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The Role of FGF21 in Pancreatic Islet Metabolism
By
Mark Yimeng Sun
A thesis submitted in conformity with the requirements for the degree of Master of Applied Science
Graduate Department of Institute of Biomaterial and Biomedical Engineering
University of Toronto
© Copyright by Mark Yimeng Sun (2011)
ii
The Role of FGF21 in Pancreatic Islet MetabolismMark Yimeng Sun
Master of Applied Science
Institute of Biomaterial and Biomedical EngineeringUniversity of Toronto
2011
ABSTRACT
The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic
FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and
triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and
function of islet �-cells, the mechanisms are currently unknown. This thesis examines
mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies
showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2)
protein expression in mouse islets. Autofluorescence microscopy showed difference in
NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets
showed significant decreased mitochondrial energetics when acutely stimulated with high
concentrations of glucose and palmitate. This decrease in energetics correlated with increased
generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state.
These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high
fat loading and reduces insulin during nutrient stress.
iii
ACKNOWLEDGEMENTS
I would like to thank my supervisors Professor Rocheleau and Professor Kilkenny for
their continued guidance, support and encouragement throughout my studies. I will always
remember to follow my interest and passion as I move on to the next stage of my career. I would
also like to thank my committee members Professor Giacca and Professor Nagai for their helpful
comments and directions.
In addition, I want to thank Professor Volchuk for the insightful suggestions on isolating
protein from pancreatic islet tissue, Svetlana Altamentova for help with animal work, and all the
Rocheleau lab members for always being there to lend a helping hand.
I want to thank my friends for helping me out during my lengthy recovery after ACL
reconstruction.
Lastly, I want to thank my family for their unconditional love. Mom and Dad, you both
have given up so much and worked so hard to provide me the wonderful opportunities to lead a
meaningful and fulfilling life. You will always be my inspiration.
iv
TABLE OF CONTENTS
Abstract ii
Acknowledgements iii
Table of Contents iv
List of Abbreviations vii
List of Tables x
List of Figures xi
CHAPTER 1 – Introduction............................................................................................................ 1
1.1 Fibroblast Growth Factor 21..................................................................................................... 1
1.1.1 FGFs and FGF Receptors ............................................................................................... 1
1.1.2 Endocrine-like FGFs....................................................................................................... 2
1.1.3 FGF21 Signalling ........................................................................................................... 3
1.1.4 FGF21 Expression and Function .................................................................................... 4
1.1.5 FGF21 and Metabolic Syndrome ................................................................................... 5
1.2 Type 2 Diabetes ........................................................................................................................ 6
1.2.1 The Glucolipotoxicity Model ......................................................................................... 6
1.2.2 FGF21 in the Pancreas.................................................................................................... 7
�����-Cell Metabolism .................................................................................................................... 8
1.3.1 NAD(P)H and GSIS ....................................................................................................... 8
1.3.2 ACC and the Regulation of Metabolism ...................................................................... 10
CHAPTER 2 – Research Rationale .............................................................................................. 12
2.1 Hypothesis............................................................................................................................... 12
2.2 Objectives ............................................................................................................................... 12
CHAPTER 3 – Materials And Methods ....................................................................................... 13
3.1 Pancreatic Islet Isolation and Tissue Culture.......................................................................... 13
v
3.2 Western Immunoblot .............................................................................................................. 13
3.3 Microfluidic Device Design.................................................................................................... 14
3.4 Microfluidic Device Fabrication............................................................................................. 15
3.5 Microfluidic Device Application ............................................................................................ 17
3.6 Redox Autofluorescence Imaging........................................................................................... 18
3.7 LipDH Redox Index................................................................................................................ 20
3.8 ImageJ Analysis ...................................................................................................................... 21
3.9 Statistical Analysis.................................................................................................................. 22
CHAPTER 4 – The Effect of FGF21 on Islet ACC Expression and Islet Metabolism................ 23
4.1 Introduction............................................................................................................................. 23
4.2 Chapter Specific Methods....................................................................................................... 24
4.2.1 pACC Western Immunoblot......................................................................................... 24
4.2.2 Palmitate Preparation.................................................................................................... 25
4.2.3 Applying the Microfluidic Device Imaging Platform .................................................. 25
4.3 Results..................................................................................................................................... 26
4.3.1 FGF21 decreases islet ACC protein levels ................................................................... 26
4.3.2 FGF21 increases islet pACC:ACC ratio at low glucose............................................... 27
4.3.3 FGF21-treated islets maintain higher NAD(P)H glucose dose response post 24 hr culture in palmitate ................................................................................................................ 28
