.5E). These

amino acids, along with Tyr and Leu are associated with human obesity and a high risk of diabetes

(Kim et al, 2010; Newgard et al, 2009). Other metabolites associated with obesity, diabetes and

metabolic syndrome were also elevated in GlcNAc treated mice, including sorbitol (Brownlee,

2001), aminoadipic acid (Wang et al, 2013), and uric acid (Johnson et al, 2013) (Figure 3.5E).

3.4.6 Oral GlcNAc Increases Complex N-Glycan Branching in Liver Glycoproteins

Glycopeptides were prepared from liver tissue of control and GlcNAc treated mice, and

differentially labelled with light and heavy stable isotope dimethyl reagents respectively. Light

and heavy labelled glycopeptides were mixed prior to mass spectrometry (MS) analysis. Intact

glycopeptides and their deglycosylated form, obtained by treatment with PNGase F, were analyzed

by LC–MS/MS (Chen et al, 2014). The combined analyses identified glycosites and composition

of N-glycosylation on glycoproteins. Specific glycosites in liver glycoproteins were compared for

GlcNAc content in control and GlcNAc treated mice. Mice supplemented with GlcNAc on 4% and

9% fat diets, in both fed and fasted conditions, displayed significantly increased global GlcNAc

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content in N-glycan structures of liver glycoproteins, which must be attributed primarily to

increased N-glycan branching (Table 3.2).

As a specific example, glycopeptides with site-specific N-glycosylation from the single

transmembrane pass glycoprotein carcinoembryonic antigen related cell adhesion molecule 1

(CEACAM1 or CD66a) were analyzed in detail. The glycosite Asn89 on CEACAM1 was

identified with high confidence, and the composition of the N-glycan structure was characterized

as tri-antennary (Figure 3.6A and 3.6C). By normalizing with the ratio of deglycosylated peptides,

this site-specific N-glycosylation was found to be 13-fold more abundant in livers from GlcNAc

treated mice (Figure 3.6B and 3.6D). Structures for hybrid bi-antennary N-glycan with

unsubstituted terminal mannose residues, and complex bi-antennary N-glycan were 0.72 and 0.70,

respectively, suggesting that they are 1.4 times less abundant in livers from GlcNAc treated mice

(Figure 3.6E-H). This result is consistent with GlcNAc incorporation into HBP and stimulation

of the N-glycan branching pathway by UDP-GlcNAc (Abdel Rahman et al, 2014). Interestingly,

CEACAM1 is found abundantly in the liver, where it regulates insulin clearance and hepatic

lipogenesis (Najjar & Russo, 2014). CEACAM1 contains multiple potential N-glycosylation sites,

and its glycans interact with lectins, including galectin-3 (Feuk-Lagerstedt et al, 1999).

3.4.7 GlcNAc Increases Nutrient Uptake and Lipid Accumulation in Cultured Cells

To explore the possibility that GlcNAc promotes increased nutrient uptake, AML12 - an

immortal mouse hepatocyte cell line was cultured in medium supplemented with GlcNAc for 20

h, which markedly increased GlcNAc-P and UDP-GlcNAc levels, over 3-fold and 6-fold

respectively, in LC-MS/MS analysis (Figure 3.7A). In a dose dependent manner, GlcNAc

treatment increased L-PHA lectin binding, a probe for Mgat5-modified complex-type tri- and tetra-

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antennary branched N-glycans (Figure 3.7B). Staining with ConA, a lectin that binds

oligomannose- and hybrid-type N-glycans, was unchanged (Figure 3.7C). This indicates that N-

glycosylation and early processing were not altered by increasing cellular UDP-GlcNAc.

Immunoblots for O-GlcNAcylated proteins did not reveal differences with GlcNAc

supplementation (Figure 3.7D). FASN level and lipid content in lipid droplets increased with

GlcNAc treatment, in a dose dependent manner (Figure 3.7E-F). Metabolites involved in fat

metabolism: citrate, Ac-CoA, malonyl-CoA, carnitine and glycerol-3P increased, while glycerol,

the immediate precursor for glycerol-3P, was depleted (Figure 3.7G). The uptake of fluorescent

glucose analog (2-NBD-Glc) and dual-isotope-labelled glutamine (15N2-Gln) was increased with

GlcNAc treatment in AML12 cells (Figure 3.7H-I).

Swainsonine (SW), an inhibitor of Golgi α-mannosidase II, blocks N-glycan-mediated

branching by Mgat2, Mgat4 and Mgat5 (Lau et al, 2007). SW reduced the GlcNAc-dependent

increase in complex-type N-glycan branching, and importantly, SW blocked the GlcNAc-induced

increase in lipid droplet accumulation in AML12 cells (Figure 3.7J-K). GlcNAc also enhanced

15N2-Gln uptake in the immortal epithelial HeLa cells, along with promoting lipid accumulation,

and increasing levels of UDP-GlcNAc and L-PHA (Figure 3.8A-E). 3T3-L1 fibroblasts induced

to differentiate into adipocytes exhibited a GlcNAc dose dependent increase in fatty acid uptake,

lipid accumulation, and Mgat5-modified N-glycans (Figure 3.8F-H). These results show that

GlcNAc induces a cell autonomous enhancement of Glc, Gln and fatty acid uptake. Thus, using

different cell types, representative of distinct lineages with physiologically relevant metabolic

functions, I show that exogenous GlcNAc elevates cellular UDP-GlcNAc level and promotes

increase in Mgat5-modified tri- and tetra-antennary complex-type N-glycan branching, nutrient

uptake, and lipid storage. Combined, these results suggest a model for GlcNAc dependent HBP

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remodeling of cell surface N-glycan branching and reprogramming of cellular metabolism to

enhance lipid accumulation (Figure 3.8I).

3.4.8 Oral GlcNAc Partially Restores Anabolic Metabolism in Mgat5-/- Mice

GlcNAc supplementation in Mgat5-/- mammary tumor cells has been shown to rescue a

deficiency in retention of TGF-β and EGF cell surface receptors and their signaling (Lau et al,

2007; Mendelsohn et al, 2007). The rescue is due to compensating increases in the activity of the

remaining Mgat branching enzymes driven by increased UDP-GlcNAc. The N-acetyllactosamine

branches of structurally related N-glycans are redundant and compensate functionally by rescuing

affinities for galectin association with glycoproteins' N-glycan branches at the cell surface (Lau et

al, 2007). Mgat5-/- mice display reduced body-weight and fat content (Lau et al, 2007), a phenotype

opposite to that observed with GlcNAc supplementation in wild-type mice. Therefore, an attempt

to rescue body-weight and metabolic insufficiency in Mgat5-/- mice with oral GlcNAc on 9% fat

diet was performed (Figure 3.9A). The Mgat5-/- mice on GlcNAc had increased fat-tissue by 53%,

compared to 26% increase in Mgat5+/+ mice (Figure 3.9B). The increase in fat-tissue mass in

Mgat5-/- mice was offset by an 11% decrease in lean-tissue mass (Figure 3.9B). Similar results for

body-weight and tissue composition were observed in female Mgat5+/+ and Mgat5-/- mice on 9%

fat diet (Figure 3.9G-I). GlcNAc supplementation lowered RER in both genotypes, implying

increased oxidation of fat relative to untreated mice (Figure 3.9C). The HBP metabolites GlcNAc-

P and UDP-GlcNAc were elevated in liver by GlcNAc treatment in both genotypes (Figure 3.9D).

Primary hepatocytes from young Mgat5+/+ and Mgat5-/- mice were cultured overnight in the

presence of GlcNAc. Lipid droplet content was 40% lower in Mgat5-/- hepatocytes, but increased

with GlcNAc treatment, reaching levels similar to untreated Mgat5+/+ hepatocytes (Figure 3.9E).

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Liver histology revealed increased lipid accumulation in GlcNAc supplemented mice of both

genotypes (Figure 3.9F). Collectively, GlcNAc treatment increased fat accumulation and fatty

acid oxidation in Mgat5-/- mice, consistent with the model of functional redundancy in N-glycan

branches. However, it appears that Mgat5 is required for normal balance of lean to fat body tissue

composition that cannot be compensated by increase in UDP-GlcNAc alone.

3.5 Discussion

GlcNAc is a commercially available dietary supplement used by people and domestic

animals, with unsubstantiated health claims (Schnaar & Freeze, 2008). Acute intravenous GlcNAc

administration in humans does not seem to affect blood glucose homeostasis or insulin secretion

(Gaulden & Keating, 1964), and insulin had no obvious effect on the disappearance rate of

circulating GlcNAc (Levin et al, 1961). GlcNAc taken up by cells is largely converted into UDP-

GlcNAc (Dennis et al, 2009). A previous publication has shown that C57/BL6 mice treated with

0.25 mg/ml GlcNAc in their drinking water for seven days displayed increased GlcNAc serum

concentrations (Grigorian et al, 2011). This result strongly suggests that GlcNAc taken orally is

absorbed in the small intestines and transferred into bloodstream. Using a time-course experiment

I demonstrate that heavy-GlcNAc administered orally is rapidly absorbed in the small intestine

and efficiently transferred into bloodstream. Furthermore, 3 hours following oral gavage heavy-

GlcNAc is incorporated into UDP-GlcNAc in tissue examined, demonstrating that GlcNAc is

systematically taken up from the circulation.