4.3.4 FGF21-treated islets exhibit lower NADPH levels at high glucose............................. 28
4.3.5 FGF21-treated islets exhibit decreased NADPH with citrate stimulation.................... 30
4.3.6 FGF21-treated islets exhibit altered processing of mitochondrial NADH during high fat and high glucose challenge............................................................................................... 31
4.4 Discussion ............................................................................................................................... 32
CHAPTER 5 – The Effect of FGF21 on Islet Mitochondrial Energetics and Insulin Secretion .. 37
5.1 Introduction............................................................................................................................. 37
5.2 Chapter Specific Methods....................................................................................................... 38
5.2.1 Rhodamine123 Imaging ............................................................................................... 38
5.2.2 UCP2 Western Immunoblot ......................................................................................... 38
vi
5.2.3 Insulin ELISA............................................................................................................... 39
5.3 Results..................................................................................................................................... 40
5.3.1 FGF21-treated islets exhibit lower mitochondrial membrane potential during high fat and high glucose challenge.................................................................................................... 40
5.3.2 FGF21 decreases islet UCP2 protein levels ................................................................. 41
5.3.3 FGF21 induces detection of UCP2 of higher than expected molecular mass .............. 42
5.3.4 FGF21-treated islets secrete less insulin during high fat and high glucose challenge . 43
5.4 Discussion ............................................................................................................................... 44
CHAPTER 6 – General Discussion .............................................................................................. 47
6.1 Discussion ............................................................................................................................... 47
6.2 Future Directions .................................................................................................................... 52
6.3 Concluding Remarks............................................................................................................... 54
CHAPTER 7 – References............................................................................................................ 56
vii
LIST OF ABBREVIATIONS
ACC – Acetyl-CoA Carboxylase
ADP – Adenosine Diphosphate
AICAR – Aminoimidazole Carboxamide Ribonucleotide
AMPK – Adenosine Monophosphate-activated Protein Kinase
ATP – Adenosine Triphosphate
BMHH – Imaging Buffer
BSA – Bovine Serum Albumin
CPT1 – Carnitine Palmitoyl Transferase I
DAG – Diacylglycerol
DCF - Dichlorofluorescein
ELISA – Enzyme-linked Immunosorbent Assay
ER – Endoplasmic Reticulum
FAO – Fatty Acid Oxidation
FAS – Fatty Acid Synthesis
FCCP – Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
FFA – Free Fatty Acids
FGF21 – Fibroblast Growth Factor 21
FGFR – Fibroblast Growth Factor Receptor
GLUT – Glucose Transporter
GSH – Glutathione
GSIS – Glucose Stimulated Insulin Secretion
HEPES – (4(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
viii
HSPG – Heparin Sulfate Proteogylcans
KLB – Klotho Beta
LipDH – Lipoamide Dehydrogenase
MAPK – Mitogen-activated Protein Kinase
mNADH – Mitochondrial NADH
NaCN – Sodium Cyanide
NAD(P)H – Aggregate of NADH and NADPH
NADH – Nicotinamide Adenine Dinucleotide
NADPH – Nicotinamide Adenine Dinucleotide Phosphate
NNT – Nicotinamide Nucleotide Transhydrogenase
PDMS – Polydimethylsiloxane
PI3 – Phosphoinositide 3
����– Peroxisome Proliferator-activated Receptor Alpha
PPP – Pentose Phosphate Pathway
Rh123 – Rhodamine123
ROS – Reactive Oxygen Species
RT – Room Temperature
RT-PCR – Reverse-Transcription Polymerase Chain Reaction
SDS-PAGE – Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SREBP1c – Sterol Regulatory Element-binding Protein 1c
TBS-T – Tris-Buffered Saline-Tween 20
TG – Triglyceride
UCP2 – Uncoupling Protein 2
ix
VEGF – Vascular Endothelial Growth Factor
WAT – White Adipose Tissue
x
LIST OF TABLES
Table 1. Autofluorescence Microscopy Summary
xi
LIST OF FIGURES Figure 1. Model of classical FGF ligand binding and activation of FGFR signalling
Figure 2. Endocrine FGF Signaling
Figure 3. KLB expression in pancreatic islets
Figure 4. Model of acetyl-CoA carboxylase �� ��������-cell metabolism
Figure 5. Y-channel microfluidic device design
Figure 6. Imaging Platform Setup
Figure 7. FGF21-dependent ACC regulation in pancreatic islets
Figure 8. FGF21 increases islet pACC:ACC ratio at low glucose
Figure 9. Glucose-stimulated NAD(P)H response of islets cultured in palmitate for 24 hrs
Figure 10. Glucose-stimulated NAD(P)H response
Figure 11. Glucose-stimulated NAD(P)H and LipDH(mNADH) responses
Figure 12. Citrate metabolism in control and FGF21-treated islets
Figure 13. Palmitate metabolism in control and FGF21-treated islets
Figure 14. Palmitate and glucose-stimulated changes in mitochondrial membrane potential
Figure 15. FGF21-dependent UCP2 regulation in pancreatic islets
Figure 16. Potential FGF21-dependent UCP2 activation in pancreatic islets
Figure 17. Palmitate and glucose-stimulated insulin secretion
1
CHAPTER 1 – INTRODUCTION
1.1 Fibroblast Growth Factor 21
Fibroblast growth factor 21 (FGF21) is a novel endocrine-like factor found to be involved
in the regulation of energy homeostasis [1]. FGF21 mitigates the early symptoms of metabolic
syndrome in animals models by significantly lowering blood glucose and triglycerides,
decreasing fasting insulin levels, and improving glucose tolerance [2]. This section will provide
background on FGF21 by reviewing classical paracrine FGF signalling, comparing paracrine and
endocrine-like FGF ligands, and discussing the relevance of FGF21 to metabolic syndrome.
1.1.1 FGFs and FGF Receptors
Fibroblast growth factors (FGFs) are a family of structurally related polypeptide growth
factors [3]. Classical FGFs initiate paracrine signalling by binding to low-affinity, high capacity
heparin sulfate proteoglycans (HSPG). The HSPGs protect FGFs from degradation, and act as a
reservoir for facilitating specific binding to high-affinity, low capacity transmembrane tyrosine
kinase fibroblast growth factor receptors (FGFRs). Ligand binding induces FGFR dimerization
and intracellular trans-autophosphorylation of tyrosine residues, leading to the initiation of
intracellular signalling activity (Figure 1) [3] [4]. Examples of FGF regulated processes include
proliferation through signalling via the MAPK cascade, as well as promotion of survival and
inhibition of apoptosis via the PI3/Akt pathway [5]. In addition, FGFs exert a role in vascular
endothelial growth factor (VEGF) regulated angiogenesis [6]. FGFs are also important in
development as FGF signaling is found to be involved in organogenesis [7]. Overall, there is a
diverse range of biological processes regulated by the classical FGF signalling pathway [8].