UDP-GlcNAc is potentially an ideal sensor of cellular metabolic status (Hardiville & Hart,

2014; Love & Hanover, 2005; Marshall, 2006). GlcNAc supplementation might model surfeit or

nutrient abundance in glucose, nitrogen and fatty-acid metabolism. Here I examined the effects of

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extended oral GlcNAc treatment on C57BL/6 mouse physiology and metabolism. In the post-

weaning rapid-growth phase, mice on a 4% fat diet showed more weight-gain with oral GlcNAc,

as well as increased hepatic levels of glycolytic, gluconeogenic and TCA metabolites, and reduced

levels of some amino acids, including branched-chain amino acids leucine/isoleucine. With

prolonged oral GlcNAc weight-gain was enhanced, with greater effect on 9% compared to 4% fat

diet, evidence of a clear interaction between GlcNAc and calorie enriched diet. GlcN at a similar

dosage did not significantly increase body-weight, suggesting a less potent contribution to UDP-

GlcNAc and down-stream effectors. Oral GlcNAc intake in rats for 13 weeks on 6% fat diet, has

also been shown to increase body-weight without increase in food intake (Takahashi et al, 2009).

Parameters that were not significantly altered by oral GlcNAc included food intake, total activity,

and energy expenditure per mouse, as determined through indirect calorimetry, by measuring O2

consumption and CO2 production. Thus GlcNAc treated mice are not indolent or lethargic, but

rather appear to utilize equivalent calories more efficiently, as measured by conversion to body-

mass and fat content.

It should be noted that in this experiment food intake and energy expenditure were

measured only during a short time interval, a week for food intake and 24 hours for energy

expenditure. Furthermore, by the time food intake and energy expenditure measurements were

taken the mice have already diverged in body-weight. Ideally, food intake and energy expenditure

should have been monitored throughout the experiment, since it is difficult to draw conclusions

regarding energy balance over long time intervals from data obtained during a short time window,

which is typically the case for calorimetry-based measures (Tschop et al, 2012). Food intake was

monitored when mice were housed individually, as well as when they were caged together, and in

both instances GlcNAc treatment decreased their food intake. Although food intake measurements

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are often considered to be easy and trivial, they are in fact fraught with difficulty, and vary greatly

in accuracy, reproducibility and practicality (Tschop et al, 2012). Not all ingested calories are

introduced into metabolism, and there is even the possibility that mice might potentially consume

their own faces, giving any residual nutrients a second chance as they transit through the digestive

system. Thus, it is possible that food intake and/or energy expenditure measures employed may

have been insufficiently sensitive or accurate.

Interestingly, in a study using mannose supplementation, mice consuming mannose in

drinking water weighted less and had decreased fat mass compared to untreated mice, but showed

no difference in energy expenditure or physical activity, and an actual increase in calorie intake of

20% (Hudson Freeze personal communication). At first glance this result appears to violate the

first law of thermodynamics, i.e. the law of conservation of energy which states that energy can be

transformed from one form to another, but cannot be created or destroyed. However upon closer

inspection a bomb calorimeter analysis revealed a significantly greater energy content in feces

from mice supplemented with mannose, as compared to untreated controls, suggesting decreased

nutrient absorption (Hudson Freeze personal communication). This result might be a cautionary

tale and be relevant to my own findings with GlcNAc, which have also been suggested to violate

the first law of thermodynamics, since I observe a body-weight increase with GlcNAc

supplementation, without difference in energy expenditure or activity, and an actual decrease in

food intake. I propose greater efficiency of nutrient uptake as the explanation for increased body-

weight and adiposity observed in GlcNAc treated mice. However at this point the energy content

of their feces has not been analyzed to test for such a difference. An additional parameter that plays

a role in body-weight regulation but was not accounted for in my experiments is adaptive

thermogenesis, used to maintain core body temperature. Reduction of core body temperature by

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0.3° to 0.5°C in male transgenic mice resulted in significant body-weight increase without

affecting food intake or physical activity (Conti et al, 2006).

Mass spectrometry analysis revealed that oral GlcNAc increases hepatic UDP-GlcNAc and

GlcNAc content in glycoprotein N-glycans, consistent with more highly branched N-glycans, as

demonstrated with CEACAM1. GlcNAc treatment was associated with elevated levels of leptin

and insulin. In control mice on the 9% fat diet, the ratio of circulating insulin to glucagon (I/G)

decreased 12-fold between fed and fasted conditions. The I/G ratio was abnormally low in fed and

high in fasted GlcNAc-treated mice; with no net change between fed and fasted. With elevated

levels of serum glucose, lactate and Gln, oral GlcNAc alters or delays the normal fasting response

on 9% fat diet. The atypical response to fasting, as revealed by hormonal profile, serum

metabolites, hepatic glycogen content, and AMPK and mTor signaling suggests a continued

abundance of nutrients and/or hyperinsulinemia, possibly as a result of autophagy in fed GlcNAc

treated mice associated with metabolic and ER stress induced by hepatic steatosis (Singh et al,

2009).

My results suggest that HBP and the N-glycan branching pathway interact, descriptively

as a thrifty genotype/phenotype (O'Rourke R, 2014). Mgat5 null mice are hypoglycemic, have

reduced fat deposits, are resistant to weight-gain on a 9% fat diet, and display hypersensitivity to

fasting manifested as faster glycogen depletion (Cheung et al, 2007); the opposite of GlcNAc fed

mice in the present study. GlcNAc treatment partially restored fat accumulation in Mgat5-/- mice

and primary hepatocytes, consistent with the model of redundancy, where increasing UDP-

GlcNAc drives compensating increases in N-glycan branching by the other Mgat enzymes (Dennis

& Brewer, 2013; Lau et al, 2007). However, Mgat5-/- mice treated with GlcNAc did not recover

normal body-weight, and in fact displayed lower lean tissue mass, suggesting an important role for

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Mgat5 in balancing fat and lean tissue with aging. Early aging in Mgat5-/- mice is associated with

an imbalance in TGF-β and growth factor signaling, and premature loss of muscle satellite cells

and osteoprogenitor bone-marrow cells (Cheung et al, 2007). In the absence of Mgat5, positive

feedback between metabolism, HBP and N-glycan branching may be insufficient to maintain

muscle satellite cells (Cheung et al, 2007).

GlcNAc supplementation also rescued glucagon receptor sensitivity in Mgat5-/- primary

hepatocytes and in vivo in mice (Johswich et al, 2014), and in this study enhanced its sensitivity in

wild-type mice. Treatment of glucose-starved hematopoietic cells with GlcNAc has been shown

to promote IL-3 receptor surface expression and signaling in a manner dependent on Golgi

remodeling of N-glycans, which in turn increased Gln uptake and utilization for energy production,

lipid biosynthesis and essential amino acid uptake (Wellen et al, 2010). In cultured cells, GlcNAc

increased Mgat5-dependent N-glycan branching, and enhanced uptake of Glc, Gln and fatty acid.

Swainsonine (SW), an inhibitor of N-glycan branching, blocked the GlcNAc-dependent increase

in lipid accumulation. Similarly, GlcNAc increased Gln uptake in an Mgat5-dependent manner in

Hek293 cells (Abdel Rahman et al, 2014). Amino acid uptake and catabolism, coupled with

glucagon-driven gluconeogenesis may play a major role in hepatic metabolic phenotypes observed

in GlcNAc wild-type and Mgat5-/- mice.

Genes in the HBP have been implicated in lipid accumulation in different species,

supporting its role in weight regulation. A polymorphism in GFAT is associated with obesity in

men (Weigert et al, 2005), and GFAT overexpression in HepG2 liver cells increased transcript

levels associated with ER stress and lipid accumulation (Sage et al, 2010). Genetic or

pharmacological suppression of GFAT inhibited lipogenesis in HepG2 (Sage et al, 2010), murine

3T3-L1 adipocytes (Hsieh et al, 2012), and in human visceral adipocytes (O'Rourke et al, 2013).

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Furthermore, transgenic mice overexpressing GFAT in the liver were heavier than non-transgenic

littermates (Veerababu et al, 2000). In the red flour beetle, down-regulation of UDP-GlcNAc

pyrophosphorylase (UAP1) results in depletion of fat-body (Arakane et al, 2011). Hepatic

Slc35B4, a Golgi UDP-GlcNAc transporter, was identified as a quantitative trait locus associated

with diet-induced obesity, insulin resistance and gluconeogenesis in mice (Yazbek et al, 2011).

Slc35B4 is also a candidate gene for obesity in humans (Chen et al, 2013), where it could provide

more effective transport of UDP-GlcNAc. Golgi UDP-GlcNAc transport activity controls UDP-

GlcNAc supply to the N-glycan branching pathway, where Mgat5 is most sensitive to its

concentration (Lau et al, 2007).

As a practical consideration, the sources and amounts of GlcNAc in our diet are unknown.

Dietary GlcNAc may interact with gene polymorphisms in the HBP and Golgi N-glycan pathway,

playing a role in the obesity epidemic. The microbiomes of obese humans and mice have lower

bacterial diversity and a significantly greater ratio of Firmicutes to Bacteriodetes (DeWeerdt,

2014). With a high abundance and variety of carbohydrate-active enzymes such as glycosidases in

the microbiota (El Kaoutari et al, 2013), the microbiome associated with obesity may release more

GlcNAc from chitin and other polysaccharides into the gut. Chitin is a long-chain polymer of

GlcNAc found widely in nature, and used as a food and feed additive (Hirano, 1996; Shahidi &

Abuzaytoun, 2005), which is obscured on labelling as carbohydrates or sugar. Oral GlcNAc may

also provide a selective advantage to certain gut microbial communities, and modulate host

metabolism and physiology. Further work will be necessary to elucidate the complex relationship

between diet, gut microbiota and the host.

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fasted 18 h fed ad libitum control GlcNAc control GlcNAc ALT (IU/L) 102.8 ± 38.98 99.20 ± 27.51 24.75 ± 2.213 41.75 ± 10.40 Triglycerides (mmol/L)

1.222 ± 0.2203 1.268 ± 0.1545 0.9860 ± 0.09563 1.046 ± 0.07019

Cholesterol (mmol/L) 2.250 ± 0.5781 3.640 ± 0.2272 * 3.500 ± 0.4528 4.100 ± 0.1483 Chloride (mmol/L) 100.4 ± 0.5099 98.80 ± 0.6633 100.6 ± 0.6782 99.00 ± 0.8367 Sodium/Potassium Ratio

20.40 ± 0.8124 21.40 ± 0.9798 21.60 ± 0.8718 20.60 ± 1.470

Glucose (mM) 4.225 ± 0.1493 6.400 ± 0.3342 ** 7.840 ± 0.3558 7.700 ± 0.4743 Insulin (pM) 39.17 ± 7.522 314.7 ± 84.37 * 334.7 ± 57.20 409.1 ± 97.21 Glucagon (pM) 33.65 ± 2.348 22.45 ± 2.548 * 18.75 ± 4.543 22.63 ± 4.146 Insulin to Glucagon Ratio

2.590 ± 1.073 14.59 ± 4.485 * 30.42 ± 4.240 14.90 ± 0.9945 *

HOMA2-IR 1.600 ± 0.8820 5.425 ± 1.276 * N/A N/A Leptin (pM) 313.1 ± 102.4 1040 ± 94.97 ** 900.5 ± 96.63 1412 ± 75.67 * Adiponectin (ng/ml) 23.35 ± 0.4644 23.50 ± 0.3549 25.62 ± 0.3820 24.12 ± 0.3151 *

Table 3.1 Phenotypic differences in serum biochemistry between GlcNAc treated and untreated mice on 9% fat diet, under fasted and fed conditions.

At sacrifice mice were 10 months old (n=4-5 per group), and have been on 0.5 mg/ml oral GlcNAc supplementation for 30 weeks, or 7 months. Data are mean ±SEM. *p<0.05 versus fasted or fed ad libitum control mice on 9% fat diet (2-tailed, unpaired Student's t-test). ALT alanine aminotransferase, HOMA2-IR homeostatic model assessment-insulin resistance.

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Diet

GlcNAc content (GlcNAc treated/control)

Glycopeptides with N-glycans

p-values

4% fat 1.54 361 <0.0001

4% fat, fasted 1.99 394 <0.0001

9% fat 1.52 292 <0.0001

9% fat, fasted 1.54 218 0.2785

Table 3.2 Global analysis of relative GlcNAc content in liver N-glycans.

N-glycans from liver glycopeptides, from control and GlcNAc treated mice, were identified by differential labelling with stable light and heavy isotope dimethyl labelling followed by LC-MS/MS. Liver was isolated from mice on 4% and 9% fat diets, in fasted and fed conditions. Sign test with probability of 0.5 and two-tail p-value was performed.

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Figure 3.1 Oral GlcNAc is rapidly absorbed by gut to enter bloodstream and be taken up by tissue from circulation.

(A) Change in body-weight for wild-type C57BL/6 male mice on diets containing different percentages of fat. Data shown are mean ± SEM, n=8, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared with 4% fat diet, with significant differences indicated as #p<0.05 and ##p<0.01 versus 9% fat diet, and *p<0.05 and **p<0.01 versus 22% fat diet. (B) Respiratory exchange ratio (RER) of mice fed different percentage fat diets for 50 weeks, (C) quantification of data in (B) for night and day. Data shown are mean ±SEM, analyzed by one-way ANOVA followed by Tukey's multiple comparison test, with significant differences indicated as *p<0.05, **p<0.01, and ***p<0.001. (D) Time-course of relative abundance of 13C6-GlcNAc in serum of mice orally gavaged with a bolus administration of 13C6-GlcNAc at 20 µg/g of mouse. (E) Time-course of relative abundance of Glc-d7 in serum of mice orally gavaged with a bolus administration of Glc-d7 at 50 µg/g of mouse. (F) At 180 minutes following oral gavage with 20 µg/g 13C6-GlcNAc, UDP-13C6-GlcNAc was detected as a strong peak in different mouse tissues.

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Figure 3.2 Oral GlcNAc increases UDP-GlcNAc level and promotes weight-gain in mice.

(A) Analysis of change in body-weight of wild-type C57BL/6 male mice on low fat diet over 90 days supplemented with oral GlcNAc. (B) Relative abundance of liver metabolites in the distal portion of HBP measured by LC-MS/MS in 90 day GlcNAc treated mice on low fat diet. (C) Principle component analysis of all measured liver metabolites in mice on low fat diet and GlcNAc supplied in drinking water at 0.5, 5.0 and 15 mg/ml. Steady-state liver metabolites in the glycolysis and gluconeogenesis pathways (D), tricarboxylic acid (TCA) cycle (E), amino acids (F), and oxidized and reduced forms and ratios of glutathione and nicotinamide adenine dinucleotides (G). All metabolites measured by LC-MS/MS and expressed as fold change in 90 day GlcNAc treated mice on low fat diet. Relative levels of specific metabolites were normalized to liver weight. Metabolomic data shown are mean ±SEM, n=10, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared with vehicle control of 0 mg/ml GlcNAc, with significant differences represented as *p<0.05, **p<0.01, and ***p<0.001.

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Figure 3.3 Oral GlcNAc promotes weight-gain and lipid accumulation.

(A) Change in body-weight for wild-type C57BL/6 male mice on 4% and 9% fat diet over weeks of oral GlcNAc or GlcN supplied in drinking water at 0.5 mg/ml. Data shown are mean ± SEM, n=10, analyzed by 2-tailed unpaired Student's t-test, with significant differences indicated as #p<0.05 for 4% fat versus GlcNAc 4% fat, and *p<0.05 for 9% fat versus GlcNAc 9% fat. (B) Body-weight and (C) calorie intake per body-weight per day at 34 weeks of age, or following 20 weeks of GlcNAc treatment. Terminal body-weight (D), tissue composition (E), and epidydymal-fat and liver weight normalized to body-weight (F) at sacrifice. Error bars represent ±SEM, n=10, *p<0.05 or **p<0.01 GlcNAc treated versus control, on either 4% or 9% fat diet (2-tailed, unpaired Student's t-test). Serum free fatty acids (FFA) (G), triglycerides (TG) (H), and significant steady-state metabolite changes (I) in mice on 9% fat diet and supplemented with 0.5 mg/ml oral GlcNAc for 90 days. Error bars represent ±SEM, n=5, *p<0.05, or **p<0.01 versus control (2-tailed, unpaired Student's t-test). (J) Western blot analysis of metabolic signaling pathways in liver lysates from mice maintained on 9% fat diet supplemented with GlcN or GlcNAc, probed for phosphorylated AMP-Activated Protein Kinase (AMPK-α), Acetyl-CoA Carboxylates Kinase (ACC) and ribosomal protein S6 (S6). Tubulin was used as a loading control.

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Figure 3.4 Oral GlcNAc promotes fatty acid oxidation without affecting activity or energy expenditure.

(A) Analysis of total activity, (B) oxygen consumption rate, (C) carbon dioxide emission rate, and (D) Respiratory Exchange Ratio (RER=VCO2/VO2) over 20 hour period, (E) quantification of data in (D) for night and day, (F) energy expenditure, and (G) food and (H) water intake. Liver free fatty acids (FFA) (I) and triglycerides (TG) (J) in mice on 9% fat diet and orally supplemented with 0.5 mg/ml GlcNAc. Error bars represent ±SEM, n=5, *p<0.05, or ** p<0.01 versus control (2-tailed, unpaired Student's t-test). (K) Representative images of liver histology sections stained with oil red O to detect TG and neutral lipids in mice on 9% fat diet, fed ad libitum or fasted for 18 h, and supplemented with 0.5 mg/ml GlcNAc in drinking water for 30 weeks.

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Figure 3.5 Oral GlcNAc alters fasting liver metabolism.

(A) Representative images of liver histology sections stained with periodic acid-Schiff, with glycogen deposits detected as purple-magenta areas, obtained from mice on 9% fat diet mice, fed ad libitum or fasted for 18 h, and supplemented with 0.5 mg/ml GlcNAc in drinking water for 30 weeks. (B) Liver glycogen content. Error bars represent ±SEM, n=5, *p<0.05 versus control (2-tailed, unpaired Student's t-test). (C) Immunoblot analysis of metabolic signaling pathways with FASN, and phosphorylated versions of GS, Akt kinase, ribosomal protein S6, AMPK-α, and ACC in liver lysates of fasted mice maintained on 9% fat diet and treated with GlcN or GlcNAc. (D) Quantification of immunoblots using tubulin as loading control. (E) Steady-state relative abundance of metabolites in blood serum from mice maintained on 9% fat diet and GlcNAc for 30 weeks. (F) Intraperitoneal glucagon tolerance test, and (G) intraperitoneal glucose tolerance test, with Area Under the Curve (AUC) quantification in mice treated with 0.5 mg/ml oral GlcNAc for 23 weeks. Error bars represent ±SEM, n=10, analyzed with 2-tailed unpaired Student's t-test *p<0.05 versus control on the same diet.