2
Figure 1. Model of classical FGF ligand binding and activation of FGFR signalling [4]. Paracrine secreted FGF (fibroblast growth factor) ligands are sequestered by the heparin sulfate proteoglycans in the extracellular matrix (ECM). The ECM localized ligands bind to FGFRs (fibroblast growth factor receptors) causing dimerization, cross phosphorylation of the intracellular tyrosine kinase domains, and activation of signal transduction.
1.1.2 Endocrine-like FGFs
Recently, the FGF19 subfamily (including FGF19, FGF21, and FGF23) has emerged as a
novel class of FGFs shown to exert hormone-like function to maintain various metabolic
processes (Figure 2) [9]. For instance, FGF23 has been found to act as a regulator of phosphate
and vitamin D metabolism [10] and FGF19 plays a role in the maintenance of bile acid
homeostasis [11]. Structurally, the heparin binding regions of the FGF19 subfamily distinctly
differ from those of the classical FGFs resulting in a significant reduction in the heparin sulfate
proteoglycans (HSPG) binding affinity. In addition, endocrine FGFs require a co-receptor from
the klotho receptor family to promote FGFR dimerization and subsequent activation of signalling
cascades (Figure 2). Overall, the weak affinity of the FGF19 subfamily to HSPG allows these
ligands to escape the extracellular matrix reservoir, and the tissue specific expressions of FGFRs
3
and relevant klotho co-receptors allows the FGF19 subfamily of ligands to function like
endocrine factors [9].
Figure 2. Endocrine FGF Signaling [9]. FGF15/19 is secreted by the ileum to regulated bile acid homeostasis by decreasing liver bile acid synthesis, increase gallbladder filling and decrease ileum transport. FGF21 is secreted by the liver to regulate energy homeostasis by inducing increased glucose uptake and lipolysis in white adipose tissue. FGF23 is secreted from bone to regulated phosphate and vitamin D homeostasis through increased phosphate excretion and decreased vitamin D synthesis in the kidney.
1.1.3 FGF21 Signalling
FGF21 is a member of the FGF19 subfamily of endocrine FGFs. Similar to the classical
FGF ligands, FGF21 binds to tyrosine kinase FGFRs to activate downstream signalling cascades
such as the extracellular-signal-regulated-kinase1/2 (ERK1/2) pathway [12]. Early attempts to
stimulate FGFR with FGF21 failed to demonstrate direct activation of downstream signal
pathways [1]. Further investigation identified a single-pass transmembrane protein in the klotho
family �������������� (KLB), and confirmed its role as a cofactor in successful FGF21 binding to
FGFR1 [13]. As mentioned previously, although FGFR1 is expressed ubiquitously throughout
the body, FGF21 exerts its regulatory function only at tissues that express both FGFR1 and the
4
relevant co-receptor KLB. Specifically, FGF21 has been shown to be involved in regulating
metabolic tissues such as the adipose, the liver and the pancreas, all of which express both
FGFR1 and KLB [14]. The Rocheleau lab has demonstrated the expression of FGFR1 in mouse
islets [3], and recently confirmed KLB expression both at the gene and protein levels in mouse
islets using RT-PCR and immunofluorescence labelling respectively (Figure 3).
Figure 3. KLB expression in pancreatic islets. (A) The cDNA from two separate mouse islet preparations (Islets 1 and 2) were amplified using oligonucleotide primers designed to recognize the N- (KLB-front) and C-terminal (KLB-��������������������-actin cDNA was amplified to ensure sample integrity, and water was included as a no-DNA negative control. (B) Pancreatic islets were fixed and immunofluorescently labeled using an antibody specific for the extracellular portion of KLB. Immunostaining with no primary (No Primary) was included as a negative control; the scale bar represents 50 μm.
1.1.4 FGF21 Expression and Function
FGF21 expression is regulated in a circadian manner [15]. Circulating FGF21 rise at
midnight peaking in the early morning and declines to basal concentrations early in the afternoon
[16]. The time dependent oscillatory expression of FGF21 mRNA followed that of free fatty
acids (FFA) and opposed patterns observed in blood insulin and glucose [16].
In the liver, FGF21 expression is induced via activation of peroxisome proliferator-
���������� ���� ���� �� ��� !���� [17]. During fasting or high nutrient challenge, hepatic
breakdown of fatty acids is activated. The metabolised fatty acids in turn activate the nuclear
receptor ���� to stimulate the expression of FGF21 mRNA and subsequent secretion of the
5
protein [17]. Secreted FGF21 further stimulates the liver to induce the expression of peroxisome
proliferator-���������� ���� ���� "� ������������ ������-�� !�#$-�� leading to increases in the
transcription of genes involved in fatty acid oxidation, TCA cycle flux and mitochondrial
oxidative phosphorylation [18]. Furthermore, FGF21 increases the expression of the GLUT1
transporter [19] and stimulates lipolysis in white adipocytes [2]. Overall, these results
demonstrate that FGF21 plays a role in the adaptive metabolic response to starvation and high
nutrient loading in normal physiology.
1.1.5 FGF21 and Metabolic Syndrome
The motivation in testing FGF21 in obese/diabetic animal models began with the
identification of FGF21’s ability to enhance glucose uptake in adipocytes [1]. Systemic
administration of FGF21 or transgenic over-expression of FGF21 in obese animals led to
significantly lower blood glucose and triglycerides, decreased fasting insulin levels, and
improved glucose tolerance [2]. Administration of FGF21 to diabetic rhesus monkeys over a 6
week period resulted in reduced fasting plasma glucose, triglyercide, insulin, and glucagon levels
without evidence of mitogenicity, hypoglycemia or weight gain [1]. In contrast, it has been
shown that ablation of FGF21 in a FGF21-KO model led to development of fatty liver,
hypertriglyceridemia, increased serum FFAs and attenuated glucose handling and insulin
sensitivity [20]. As well, it has been shown that the circadian regulation of FGF21 is disrupted
in obese individuals with a higher baseline value and blunted rise in nocturnal circulating
concentrations [16]. Overall, these studies show that FGF21 has mitigating effects on metabolic
syndrome and the disruption of FGF21 regulation is relevant in the development of the disease.