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Figure 3.6 Oral GlcNAc increases tri-antennary N-glycan structures on glycosite Asn89 of CEACAM1 hepatic transmembrane glycoprotein.

(A) By matching the Y1 ion (peptide + GlcNAc) from the MS/MS spectrum to the list of deglycosylated peptides identified by Mascot database search with accurate molecular weight and retention time, the peptide sequence was identified as Asn89 of CEACAM1 transmembrane glycoprotein. (B) The extracted ion chromatogram peak area for control (light) and GlcNAc (heavy) labelled precursor from deglycosylated CEACAM1 peptides. With a ratio of GlcNAc to control of 1.68, no significant difference was found, which demonstrates that the increase in abundance of intact glycopeptides with tri-antennary complex structure was due to increased branching of complex N-glycans. (C) Annotated MS/MS spectrum of heavy-labelled intact glycopeptide identified with complex tri-antennary N-glycan structure. Terminal sialylation could be verified by the existence of oxonium ion with m/z 292 and 274. (D) The extracted ion chromatogram (XIC) of control (light) and GlcNAc (heavy) labelled peptide precursor from full MS scan indicates the abundance of tri-antennary glycopeptide being much higher in liver lysates from GlcNAc treated mice, with a ratio of 21 in GlcNAc to control. (E) Hybrid bi-antennary N-glycan structure with unsubstituted terminal mannose residues. (F) XIC from control and GlcNAc bi-antennary hybrid N-glycan with mannose, with a peak area ratio of GlcNAc to control of 1.21. (G) Complex bi-antennary N-glycan structure. (H) XIC from control and GlcNAc complex bi-antennary N-glycan, with a peak area ratio of GlcNAc to control of 1.17.

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Figure 3.7 GlcNAc increases UDP-GlcNAc, β1,6-GlcNAc branched N-glycans and lipid accumulation.

(A) Fold changes of distal HBP metabolites upon GlcNAc treatment for 20 h. (B) Analysis of Mgat5-mediated β1,6-GlcNAc branched complex-type N-glycans on cell surface glycoproteins quantified with fluorophore conjugated lectin L-PHA. (C) Analysis of oligomannose-type and hybrid-type N-glycans on cell surface glycoproteins with fluorophore conjugated lectin ConA. (D) Immunoblot analysis of O-GlcNAcylation detected with monoclonal-antibody CTD110.6. (E) Protein expression level by western blot analysis of FASN enzyme and loading control tubulin, used for relative quantification of immunoblot. (F) Intracellular lipid droplets, quantified microscopically with lipophilic fluorescent probe BODIPY 493/503. (G) Fold change in specific metabolites involved in fat metabolism, normalized to cell number. (H) Glucose uptake in cells treated with GlcNAc for 20h, grown in the presence of fluorescent glucose analog 2-NBD-Glc for 1h, and quantified using flow cytometry as mean fluorescent intensity (MFI). (I) Analysis of heavy-isotope labelled 15N2-Gln uptake in cells treated with GlcNAc for 20 h, pulsed with 15N2-Gln for designated times, and quantified using mass spectrometry. (J) Tri- and tetra-antennary Mgat5-modified N-glycans, and (K) intracellular lipid accumulation in the absence and presence of GlcNAc and/or Swainsonine (SW), quantified microscopically with fluorophore-conjugated lectin L-PHA or BODIPY 493/503. Data shown are mean ±SEM, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared with vehicle control (A-C, E-G, J-L), with significant differences represented as *p<0.05, **p<0.01, and ***p<0.001.

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Figure 3.8 HBP and N-glycan dependent regulation of cellular metabolism.

(A) Analysis of 15N2-Gln uptake and its downstream metabolites (B) glutamate (15N-Glu) and (C) 15N-Gln in HeLa cells treated with GlcNAc for 20 h, pulsed with 15N2-Gln, and quantified using LC-MS/MS. (D) Analysis of intracellular lipid in HeLa cells. (E) Fold change (FC) in L-PHA and UDP-GlcNAc as a function of GlcNAc treatment in HeLa cells. (F) Kinetic reading monitoring uptake of non-esterified long-chain-fatty-acid-analog (BODIPY-FA) quantified as Area Under the Curve (AUC) for Relative Fluorescence Units (RFU). (G) Analysis of intracellular lipid and (H) complex-type N-glycans in 3T3-L1 adipocytes. Data are mean ±SEM, analyzed by t-test or one-way ANOVA followed by Dunnett’s test, significant differences represented as *p<0.05, **p<0.01, and ***p<0.001. (I) GlcNAc salvaged by HBP increases UDP-GlcNAc, the substrate for N-acetylglucosaminyltransferase enzymes (Mgat1, 2, 4, 5) acting on glycoprotein acceptors trafficking through Golgi en route to the cell surface. Km values for UDP-GlcNAc decline from Mgat1, Mgat2, Mgat4 to Mgat5, making biosynthesis of tri- and tetra-antennary N-glycans sensitive to UDP-GlcNAc levels. N-glycan branching increases the affinity of glycoproteins for galectins, which cross-link and oppose loss of receptors and transporters to endocytosis. This increases cell surface residency of glucose (Glc), glutamine (Gln), and fatty-acid (FA) transporters (Glut, SLC and CD36 respectively), and other transmembrane glycoproteins such as CEACAM1. The improved cell surface retention allows more nutrients to enter the cell and contribute to increased lipid accumulation via FASN. A positive-feedback loop is formed by increasing uptake and flux of Glc, Gln and Ac-CoA through de novo HBP to UDP-GlcNAc and N-glycan branching on receptors and transporters.

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Figure 3.9 Oral GlcNAc promotes lipid storage in male and female Mgat5 wild-type and null mice.

(A) Change in body-weight for Mgat5 wild-type (wt) and null male mice on 9% fat diet supplemented with 0.5 mg/ml oral GlcNAc in drinking water for 30 weeks. (B) Fat and lean tissue mass, as measured by magnetic resonance imaging. (C) Diurnal and nocturnal respiratory exchange ratio (RER). (D) Relative abundance of liver GlcNAc-P and UDP-GlcNAc determined using LC-MS/MS and expressed as fold change. Error bars represent ±SEM, n=4-5, with statistical significance indicated as *p<0.05 and **p<0.01 versus control for the same genotype (2-tailed, unpaired Student's t-test). (E) Analysis of intracellular lipid accumulation in lipid droplets as a function of overnight exogenous GlcNAc supplementation in primary hepatocytes obtained from Mgat5 wt and null mice, quantified microscopically using the lipophilic fluorescent probe BODIPY 493/503. Data shown are mean ±SEM, analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test compared to 0 mM GlcNAc control of respective genotype, with significant differences indicated as *p<0.05, **p<0.01, and ***p<0.001. (F) Histological analysis of liver tissue sections stained with oil red O, identifying lipid deposits as red-stained areas. (G) Change in body-weight of Mgat5 wt and null female mice on 9% fat diet supplemented with 0.5 mg/ml oral GlcNAc in drinking water for 21 weeks. (H) Fat-tissue to body-weight (g/g) ratio, and (I) lean-tissue to body-weight (g/g) ratio, as measured by DEXA. (J) Serum concentration of leptin. Error bars represent ±SEM, with 4 to 6 mice per group, *p<0.05 versus control for the same genotype (2-tailed, unpaired Student's t-test).

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Chapter 4

Discussion and Future Directions

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4.1 GlcNAc and N-Glycan Branching in Mgat5 Null Mice

Mammalian glycans are involved in molecular and cellular mechanisms that control health

and disease, and changes in glycosylation have been observed in both genetic and acquired disease

states (Dennis et al, 2009; Freeze et al, 2015; Ohtsubo & Marth, 2006). The HBP and Golgi N-

glycan branching pathway are linked to metabolic homeostasis (Dennis et al, 2009; McClain,

2002). Null mutations in genes encoding N-glycan branching enzymes Mgat4a and Mgat5, which

are sensitive to UDP-GlcNAc levels from the HBP, disrupt mouse glucose homeostasis and result

in abnormal body-weight (Dennis et al, 2009). Mgat5-/- mice are leaner, smaller in size, and exhibit

reduced fat pad depots despite maintaining daily calorie intake and physical activity similar to

wild-type mice, who show large abdominal fat deposits on the same diet (Cheung et al, 2007).

In my studies, oral GlcNAc supplementation partially restored fat accumulation in Mgat5-

/- mice and primary hepatocytes. This result is consistent with functional redundancy of N-glycan

branches, where increased levels of UDP-GlcNAc drive compensating increases in N-glycan

branching by other Mgat enzymes (Dennis & Brewer, 2013). Indeed, even though Mgat5 enzyme

is absent and hence no β1,6-linked GlcNAc can be added, there is evidence of functional

redundancy or compensation in the remaining structurally related N-glycan branches to maintain

branch complexity on glycoproteins for cross-linking endogenous galectins at the cell surface

(Dennis & Brewer, 2013; Johswich et al, 2014). This can occur provided there is an abundant

supply of the common substrate UDP-GlcNAc, since the Golgi N-glycan branching pathway is

sensitive to its levels, especially for generating tri- and tetra-antennary N-glycans (Dennis et al,

2009). In fact, GlcNAc supplementation in Mgat5-/- cells rescued levels of EGF and TGF-ß cell

surface receptors and their signaling by restoring the glycan-galectin lattice (Lau et al, 2007).

Therefore it was reasonable to assume that additional metabolic supply of UDP-GlcNAc, through

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GlcNAc supplementation to HBP, may rescue fat accumulation defects observed in Mgat5-/- mice,

as it partially did, supporting the idea of functional redundancy between N-glycans. Also, GlcNAc

supplementation rescued glucagon receptor sensitivity and signaling in Mgat5-/- primary

hepatocytes and in vivo in Mgat5-/- mice in the glucagon tolerance test (Johswich et al, 2014). In

my study, we further show that GlcNAc enhanced glucagon sensitivity in wild-type mice in the

glucagon tolerance test, which might be indicative of enhanced glucagon receptor activity through

increased N-glycan branching.

4.2 HBP and N-Glycan Branching Reprogram Metabolism to Promote Fat Accumulation

Experimental evidence suggests that UDP-GlcNAc concentration is at least 20-fold higher

in the Golgi than in the cytoplasm (Waldman & Rudnick, 1990). This concentration gradient is

established by the Golgi UDP-GlcNAc antiporters, which exchange uridine monophosphate

(UMP) for UDP-GlcNAc, thus forming a direct proportionality between the steady-state amounts

of UDP-GlcNAc inside the Golgi and the cytosol (Lau et al, 2007). The Vmax of UDP-GlcNAc

transport is on the order of ~0.2 mM/sec, which corresponds to mM concentration of UDP-GlcNAc

in the Golgi (Lau et al, 2007). Previous calculations used to establish a computational model of

Golgi N-glycan branching ultrasensitivity estimate the basal physiological Golgi concentration of

UDP-GlcNAc at ~1.5 mM (Lau et al, 2007). Moreover, a recent publication reported that the UDP-

GlcNAc antiporter SLC35A3 forms a complex with Mgat5 enzyme in the Golgi membrane, and

thus augments its catalytic activity by proximity (Maszczak-Seneczko et al, 2015). SLC35A3

ensures a localized subcellular supply of substrate for the late Mgat enzymes to regulate N-glycan

branching pathway. Indeed, cells deficient in SLC35A3 activity displayed reduced amount of

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highly branched tri- and tetra-antennary N-glycans (Maszczak-Seneczko et al, 2013). Thus, UDP-

GlcNAc distribution in the Golgi does not appear to be homogenous, but is rather highly localized.

A relatively restricted localization of UDP-GlcNAc transporters within the Golgi suggests that this

common donor substrate may be preferentially supplied to specific Golgi compartments where

Mgat branching enzymes reside. GlcNAc supplementation and Mgat5 overexpression displayed

synergistic increase in branching, consistent with Mgat5’s high Km value and pathway

ultrasensitivity to UDP-GlcNAc (Abdel Rahman et al, 2015; Lau et al, 2007).

Numerous lines of evidence indicate that metabolic flux through the HBP and availability

of intracellular UDP-GlcNAc regulate activities of Golgi Mgat enzymes, and control the N-glycan

branching pathway and cell surface retention of transmembrane glycoproteins (Dennis et al, 2009;

Johswich et al, 2014). In cultured cells, GlcNAc directly enters the HBP to increase total cellular

UDP-GlcNAc pool, enhance N-glycan branching, and improve the association of glycoprotein

receptors and transporters with galectins, thereby increasing their cell surface retention and

sensitivity to extracellular factors (Abdel Rahman et al, 2015; Dennis et al, 2009; Johswich et al,

2014). I hypothesized that GlcNAc supplementation to the HBP could increase the surface level

of nutrient transporters to facilitate efficient nutrient uptake, i.e. transport more fatty acids, glucose

and/or glutamine, and synthesize and store more lipids, via an Mgat5-mediated N-glycan

dependent mechanism (Abdel Rahman et al, 2015).

I examined the role of increasing UDP-GlcNAc pool through GlcNAc supply in order to

elucidate its effects in vitro and in vivo at the molecular, cellular and physiological levels of

analysis. GlcNAc supplemented to mice, delivered by ad libitum drinking water, resulted in

increased hepatic UDP-GlcNAc pool, indicating that the salvage pathway has the capacity to

elevate UDP-GlcNAc levels in vivo. I show that extended GlcNAc supplementation in mice

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increased their body-weight without affecting calorie-intake, activity, or energy expenditure.

These results show that GlcNAc treated mice are not indolent or lethargic, but rather that chronic

GlcNAc ingestion promotes weight-gain. Moreover, oral GlcNAc increased GlcNAc content and

N-glycan branching in liver glycoproteins. I conclude that this effect of GlcNAc is due to

modification or reprogramming of cell surface and cellular metabolism through HBP and

downstream N-glycan branching pathway, to promote more nutrient uptake and consequent

increased fat accumulation. In cultured cells, GlcNAc enhanced nutrient uptake, and increased

synthesis and lipid accumulation, while inhibition of N-glycan branching blocked GlcNAc-

dependent lipid accumulation. These results suggest the possible involvement of HBP and N-

glycan branching in conditions characterized by increased capacity to store fat such as obesity.

4.3 Consequences of Long-Term Daily Oral GlcNAc Intake

GlcNAc supplemented wild-type mice had more fat, were heavier, and displayed a delay

in normal fasting response, as determined by their glycaemia, insulin, glucagon and leptin levels,

as well as liver glycogen content during fasting. Indeed, the glycogen and lipid metabolism in

livers of GlcNAc treated mice was altered, and the livers were larger, fatty, and suggestive of

hepatic steatosis. Since increased body-weight and obesity are risk factors for diabetes and the

metabolic syndrome, it is of interest to determine if mice supplemented with GlcNAc on a daily

basis for an extended period of time show disease associated biomarkers or develop insulin

resistance. After 7 months of oral GlcNAc, elevated serum levels of amino acids Gln, Phe, Ile, Tyr

and Leu were observed. These amino acids are associated with obesity and a high risk of diabetes

(Kim et al, 2010; Newgard et al, 2009). Additional serum metabolites linked to obesity, diabetes

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and the metabolic syndrome were also elevated, including sorbitol, aminoadipic acid, and uric acid

(Brownlee, 2001; Johnson et al, 2013; Wang et al, 2013).

A clinical index based on paired concentrations of fasting insulin and glucose, the

homeostatic model assessment for estimation of insulin resistance (HOMA2-IR), showed an

elevated score for GlcNAc treated mice, suggestive of insulin resistance (Minor et al, 2011;

Wallace et al, 2004). However no insulin tolerance test was performed on these mice to definitively

answer this question. Combined, these findings might be indicative of inception of hepatic insulin

resistance in GlcNAc treated mice, which causes impaired suppression of glucose production by

insulin in the liver, leading to hyperglycemia, Indeed, hyperglycemia was observed in fasted

GlcNAc treated mice, despite lowered glucagon level. Hypersensitivity of the liver to glucagon,

through enhanced cell surface residency of the glucagon receptor, could suppress the inhibitory

effect of elevated insulin on glucose production by the liver, and thus might account for these

effects of GlcNAc. However, it should be pointed out that all these observations might not have

been a direct result of GlcNAc, but rather secondary to the increased body-weight promoted by

GlcNAc in C57BL/6 mice. The C57BL/6 mice are widely used as a model for human type 2

diabetes, due to being prone to diet-induced obesity and insulin resistance. Consequently, it would

be interesting to determine if GlcNAc supplementation in a different strain of mice, or another

model organism, would result in a similar phenotype to that observed in C57BL/6 strain.

4.4 GlcNAc Salvage into HBP

GlcNAc enters the cell without deacetylation by the process of passive diffusion or

pinocytosis, or becomes available intracellularly as a result of endogenous lysosomal degradation

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and catabolism of glycoconjugates (Dennis et al, 2009; Tesoriere et al, 1972). In the salvage

pathway the cytosolic enzyme NAGK catalyzes the phosphorylation of free cytoplasmic GlcNAc

to GlcNAc-6P, which can subsequently enter the anabolic HBP leading to formation of UDP-

GlcNAc (Dennis et al, 2009). A homozygous mutation in GNPNAT1 (GNA1), encoding the

enzyme for de novo HBP upstream of NAGK and the salvage pathway resulted in embryonic

lethality at around embryonic day 7.5, with a general proliferative delay of development (Boehmelt

et al, 2000). Mice with a severe reduction in PGM3 enzyme activity, a key step in HBP catalyzing

the interconversion of GlcNAc-6-P and GlcNAc-1-P, die between embryonic day 3.5 and 6.5,

suggesting that PGM3 is required for early embryonic development (Greig et al, 2007).

Knockout mice deficient in UAP1, gene encoding the last enzyme in the HBP, have not

been reported. Deletion of this gene should completely abolish both de novo and salvage synthesis

of UDP-GlcNAc, and considering that UDP-GlcNAc is essential for synthesis of different

glycoconjugates, this null mutation would most likely be catastrophic for early embryonic

development. NAGK null mice have also not been reported, however if such mice were viable,

which is probable since the de novo HBP to UDP-GlcNAc would still be intact, I expect that

GlcNAc supplementation to them would not result in the phenotypes observed in mice with normal

NAGK activity. If however viable NAGK knockout mice supplemented with GlcNAc did show

weight-gain, this would suggest that in vivo GlcNAc was acting through different means, probably

the microbiome, in which case GlcNAc supplementation to germ-free mice should not result in

increased body-weight.