6
1.2 Type 2 Diabetes
The beneficial effects of FGF21obesrved in obese/diabetic animal models have made it a
potential therapeutic target for the treatment of type 2 diabetes. Type 2 diabetes is a complex
metabolic disorder characterized by impaired insulin secretion and peripheral tissue insulin
resistance. The most important risk factor in the development of diabetes is obesity, and
populations of the western world are at higher risk than ever for the development type 2 diabetes
[21]. Since the function of the pancreatic islet is central to the disease of diabetes, this section
will review the glucolipotoxicity model of diabetes pathogenesis and discuss the current
understanding of FGF21’s role in the pancreas.
1.2.1 The Glucolipotoxicity Model
During the development of type 2 diabetes, the glucose-stimulated insulin response is
dampened by glucose and lipid toxicity [22]. The glucolipotoxicity model of diabetes
pathogenesis describes that elevated levels of fat and glucose from excess nutrition has
�����%������ �������� ��� ���� ��������� ���� ��������� ��� ���������� �-cells [23]. The effects of
glucolipotoxicity begin with prolonged high nutrient loading leading to endoplasmic reticulum
(ER) stress in the white adipose tissue (WAT). As the ER of adipocytes can no longer process
the excess nutrients, an efflux of free fatty acids enters the circulatory system and creates an
overload of triglycerides (TG) in other metabolic tissues including the pancreatic islet. Although
mechanistically unclear, is has been shown that prolonged exposure of long chain fatty acids
such as TGs is detrimental to �-cell health and function by increasing apoptosis and decreasing
insulin secretion [24]. Early protective responses by the islet �-cell include modified cycling of
excess nutrients. Specifically, the activation of the AMPK pathway during high nutrient loading
7
has been shown to inhibit the fatty acid synthesis (FAS) pathway and promote the activation of
the fatty acid oxidation (FAO) pathway [24]. These mechanisms potentially provide �-cells with
a release valve to nutrient stress by allowing lipid detoxification via the breakdown of fatty acids
and limiting the buildup of long chain fatty acids [24]. Furthermore, glucolipotoxicity also
creates insulin resistance in target tissues such as the liver and adipose tissue [25]. To
accommodate a loss of insulin sensitivity in target tissues, ���� �����������-cells compensate by
hyper-secreting insulin to restore normal blood glucose levels. With prolonged exposure to
glucolipotoxic stress, the �-cell’s compensatory mechanisms eventually fails and results in a loss
of �-cell mass. The result is lowered plasma insulin levels, elevated glucose concentrations, and
ultimately full blown type 2 diabetes [26]. Overall, the understanding of the mechanisms by
which the �-cell can protect itself from fuel surfeit may lead to potential new therapeutic options
for the treatment of type 2 diabetes.
1.2.2 FGF21 in the Pancreas
In 2000, Hart et al demonstrated that attenuation of FGFR1c signalling in mice by over-
expression of a dominant negative form of FGFR1 led to the development of diabetes [27].
&�����%�����'��(�����������������%(��������-cells, impaired expression of glucose transporter 2
(GLUT2), and increased pro-insulin content due to impaired expression of prohormone
convertases [27]. Furthermore, it was shown that FGF21 administration in diabetic mice
�����������������-cell mass, islet insulin content and the glucose stimulated insulin secretion [12].
The same study also demonstrated that FGF21-treated ex vivo islets cultured in high glucose and
high palmitate had �����������-cell apoptosis. These results suggest that FGF21 regulation plays
a role in promoting �-cell survival and maintaining �-cell function under high nutrient
8
environments induced by both diet and disease. However, the mechanisms of FGF21 in the
regulation of pancreatic islet metabolism are largely unknown.
1.3 �-Cell Metabolism
To examine the potential FGF21 regulation of islet metabolism during varying nutritional
������)� �� �����*� ��� �-cell metabolism is necessary. This section will introduce the basic
%���(����� ���*�+����������-cell and their regulation.
1.3.1 NAD(P)H and GSIS
The pancreatic islet is a micro-organ built to rapidly sense extracellular glucose and
maintain blood glucose through the regulated secretion of insulin by the islet �-cells. Therefore,
�����-cell is designed for large fluxes in glucose metabolism. A rise in blood glucose stimulates
�-cell metabolism and increases the ATP:ADP ratio resulting in a cascade of events including
closure of ATP-sensitive K+ channels, membrane depolarization, Ca2+ influx, and insulin
secretion [28]. ,���� �'������������ ������� ��� ��*)� ���� �-cell can also metabolise fats via the
transport of fatty acyl-CoA into the mitochondria through the carnitine/palmitoyl-transferase 1
(CPT1) transporter (Figure 4) [22]. Nicotinamide adenine dinucleotide (NAD+) is a metabolic
coenzyme involved in redox reactions [29]. Both glucose and fat metabolism initiate the TCA
cycle to generate increasing levels of NADH (the reduced form of NAD+) (Figure 4), the energy
exchange molecule of the electron transport chain (ETC). The NADH molecules ultimately
establish the proton gradient for ATP production [29]. Furthermore, when there is a surfeit of
nutrients in the form of either fat, glucose, or both, major TCA cycle intermediates such as
citrate will be shuttled into the cytosol to undergo anabolic processes. Citrate exported from the
9
TCA cycle is enzymatically converted by acetyl-CoA carboxylase (ACC) to malonyl-CoA to
initiate the fatty acid synthesis (FAS) pathway [22] (Figure 4). The FAS pathway consumes
molecules of NADPH (the reduced form of coenzyme nicotinamide adenine dinucleotide
phosphate, NADP+) (Figure 4) in the formation of palmitate from malonyl-CoA and also in the
generation of long chain fatty acids [29]. Although structurally similar to NADH, NADPH is
primarily used in anabolic processes in metabolism. The NADPH used in the FAS process is
generated by the pentose phosphate pathway (PPP), and also by NADP+-dependent cytosolic
enzymes including isocitrate dehydrogenase [30]. Overall, cells process nutrients for both short-
and long-term needs through the generation of energy intermediates NADH and NADPH.