4.5 GlcNAc Supply and Increased N-Glycan Branching Promote Fat Accumulation

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The two main factors driving chemical reactions are the concentration of substrates and the

activity of enzymes. Increased levels of enzymes Mgat4 or Mgat5 compensate for their lower

UDP-GlcNAc affinities, essentially reducing the UDP-GlcNAc requirement of the branching

pathway to tri- and tetra-antennary N-glycans. Enforced pancreatic β-cell-specific constitutive

Mgat4a expression in transgenic mice maintained proper Glut2 N-glycan branching and its cell

surface residency, thereby protecting from metabolic diseases resulting from reduction of

pancreatic Mgat4a gene expression induced by high-fat diet (Ohtsubo et al, 2011). Mgat4b was

found to be upregulated in a study designed to identify genes in the liver that control fat deposition

in fast-growing chickens (Claire D'Andre et al, 2013).

Transgenic mice overexpressing the enzyme Mgat5, generated from a C57BL/6 and

DBA/2 cross and using a β-actin promoter, had increased Mgat5 protein expression and enzyme

activity in the liver, pancreas, kidney, brain and skin (Terao et al, 2011). Interestingly, compared

to wild-type littermates, these mice displayed increased body-weight, liver weight, and liver to

body-weight ratio on normal chow (Kamada et al, 2012). Furthermore, hepatic lipogenesis was

increased in Mgat5 transgenic mice, with significant hepatic elevation of fatty acid biosynthesis-

related genes such as fatty acid synthase (FASN), acetyl-co-carboxylase-1 (ACC-1) and sterol

regulatory element binding protein-1 (SREBP-1) (Kamada et al, 2015). These findings are very

reminiscent of my own result with GlcNAc supplementation in wild-type mice on high-fat diet,

and corroborate my own conclusion. Based on previous data, including my cell line work with tet-

inducible Mgat5 overexpression described earlier, it was expected that Mgat5 overexpression

would have similar effects on the Golgi N-glycan branching pathway in mice as providing extra

supply of UDP-GlcNAc (Abdel Rahman et al, 2015). Hence, it was not surprising that both Mgat5

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overexpression and GlcNAc supplementation displayed a similar increase in body and liver weight

in vivo.

It would be of interest to determine the combined effect of GlcNAc supplementation and

Mgat5 overexpression on body and organ weight in mice, as well as the impact on their metabolic

homeostasis. In cells in culture, GlcNAc supplementation together with tet-induced Mgat5

overexpression results in a synergistic interaction, i.e. a response of increased N-glycan branching

that is greater than the sum of their individual contributions (Abdel Rahman et al, 2015). Also,

since the liver figured prominently in the phenotype in my study, perhaps overexpressing Mgat5

specifically and exclusively in the liver, and supplementing mice with GlcNAc would be sufficient

to yield the same phenotype of increased body and liver weight. Interestingly, genetic and

functional studies in mouse models for quantitative trait loci involved in diet-induced obesity and

glucose homeostasis have identified increased liver expression of Slc35B4 as promoting obesity,

insulin resistance, and gluconeogenesis in mice (Yazbek et al, 2011). Slc35B4 is a sugar-

nucleotide transporter responsible for transporting synthesized cytosolic UDP-GlcNAc into Golgi

apparatus (Ashikov et al, 2005). The increased expression of Golgi UDP-GlcNAc transporter

could increase the availability of UDP-GlcNAc for Mgat enzymes in the N-glycan branching

pathway. Furthermore, Slc35B4 is a candidate gene for obesity in humans, with increased

expression in subcutaneous adipose tissue (Chen et al, 2013; Fox et al, 2007). This is strong support

for the action of UDP-GlcNAc in a Golgi pathway as a modifier of metabolism.

4.6 Increased HBP Activity Promotes Fat Accumulation

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It should be noted that concordant with my results of GlcNAc supplementation, genes in

the HBP have been implicated in nutrient assimilation and regulation of fat accumulation.

Transgenic mice overexpressing GFAT in the liver displayed increased levels of UDP-GlcNAc,

synthesized excess fatty acids and triglycerides, and gained more weight than non-transgenic

littermates (McClain, 2002). Single nucleotide polymorphism (SNP) in the 5'-flanking region of

GFAT was associated with significantly higher body-mass index and percent body fat in men, as

well as higher intramyocellular lipid content in muscle (Weigert et al, 2005). A polymorphism in

an intron of the porcine GFAT gene was associated with lean meat percentage, back fat thickness,

and back fat at the rump (Liu et al, 2010). These SNPs could potentially change binding of

transcription factor(s) and alter transcriptional activity of GFAT. In adipocytes GFAT regulates

adipogenesis, with differentiation of 3T3-L1 pre-adipocytes being accompanied by increase in

GFAT mRNA expression (Hsieh et al, 2012). Pharmacological or siRNA suppression of GFAT

inhibited lipogenesis in murine 3T3-L1 adipocytes, as well as in human visceral and subcutaneous

primary adipocytes (Hsieh et al, 2012; O'Rourke et al, 2013). Collectively, these results suggest

that activation of the HBP is a regulator of lipogenesis in different tissues.

Genes in the HBP downstream of GFAT have also been implicated in lipid accumulation.

In the red flour beetle, reduction of UAP1 transcript, the UDP-GlcNAc pyrophosphorylase

responsible for synthesizing UDP-GlcNAc by catalyzing its formation from GlcNAc-1P and UTP,

results in depletion of fat-body in adults (Arakane et al, 2011). This result suggests a physiological

function for UAP1 in fat body formation, and authors speculate that it might be due to a failure of

glycosylation of glycoproteins, which could lead to defects in absorption of nutrients (Arakane et

al, 2011). In cattle bulls a significantly higher level of expression of liver UAP1 was identified in

Charolais compared to Holstein breed (Schwerin et al, 2006). The Charolais beef cattle are

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characterized by their ability to deposit nutrients and metabolites as meat and fat, while the German

Holstein-Friesian dairy breeds are especially apt at secreting metabolized feed components into

milk (Schwerin et al, 2006). These results clearly indicate that the HBP regulates fat mass in

different species.

Increased flux through HBP correlates with increased lipogenic gene expression and

energy storage as lipid. Microarray analysis performed on NMuMG mouse mammary epithelial

cells supplemented with GlcNAc revealed an increase in transcripts in the fatty acid biosynthesis

pathway (Lau et al, 2008a). In isolated primary adipocytes, glucosamine (GlcN), which directly

enters the HBP at the level of GlcN-6P, up-regulated mRNA transcripts for lipogenic enzymes

FASN and ACC-1 in a dose-dependent manner (Rumberger et al, 2003). Overexpression of GFAT,

or treatment with GlcN in human HepG2 liver cells resulted in increased transcript levels of

lipogenic genes FASN, ACC-1 and SREBP-1, and these effects were abolished by treatment with

a GFAT inhibitor (Hirahatake et al, 2011; Sage et al, 2010). GlcN also induced accumulation and

increased mRNA expression of lipogenic genes in C2C12 myoblasts in culture, and in rat skeletal

muscles following transient GlcN infusion (Fujita et al, 2005). Furthermore, study with

hyperinsulinemic rats in association with short-term GlcN infusion to mimic nutrient excess

revealed a down-regulation of transcripts encoding products involved in fatty acid oxidation in

skeletal muscles (Obici et al, 2002). Collectively, these findings imply that HBP flux leads to a

coordinated response whereby nutrient uptake is increased and calories preferentially stored as fat,

suggesting that genes in the HBP might be involved in the thrifty genotype/phenotype (McClain,

2002; O'Rourke R, 2014).

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4.7 HBP and the Thrifty Genotype/Phenotype Hypothesis

Based on the evidence that increased activity in the HBP promotes fat accumulation, and

that GlcNAc supplementation and Mgat5 overexpression result in the same, I suggest that the HBP

and Golgi N-glycan branching pathway interact in a manner suggestive of a thrifty

genotype/phenotype (O'Rourke R, 2014). Mgat5-deficinet mice were hypoglycemic and displayed

hypersensitivity to fasting, manifested as faster glycogen depletion (Cheung et al, 2007). They also

had reduced fat depots and were resistant to weight-gain on high-fat diet, although their food intake

was equivalent to wild-type littermates (Cheung et al, 2007). These phenotypes are opposite to

those exhibited by mice supplemented with GlcNAc in the present study, suggesting that increased

HBP activity and N-glycan branching are good candidates to promote a thrifty phenotype, i.e.

efficiency in uptake and storage of nutrients. Swainsonine, an inhibitor of Golgi α-mannosidase II

that blocks N-glycan branching was discovered as an alkaloid present in locoweed (Stegelmeier et

al, 1999). Locoweed consumed by grazing animals over several weeks resulted in weight-loss,

along with behavioural changes and even death (Stegelmeier et al, 1999).

Historically, the thrifty genotype or phenotype conferred an evolutionarily advantage by

promoting the efficient energy storage of fuel as fat during rare periods of nutritional abundance,

which in turn increased the likelihood of survival during periods of famine or times of varying

food scarcity (McClain, 2002; O'Rourke R, 2014). However, this tendency to store fat by

individuals harboring thrifty genes would be maladaptive or detrimental in environments with

calorie rich and stable food supply, as is the case in modern society or in ad libitum fed

experimental animals (McClain, 2002; O'Rourke R, 2014). In fact, it has been proposed that the

large increase in prevalence of conditions such as obesity, diabetes and the metabolic syndrome is

a result of evolutionary adapted biological modules such as the HBP being pushed into overdrive

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by environmental changes of chronic excess calorie consumption and reduced physical activity

(McClain, 2002).