Therefore, studying the changes in cellular levels of NADH and NADPH provides insight into
the understanding of metabolism during different nutritional states.
Figure 4. Model of acetyl-����������� �������������-cell metabolism
10
1.3.2 ACC and the Regulation of Metabolism
A key enzyme in the regulation of metabolic partitioning is Acetyl-CoA Carboxylase
(ACC). ACC catalyzes the conversion of citrate to malonyl-CoA, the substrate for FAS.
Malonyl-CoA also acts as an inhibitor of fatty acid oxidation (FAO) by blocking the CPT1
transporter [29]. Therefore, ACC activity is important in determining the partitioning between
FAS versus FAO.
The activity of ACC is regulated by changes in cytosolic citrate concentration. Citrate
acts both as an allosteric activator of ACC, and a precursor of ACC’s substrate, acetyl-CoA [22].
Furthermore, ACC is also regulated by the fuel sensing AMP kinase (AMPK). AMPK is found
in the cytosol and becomes activated at low energy states by sensing increases in the AMP/ATP
ratio reflecting either increased cellular energy expenditure or fuel deprivation. AMPK
activation phosphorylates ACC to inhibit its function and decreases the synthesis of ACC at the
transcriptional level [22].
In the context of the glucolipotoxicity model in �-cells, AMPK regulation of ACC
suggests that high concentrations of glucose and fat will lead to reduced AMPK activity and
increased malonyl-CoA levels. As a result, the �-cell will partition towards FAS and build up
potentially toxic long chain fatty acids [22]. Results from recent studies suggest that changes in
malonyl-CoA signalling are �% �������������� ���� �����������-cell dysfunction, and ultimately
type 2 diabetes [22]. Pharmacological agents such as AICAR and metformin (both activators of
AMPK which in turn inhibits ACC) have been shown to prevent glucoliptoxicity-induced
apoptosis in INS832/13 cells [31]. Furthermore, AICAR and metformin were shown to
ameliorated the consequences of over-expressing sterol regulatory element-binding protein 1c
!-.������ ��� �-cells including activation of fatty acid synthase gene expression, triglyceride
11
(TG) buildup, and profound inhibition of glucose stimulated insulin secretion (GSIS) [32, 33].
Overall, these studies indicate that the ACC-malonyl-CoA �+���%� ����� ��+� ��������� ��� �����-
cell’s nutrient sensing mechanisms, and disruption of these mechanisms has direct implications
����-cell metabolism.
12
CHAPTER 2 – RESEARCH RATIONALE
2.1 Hypothesis
Islet dysfunction is believed to begin at the first signs of insulin resistance, and
dysfunction of islet metabolism plays a vital role in the progression and pathogenesis of type 2
diabetes [34]. FGF21 has shown promise as a therapeutic for type 2 diabetes; however, the
mechanisms of FGF21 regulation of pancreatic islet metabolism are currently unknown. This
thesis examines the effect of FGF21 on islet metabolism. Recent studies have demonstrated that
FGF21 stimulation causes a reduction in ACC protein expression in both liver and WAT [35,
36]. ACC plays a central role in the regulation of nutrient partitioning in cellular metabolism
[22]. Therefore, I hypothesized that (1) FGF21 stimulation will decrease islet ACC protein
expression and modulate islet metabolism during high glucose and high fat loading, and (2)
FGF21 stimulation will modulate islet mitochondrial energetics and insulin secretion.
2.2 Objectives
To address the first hypothesis:
1. Measure the effect of FGF21 on islet ACC protein expression.
2. Determine the effect of FGF21 on the metabolism of substrates up- and down-stream of
ACC.
To address the second hypothesis:
1. Determine the effect of FGF21 on islet mitochondrial membrane potential.
2. Determine the effect of FGF21 on islet insulin secretion.
13
CHAPTER 3 – MATERIALS AND METHODS
3.1 Pancreatic Islet Isolation and Tissue Culture
To examine the effect of FGF21 on islet ACC levels, I conducted ex vivo culturing of
mouse islets in the presence and absence of FGF21. Animal procedures were approved by the
Animal Care Committee of the University Health Network, Toronto, Ontario, Canada in
accordance with the policies and guidelines of the Canadian Council on Animal Care (Animal
Use Protocol #1531). The C57BL6 mice strain is a common inbred strain widely used for
models of human disease. Therefore, this strain was chosen due to its availability and
robustness. Pancreatic islets were isolated from 8- to 12- week-old C57BL6 male mice by using
collagenase digestion (Roche) [37]. Islets were subsequently cultured for 48 hours in the
presence or absence of FGF21 (100 ng/ml) in islet media (full RPMI 1640 medium
supplemented with 11 mM glucose, 10% FBS, and 5 U/ml penicillin-streptomycin). The
stimulation time was chosen based on previous studies in the liver and adipose tissue indicating
changes in ACC protein level were detectable post 48 hours of culture [35].