4.8 N-Glycan Branching and Nutrient Uptake

My studies provide evidence that N-glycan branching regulates metabolism, and that

GlcNAc supplementation to UDP-GlcNAc and N-glycan branching enhances nutrient uptake and

reprograms cellular metabolism to promote lipid storage and body-weight increase. I also observed

that UDP-GlcNAc levels are sensitive to extracellular Glc and Gln supply, and that induced Mgat5

overexpression enhanced cell growth under limiting nutrient conditions in a cell-autonomous

manner (Abdel Rahman et al, 2015; Abdel Rahman et al, 2013). Future studies could include in

vivo analysis of nutrient uptake in GlcNAc supplemented Mgat5 wild-type and null mice using

intraperitoneal injection of glucose, glutamine or fatty acids labelled with tritium (2-deoxy-D-[1-

14C]glucose analog) or non-radioactive stable heavy isotopes (U-13C6 D-Glucose or 13C5 L-

Glutamine), and subsequent determination of incorporation into tissues of interest.

GlcNAc supplementation increased N-glycan branching in cell culture and in liver

glycoproteins. Since the Golgi N-glycan pathway regulates multiple surface glycoproteins, and the

number of glycoproteins known to carry N-glycans is vast, the phenotype observed is most likely

due to a complex interaction involving different glycoproteins. The increased uptake of Glc, Gln

and FA suggests that increased Mgat5-mediated N-glycans and enhanced cell surface residency of

nutrient transporters such as Gluts, Slc3a2 and/or CD36 could be involved. This will however

require further testing and validation in terms of glycan-galectin lattice formation, glycoprotein

retention and half-life at the cell surface, and facilitated nutrient transport into the cell. In addition

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to solute transporters, cell surface receptors for growth factors, cytokines and hormones are also

glycoproteins bearing N-glycans, and hence could be involved in the phenotype reported. At this

point it remains to be determined if this phenotype is mediated by a strong contribution from a few

cell surface glycoproteins, or a weak effect arising from many, both of which could ultimately

result in the same outcome. Establishing which specific glycoprotein is responsible might prove

difficult. However, it is feasible to perform studies on individual transmembrane glycoproteins,

for instance CD36, or a specific glucose or glutamine transporter, and determine how its N-glycan

branching, cell surface residency, transport capacity and intermediary metabolism are affected by

GlcNAc supplementation and modification of N-glycan branching via inducible gene knockout or

overexpression. To gain insight, cell culture studies could be performed aimed at determining

transporter glycosite specific N-glycan branching, cell surface dynamics, nutrient transport, and

cellular metabolism. Introducing sequon-specific mutations that prevent N-glycan formation in

such a transporter, and determining if cell surface residency is reduced and nutrient uptake

decreased would also provide valuable information.

4.9 HBP Interacts with Gene Polymorphisms in N-Glycan Branching Pathway

In human disease the complete deficiency of a particular gene is relatively rare, while

partial deficiencies in multiple genes and abnormal inductions of some genes appear more common

(Lauc et al, 2010; Zoldos et al, 2013). The human population harbors numerous genetic variants.

The majority of human variability originates from SNPs that individually do not have visible

phenotypes, but if present in specific combinations within the same individual can give rise to

significant phenotypic effects (Lauc et al, 2010; Zoldos et al, 2013). Due to numerous genes

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required to generate and modify N-glycans, N-glycosylation might be particularly prone to this

type of variability (Lauc et al, 2010; Zoldos et al, 2013). To investigate the association of common

genetic variation with plasma levels of fatty acids, genome-wide association study (GWAS)

conducted in different cohorts of individuals of European ancestry identified variant alleles of

SNPs in ALG14 gene to be associated with higher circulating levels of palmitic acid (Wu et al,

2013). The protein encoded by ALG14 gene heterodimerizes with protein encoded by ALG7 or

ALG13 gene to form a bipartite UDP-GlcNAc transferase responsible for catalyzing the first two

committed steps in the biosynthesis of oligosaccharides in the ER that are essential for N-linked

glycosylation of proteins. Mutations in genes ALG7, ALG14, ALG2 or GFAT1 result in

congenital myasthenic syndrome, which impairs signal transmission at the neuromuscular synapse

(Freeze et al, 2015). This syndrome is part of a wider spectrum of congenital disorders of

glycosylation caused by genetic defects affecting primarily N-linked glycosylation (Freeze et al,

2015). Interfering RNA silencing of ALG14 expression resulted in reduced cell surface expression

of muscle acetylcholine receptors (Cossins et al, 2013). Furthermore, myotubes derived from

patients with GFAT1 mutations, or inhibition of GFAT1 enzymatic activity or RNA silencing of

GFAT1 expression reduced cell surface expression of muscle acetylcholine receptors (Zoltowska

et al, 2013). This demonstrates that multiple genes encoding components of the hexosamine

biosynthetic and N-linked glycosylation pathways have the potential to harbor mutations that cause

this syndrome, and suggests a general framework that might be operational in other diseases and

disorders.

In a GWAS carried out in individuals from diverse European populations a SNP located

downstream of the Mgat1 gene was shown to be significantly associated with body-weight

(Jacobsson et al, 2012; Johansson et al, 2010). Furthermore, two other SNPs in the Mgat1 gene

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were found to be nominally associated to body-weight (Johansson et al, 2010). The molecular

mechanisms by which these SNPs may contribute to the observed associations are unclear. Genetic

variants can result in changes in expression, or SNPs in the HBP and Mgat genes can affect the

functional efficacy of the corresponding enzymes in the HBP and Golgi N-glycan branching

pathway. For instance, missense mutations that dramatically increased the apparent Km value of

Mgat1 for both UDP-GlcNAc and the glycoprotein acceptor were identified in CHO mutant cells

(Chen et al, 2001). Cells harboring a genetic variant of this kind synthesized hybrid and complex

type N-glycans on cell surface glycoproteins, albeit in reduced amounts compared to parental CHO

cells (Chen et al, 2001). This alteration might in turn translate into functional consequences for

growth factor signaling and nutrient transport.

Human multiple sclerosis patients frequently show partial deficiencies in Mgat5 modified N-

glycans, which lowers their T-cell sensitivity to autoimmune activation (Grigorian et al, 2009).

Disease associated human Mgat1 SNPs showed a gain-of-function mutation that increased Mgat1

mRNA and protein levels, which in turn increased Mgat1 activity and decreased Mgat5-mediated

β1,6-GlcNAc-branched N-glycans (Mkhikian et al, 2011). When metabolism limited substrate

availability in the form of UDP-GlcNAc, the Mgat1 gain-of-function haplotype lowered N-glycan

branching by limiting UDP-GlcNAc availability to downstream Mgat branching enzymes

(Mkhikian et al, 2011). In contrast, when UDP-GlcNAc supply increased, as occurs in presence of

high glucose or supplementation with GlcNAc and uridine, the Mgat1 haplotype had the opposite

effect on late N-glycan branching and glycoprotein surface residency (Mkhikian et al, 2011). These

studies demonstrate that small changes in few genes and/or metabolites affecting a common

biochemical pathway can have drastic effects on N-glycosylation, and provide a framework for

understanding how genetic and environmental effects converge and interact at the molecular level.

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Mgat5-deficient mice display lower thresholds to T-cell receptor clustering, T-cell

activation and autoimmune disease (Grigorian et al, 2009). However these phenotypes of Mgat5-/-

mice appear more penetrant on certain mouse genetic backgrounds than others, suggesting the

existence of strain-dependent modifier genes or strain-specific SNPs in the HBP and/or N-glycan

branching pathway for these phenotypes (Dennis et al, 2009; Grigorian et al, 2009). The PL/J strain

of mice, which show reduced Mgat activities, is hypersensitive to spontaneous demyelinating

autoimmune disease, a model of multiple sclerosis (Grigorian et al, 2009). Multiple sclerosis

increases in frequency and onset in PL/J Mgat5-/- mice, while the 129/sv Mgat5-/- mice do not display

the clinical or histopathological symptoms of this autoimmune disease at any age (Grigorian et al,

2009). Importantly, GlcNAc supply to the HBP and N-glycan branching regulates PL/J T-cell

hypersensitivity and susceptibility to autoimmune disease in vitro and in vivo, suggesting that some

of these polymorphic differences are amenable to environmental influences through metabolism

(Grigorian et al, 2009; Mkhikian et al, 2011). This implies that genetic variation and conditional

regulation via environmental factors that influence metabolic pathways interact through the HBP and

Golgi N-glycan branching pathway to regulate cellular homeostasis (Dennis et al, 2009; Mkhikian et

al, 2011).

It would be of interest to determine if variant alleles in the HBP and/or Golgi N-glycan

enzymes could be linked with body-weight regulation, obesity, or other metabolic diseases. The

C57BL/6 strain of mice used in the GlcNAc supplementation experiments is sensitive to

development of diet-induced obesity and insulin resistance. It would be interesting to find out if

GlcNAc supplementation in a different strain of mice, such as PL/J or 129/sv, would generate a

similar phenotype to that observed in C57BL/6 mice. Future studies could also include direct

sequence comparison of genes in the HBP and N-glycan branching pathway to identify genetic

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polymorphisms between strains of mice exhibiting different body-weight and/or composition, or

in human populations displaying obesity and severe body-weight phenotypes.