3.2 Western Immunoblot
Post 48 hour culture, islets were pipette-picked into microfuge tubes containing islet
media and collected by centrifugation (3000 rpm; 3 min). Islets were re-suspended in lysis
buffer [1% Triton X-100, 100 mM NaCl, 50 mM HEPES, 5% glycerol, 1 mM sodium vanadate,
���� ������������(�����%�'�!������ �����-�������/���0�1�����2������+���������������34�%��������
Whole-islet protein lysates (~20 islets/lane) were separated by 8% SDS-PAGE and transferred to
Tran-Blot nitrocellulose membranes (Bio-Rad). Membranes were blocked by incubating with
5% non-fat dry milk powder in Tris-buffered saline-Tween20 (TBS-T) for 1 hour at room
14
temperature (RT). Proteins of interest were detected by overnight incubation at 4°C in 5%
BSA/TBS-T containing the following antibodies at manufacturer’s recommendations: acetyl-
$������(�'+����� !�$$�� 5$����-� ����� �&������� +� !$-&�)��6�7772)��-actin [(CST); 1:1000].
Blots were subsequently incubated with anti-rabbit peroxidase-linked antibody [(CST), 1:2000]
diluted in 5% milk/TBS-T (45 min RT), and proteins were detected by enhanced
chemiluminescence (Pierce, Thermo Scientific Inc.). Protein levels of control and FGF21-
treated islets were quantified using blot band densitometry measurements.
3.3 Microfluidic Device Design
The pancreatic islet is a micro-organ with a size varying around 100 μm. Therefore, the
consistent manipulation of the tissue during microscopy experiments is difficult to perform in a
dish format. As a result, I designed a PDMS based microfluidic device to hold islets stationary
in a micro-channel. This device allowed easy change of stimulation media to the loaded ex vivo
islets during microscopy experiments.
A simple Y-channel design was employed to allow loading of islets from one inlet and
subsequent media flow from the second inlet. A drop in main channel height was implemented
to block islets from flowing out of the channel. Channels were designed using Adobe Illustrator
CS4. Channels were drawn to actual size using Adobe Illustrator in white color, and the
background was kept in black color. Designs were printed to acetate film. Separate films were
printed for the 25 μm layer and 125 μm layer (Figure 5).
15
Figure 5. Y-channel microfluidic device design. The 100 μm and 25 μm layer masks were printed on acetate film paper and used in the fabrication of device masters slides. The masks were placed over-top the SU8 photo-resist toallow UV-induced polymerization of the exposure channel designs. The hatch marks on the corner of each mask were used to assist proper alignment of the two layers.
3.4 Microfluidic Device Fabrication
Device masters were fabricated at the University of Toronto Microfluidics Foundry.
Corning glass slides were used as the master substrate. The slides were washed in the order of
isopropanol, acetone, isopropanol, and immediately air gun dried. The cleaned glass slides were
placed on a 65oC hot plate for 15 minutes to dry. SU8 photo-resist was used to coat the glass
substrate slides. SU8-2025 and SU8-2100 were used for the 25 μm and 125 μm layers,
respectively. The 25 μm layer was spun first using the spincoater programmed with step 1 at
500 rpm, ramp 5 seconds, dwell 10 seconds, and step 2 at 3000 rpm, ramp 5 seconds, dwell 30
seconds. The SU8-2025 coated glass substrate was soft baked at 65oC for 3 minutes, 95oC for 6
minutes, 65oC for 3 minutes, and cooled at room temperature for 10 minutes. Next, the 25 μm
master film negative was placed over the SU8 coated glass slide, and channel features were
exposed to UV for 3.8 seconds at 22 mW power. The post-UV exposure bake was done at 65oC
for 3 minutes, 95oC for 6 minutes, 65oC for 3 minutes, and cooled at room temperature for 30
minutes. Subsequently, the 125 μm layer was spun using SU8-2100 at settings, step 1 at 500
16
rpm, ramp 5 seconds, dwell 10 seconds, and step 2 at 2,500 rpm, ramp 5 seconds, dwell 30
seconds. The subsequent SU8-2100 coat was soft baked at 65oC for 5 minutes, 95oC for 30
minutes, 65oC for 5 minutes, and cooled at room temperature for 10 minutes. The 100 μm
features were added by applying the corresponding 100 μm master film negative, with channel
features exposed to UV for 7.8 seconds with 22 mW of power. The post-UV exposure bake was
done at 65oC for 5 minutes, 95oC for 12 minutes, 65oC for 5 minutes, and cooled at room
temperature for 30 minutes. The UV exposed channel features of the various SU8 coating were
thermally cross linked, and rendered insoluble to the MicroChem SU8 liquid developers. The
masters were finally developed by soaking in the SU8 developer solution in glass trays agitated
on an orbital shaker for 15 minutes. The masters were left to dry in room temperature overnight
before use in PDMS device fabrication.
Microfluidic devices were fabricated using elastomer polydimethylsiloxane (PDMS)
(Dow-Corning) [28]. PDMS and curing reagent were poured into P100 dishes at a 10:1 ratio (35
g to 3.5 g), mixed in a fume hood, and vacuum desiccated for removal of bubbles. The mixtures
were subsequently poured over top of the master slides containing positive moulds of channels.
The dishes were desiccated again to remove bubbles and baked at 80oC for 3 hours in a vacuum
oven for curing. The PDMS devices were peeled off from the master moulds gently, and cut into
appropriate sizes for bonding onto No. 1 thickness 24 × 50-mm coverslips (VWR Scientific).
Cover slips were cleaned using methanol, and dried using beta wipes. PDMS devices were first
hole-punched at the ends of channels. The hole-punched devices were placed channel side up on
a glass slide, and clear tape was used to clean the channel side while pushing the top side of the
PDMS against the glass slide. The cleaned PDMS device and dried cover slip were
simultaneously oxygen plasma treated [Harrick Plasma Cleaner] at high power for 1 minute.