Variants of the gene glucosamine-6-phosphate deaminase 2 (GNPDA2), which codes for

an enzyme participating in the HBP, is strongly linked to BMI and obesity in children and adults

from different human populations (Renstrom et al, 2009; Willer et al, 2009; Wu et al, 2010; Zhao

et al, 2009). GNPDA2 transcript levels were down-regulated in the hypothalamus of high-fat diet

fed rats (Gutierrez-Aguilar et al, 2012). GNPDA2 catalyzes the reversible reaction converting

GlcN-6P back to Fru-6P and ammonium, thus opposing the function of GFAT which catalyzes the

conversion of glutamine and Fru-6P to GlcN-6P and glutamate at the start of the HBP. The function

of GNPDA2 has not been explored but it is reasonable to assume that a deletion or deleterious

mutation in this gene would result in enhanced flux through the HBP and increased UDP-GlcNAc

formation. Such an outcome would support my findings of increased body-weight observed with

GlcNAc supplementation experiments, and relate it to the epidemiological findings linking SNPs

in GNPDA2 with obesity. The caveat however is that there is also a GNPDA1, which could be

redundant in function, and that both enzymes GNPDA and GFAT are allosterically controlled by

GlcNAc-6P, in a positive and negative manner respectively (Broschat et al, 2002; Lara-Lemus &

Calcagno, 1998).

4.10 GlcNAc Supplementation and O-GlcNAcylation

Most studies where the HBP is supplemented to increase UDP-GlcNAc levels are very

selective in terms of which downstream pathway utilizing UDP-GlcNAc is analyzed, either

pursuing O-GlcNAcylation, N-glycosylation, or hyaluronan synthesis. However the fact remains

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that any of these could be impacted by increased activity in the HBP. In theory any glycoconjugate,

be it a glycoprotein, glycolipid, or glycosaminoglycan, that utilizes GlcNAc from UDP-GlcNAc

could potentially be affected. The complex nature of the effect of increasing flux in the HBP, with

few potential targets involved, makes it difficult to unambiguously dissect its role. I focused on N-

glycosylation, for numerous valid reasons described above and Dennis laboratory’s long-term

commitment to N-glycan branching, but given that the role of O-GlcNAcylation is firmly

established in metabolism and energy homeostasis it is reasonable to assume that it could also have

been a contributing factor to the metabolic phenotype observed with GlcNAc supplementation

(Hardiville & Hart, 2014). For instance, O-GlcNAcylation is involved in short-term fasting

response. In response to glucagon and cAMP, CRTC2 is dephosphorylated and O-GlcNAcylated

at the same site, which promotes its translocation into the nucleus, binding to CREB, and induction

of gluconeogenic gene expression (Dentin et al, 2008). In this study no major changes were

detected in O-GlcNAcylation at the level of western-blot analysis probed with the O-β-GlcNAc-

specific monoclonal antibody CTD 110.6, a reagent heavily used in O-GlcNAcylation research

(Hardiville & Hart, 2014). However, it is reasonable to argue that one limitation of this approach

was the coarse level of resolution employed in my analysis of O-GlcNAcylation. Thus, I cannot

exclude the possibility that more subtle changes in O-GlcNAcylation, such as that occurring on

CRTC2, did take place on specific signaling proteins, transcription factors, metabolic enzymes, or

even histones, which would have eluded the analysis performed. Increasing however, increasing

O-GlcNAcylation globally by using a selective inhibitor of O-GlcNAcase for a few months did

not affect body-weight, or glucose or lipid metabolism in rodents (Macauley et al, 2010a;

Macauley et al, 2010b).

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4.11 GlcNAc Supplementation and Gut Microbiota

It has been shown before, and confirmed and extended in my studies herein, that GlcNAc

supplementation in cells in culture contributes to UDP-GlcNAc pool and increases β1,6-GlcNAc

branching (Abdel Rahman et al, 2015; Abdel Rahman et al, 2013; Dennis et al, 2009; Johswich et

al, 2014; Wellen et al, 2010). My results show that GlcNAc supplementation affects metabolism

in a cell-autonomous manner in cell culture (Abdel Rahman et al, 2015). In mammalian cells in

culture, salvaged GlcNAc does not appear to re-enter glycolysis to be used as a fuel source for

energy production (Abdel Rahman et al, 2013; Chertov et al, 2011; Wellen et al, 2010). However

in vivo the scenario could be much different, with the gut microbiota acting as a confounding

variable. The microbiome is a diverse ecosystem containing trillions of microbes such as bacteria,

fungi, archaea, protozoa, and viruses, living in the host’s digestive system (Greiner & Backhed,

2011). There is a complex relationship and an intimate connection between the host and the

microbiome, primarily though diet, and more and more studies are being published describing how

dietary interventions affect the host metabolism and physiology in health and disease via their

impact on the microbiome (Greiner & Backhed, 2011). Since nutrients affect the composition of

the gut microbiota, such changes are possible to occur over the course of dietary GlcNAc

supplementation.

The gut microbiota contributes to host metabolism by several mechanisms including

increased inflammatory tone, increased energy harvest from the diet, modulation of lipid

metabolism, and altered endocrine function (Greiner & Backhed, 2011). Recent data have revealed

that the gut microbiota has a strong effect on energy homeostasis, and thus could be considered an

environmental factor that promotes adiposity, and contributes to obesity and other metabolic

diseases (Fang & Evans, 2013; Greiner & Backhed, 2011). Studies in rodents and humans have

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shown that the microbiome of obese has lower bacterial diversity and a significantly greater ratio

of the phylum Firmicutes to Bacteriodetes (Fang & Evans, 2013; Greiner & Backhed, 2011). It is

possible that GlcNAc supplementation in mice promoted selective advantage to certain microbial

communities, thereby altering the composition of the gut microbiome to modulate the host

metabolism, physiology and homeostatic regulation. Experiments are currently underway to

examine the effect of GlcNAc supplementation on gut microbiota in mice. A recent study reported

that supplementation with specific human breast milk oligosaccharides influenced the selective

intestinal bacterial colonization pattern in mice, as indicated by changes in the abundance of

specific strains (Weiss et al, 2014). The sources and amounts of GlcNAc available to the host

and/or the gut microbiome have not been studied. However GlcNAc residues are common

constituents of glycoprotein N-glycans, mucus glycans, glycosaminoglycan polysaccharides of

extracellular matrix, milk oligosaccharides, fungal and exoskeleton chitin, and bacterial

peptidoglycan (Konopka, 2012; Koropatkin et al, 2012). Thus, in theory, GlcNAc can be obtained

from the host glycocalyx, dietary sources, and the microbiome itself. The microbiome has a variety

of carbohydrate-active enzymes, such as the glycosidases, that may release GlcNAc from all of

these sources during metabolic breakdown in the gut, and perhaps even share it with the host

(Koropatkin et al, 2012). Indeed, the microbiome associated with obesity might have more

glycosidase activity, and hence may liberate more GlcNAc to be used by the host.

In many microorganisms GlcNAc has a significant role in cell signaling (Konopka, 2012).

For example, GlcNAc stimulates the human fungal pathogen Candida albicans to undergo changes

in morphogenesis and expression of virulence genes (Konopka, 2012). GlcNAc also regulates

virulence properties of pathogenic bacteria such as E. coli, and stimulates soil bacteria to sporulate

and produce antibiotics (Konopka, 2012). Bacteroidetes, Firmicutes and a variety of other bacterial

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taxa actively grow in the presence of GlcNAc (Tada & Grossart, 2014). Therefore, unlike

mammalian cells, bacterial cells are well adapted at utilizing GlcNAc as a carbon and energy

source in glycolysis (Brigham & Malamy, 2005; Rigali et al, 2006; Tannock, 1977). Bacteria

fermenting GlcNAc as a carbon source remove the acetyl group and excrete acetate and propionate

(Kotarski & Salyers, 1981). In this study the serum level of propionate was actually lower in

GlcNAc supplemented mice, suggesting that perhaps GlcNAc utilization by the gut bacteria was

not a significant factor.

Future in depth experiments would be necessary to determine what effects, if any, GlcNAc

supplementation has on gut microbiota, and its consequences on the host metabolism and

physiology. It should be noted that many of these bacteria reside in the cecum, the beginning of

the large intestine, while monosaccharides and disaccharides are primarily absorbed much earlier

in the small intestine. Hence, the majority of orally ingested GlcNAc could have already been

absorbed by the host, with little spared for the gut bacteria. To address this experimentally, the

effect of GlcNAc supplementation on the production of bacterial metabolites such as the short

chain fatty acids acetate, propionate, and butyrate should be measured in the colon fluid. Changes

in the production of these metabolites following GlcNAc supplementation would support a

contribution of intestinal microbes in the regulation of nutrient uptake and energy homeostasis. In

the future, in order to bypass the possible issue of GlcNAc and microbiota interaction altogether,

germ-free mice might be used to perform the same experiment. Alternatively, instead of oral

supplementation, GlcNAc might be delivered parenterally, i.e. administered via a route other than

through the digestive tract, i.e. intraperitoneally, intramuscularly or subcutaneously. Such

approach would also provide precise knowledge of the dose delivered to the animal.

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Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi … › bitstream › 1807 › 71313 › ... · 2016-02-19 · balance, and promoted excess lipid storage, as well asbody-weight