17
The irreversible bonding was performed immediately following oxygen plasma treatment, and
the bonded device was left on an 80oC hotplate for 5 minutes. Finally, Tygon tubing was
inserted into the channel ends at punch holes.
3.5 Microfluidic Device Application
The PDMS microfluidic devices were employed in all imaging experiments to hold islets
stationary in a channel and allow subsequent controlled flow of stimulation media.
Prior to imaging, islets were loaded into the microfluidic device (Figure 6A), and the
steps performed were as follows: First, the device was mounted into the 37oC temperature
controller chamber and taped in place (Figure 6B, 6C). The inlet and outlet tubings were fed
through the exit holes on the side of the chamber, and the chamber was mounted onto the
microscope stage over the 40X oil immersion objective. The channel was brought into focus
using the translight. Prior to loading islets, the channel was washed using 2 mM glucose BMHH
media (BMHH imaging media: 125 mM NaCl, 5.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 10
mM HEPES, and the indicated glucose concentration, pH 7.4) from the outlet towards the inlet
using a 22 mm gauge blunt end needle tipped syringe. The buffer flowed through the inlet, and
also filled the well. After three washes, the presence of bubbles in the channel was checked by
visual examination through the eyepiece. Next, the inlet tubing was placed into the P35 dish
containing islets. The outlet tube was disconnected from the syringe tip, and allowed to hang
slightly above the base of the microscope. By placing the inlet tubing slightly above floating
islets, the pressure difference induced suction of the islet into the tubing and allowed it to flow
into the device channel. The islet’s travel time in the tubing was monitored visually, and the
outlet tube was immediately clamped upon islets reaching the dam wall. During loading, the
18
wells were kept from drying to prevent bubble formation. As well, it was important not to
overload the channel with islets, or allow gravity flow to continue for an extended period of time
as loaded islets could be pushed underneath the drop wall. Finally, the outlet tube was connected
to a pre-mounted syringe on the digital syringe pump (Figure 6D). The clamp on the outlet tube
was swapped to the inlet tube so solution could be pulled by the syringe pump from the
stimulation well. Loaded islets were subjected to a 5 minute reset flow to allow reorientation in
the channel.
Figure 6. Imaging Setup. (A) PDMS based microfluidic devices, (B) Microfluidic device mounted into a 37oCtemperature control chamber on microscope stage, (C) Microfluidic device mounted above the 40x objective, (D)Full setup with syringe pump attached to the outlet tubing of microfluidic device mounted.
3.6 Redox Autofluorescence Imaging
A novel method to monitor metabolic trends in live islets is the use of autofluorescence
microscopy [28]. Two-photon excitation microscopy is a technique which uses two photons of
lower energy level to simultaneously excite a fluorophore in one quantum event [38]. The use of
lower energy infrared light minimizes light scattering in the tissue and allow deeper tissue
penetration with reduced photo-toxicity [38]. Cellular energy intermediates NADH and NADPH
19
are both autofluorescent under two-photon excitation at 705 nm and excitation will yield an
� �� ����8�9!��:��� ������&�������'� �������������-cells increases during glucose metabolism
and the response can be assayed using two-photon excitation NAD(P)H microscopy [28].
However, the aggregate NAD(P)H signal from two-photon excitation does not distinguish
between NADH and NADPH. To isolate the NADH and NADPH trends from the aggregate
signal, the confocal imaging of the lipoamide dehydrogenase (LipDH) flavoprotein is required.
LipDH shuttles pyruvate into the mitochondria and its redox state is in direct equilibrium with
the mitochondrial NADH pool [28]. This equilibrium allows a distinction between the nutrient
stimulated NADH and NADPH responses by comparison of the aggregate NAD(P)H and LipDH
autofluorescence signals [28].
For each imaging experiment, islets were first suspended in BMHH media (described in
Section 3.5) supplemented with the indicated concentration of glucose. Prior to imaging, the
islets were loaded into microfluidic devices. Two-photon NAD(P)H imaging was done using a
40× 1.3 NA oil immersion objective lens of a LSM710 microscope (Zeiss). Each sample was
excited using a Ti:Saph laser tuned to 705 nm and attenuated to ~3 mW (Coherent). Images
were collected with pixel dwell time of 12.6 μs. Epifluorescence was directed through a custom-
built IR-blocked band pass filter (385-550 nm, Chroma) to a non-descanned external detector
[28]. Using two photon excitation at 705 nm, endogenous NADH and NADPH are both
autofluorescent with an emission spectra of 380-550 nm, giving off a signal called the aggregate
NAD(P)H signal (Summarized in Table 1).
Confocal imaging of Lipoamide dehydrogenase (LipDH) autofluorescence was done
using 458 and 488 nm excitation and long pass 505 nm emission filters (Table 1 and [28]).
Pinhole size was set to 3.09 AU and images were collected with pixel dwell time of 12.6 μs [28].
20
The fluorescence spectrum of LipDH is red-shifted compared to other flavin autofluorescence
[39], and a ratio of the images collected at 458 and 488 nm excitation was used to effectively
normalize the red-shoulder-excited LipDH signal (488 nm) to the total flavin signal (458 nm).
The detector gain was set using the highest signal sample (i.e. islets at 2 mM glucose) such that
the brightest non-responding regions were just below detector saturation, and this level was
maintained for the duration of each experiment.
LipDH is responsible for shuttling pyruvate into the mitochondria for TCA cycle
metabolism and is in direct equilibrium with mitochondrial NADH [28, 39]. Therefore, the
LipDH(mNADH) redox index measures the mitochondrial NADH signal and can be used for
comparison to the aggregate NAD(P)H response [28]. Overall, by combining the LipDH tracked
mitochondrial NADH, and the aggregate NAD(P)H signal, the trends of both NADH and
NADPH can be tracked in live islets.
Table 1. Autofluorescence Microscopy SummaryTechnique Excitation (nm) Emission (nm) Autofluorescence signal2-photon 705 380-550 NAD(P)H = NADH + NADPH
Confocal488 505 Long Pass (488LP) LipDH = 458LP/488LP
�� 9:��%���8�9:458 505 Long Pass (458LP)
3.7 LipDH Redox Index
The LipDH(mNADH) redox index was established by using pharmacological treatments
to maximize and minimize the redox state of the LipDH flavoprotein. FCCP (Carbonyl Cyanide
p-Trifluoromethoxyphenylhydrazone) was used as a mitochondrial proton uncoupler to increase
LipdH oxidization state [28]. FCCP treatment at 2 mM glucose maximizes the 458 and 488 nm
intensities by pushing the LipDH oxidization state to a maximum. Islets were stimulated for 3
minutes with FCCP at a working concentration of 2 μM and immediately imaged. NaCN
21
(Sodium cyanide) was used to block mitochondrial cytochrome oxidase in the electron transport
chain [28]. Treatment with NaCN at 20 mM high glucose minimizes the 458 and 488 nm
intensity by reducing LipDH. Islets were stimulated for 5 minutes with NaCN at a working
concentration of 3 mM and immediately imaged. The LipDH measurements obtained from islets
treated with FCCP and NaCN treatments were used to establish the LipDH redox index.
3.8 ImageJ Analysis
To quantify the changes in auto-fluorescence intensity under various treatments, the mean
intensity of NAD(P)H (705 nm) images was obtained by selecting and measuring the intensity of
20 small circular regions of interest on each islet. Regions were selected at random while
avoiding nuclear regions and saturated pixels. Five regions were also measured at a distance
from each islet to obtain average background intensity. The mean NAD(P)H intensity of each
islet was obtained by averaging the 20 intra-islet measurements and subtracting the average
background. The LipDH images (458 and 488 nm) were analyzed in a similar manner. Bright
non-responsive lipofusion deposits visible in the 458/488 nm images and were avoided during
region selection. An ImageJ macro was used to measure the same region of interest in both the
458 and 488 nm images of the same islet in the image stack.
Furthermore, ImageJ was also used to quantify the Rhodamine 123 (Rh123) intensity of
dye labelled islets under different treatments. A threshold value was visually set to encompass
all islet regions excluding only the nucleus. The mean islet intensity was measured using the
average threshold intensity value subtracted by the average of four background measurements.
22
3.9 Statistical Analysis
For analysis of Western blot experiments, the blot band densitometry values from all
experiments were used in a Student’s t-test to assess whether differences between control and
FGF21-treated islets were statistically significant at a P value of 0.05.
For experiments with multiple treatments, a one-way ANOVA followed by the Tukey
multiple comparisons test was used to assess statistical significance. Parings with p values <
0.05 were accepted as statistically significant.
23
CHAPTER 4 – THE EFFECT OF FGF21 ON ISLET ACCEXPRESSION AND ISLET METABOLISM
4.1 Introduction
Acetyl-CoA Carboxylase (ACC) is a key regulatory enzyme involved in fatty acid
synthesis (FAS) and fatty acid oxidation (FAO). ACC catalyzes the conversion of citrate to
malonyl-CoA, the substrate for FAS and the inhibitor of CPT1 transporter for FAO [29].
Therefore, ACC activity ultimately determines the partitioning of fatty acid metabolism. Recent
studies have demonstrated that FGF21 stimulation causes a decrease in ACC protein levels in
both the liver and WAT leading to increased fatty acid oxidation [35, 36]. However, the effect of
FGF21 on the expression of ACC in the pancreatic islet is currently unknown. Therefore, we
investigated islet ACC protein expression post-culture in the presence of FGF21 by using the
western immunoblotting technique.
To examine potential differences in live islet metabolism between control and FGF21-
treated islets, we carried out a number of studies using live cell imaging of pancreatic islet redox
state using autofluorescence imaging in a microfluidic device. The first imaging experiment in
this chapter examined the glucose stimulated NAD(P)H response in FGF21-treated islets post 24
hour culture in high fat. I defined the 24 hour culture in the presence of 0.4 mM palmitate as
chronic high fat treatment, and used the NAD(P)H response to subsequent glucose challenge as a
readout of islet function. This would be the only experiment examining differences after a
chronic culture. The purpose of this experiment was to test the microfluidic device imaging
������%����������������%�*���� ����������������*�� �;#;<�� ��������������-cell function post
chronic high fat culture [12]. -����� ������ �-cell dysfunction begins at the early stages of high
nutrient loading, it is important to understand the mechanisms of FGF21 protection in
24
maintaining the tissue in its healthy state. Islets cultured chronically in the presence of toxic
levels of nutrients are already in a pathophysiological state, and thus the subsequent imaging
experiments in this section applied the microfluidic imaging platform to investigate potential
differences in live islet metabolism between healthy control and FGF21-treated islets. The
second imaging experiment examined the glucose stimulated NAD(P)H response of islets in
normal culture. This experiment was conducted to explore potential differences between control
and FGF21-treated islets in terms of glucose metabolism extending from subphysiological to
supraphysiological concentrations. Furthermore, the third imaging experiment examined the
metabolism of exogenous citrate challenge. Since citrate is upstream of ACC in the FAS
pathway, potential differences between control and FGF21-treated islets were expected. Finally,
the fourth imaging experiment examined the metabolism of islets during high fat and high
glucose loading. Overall, this section aimed to explore potential differences in the metabolism of
healthy control and FGF21-treated islets under various states of nutrient challenge.
4.2 Chapter Specific Methods
General methods were described previously in chapter 3. Methods specific to this
chapter are described below.
4.2.1 pACC Western Immunoblot
Islet ACC activation was investigated by measuring the phosphorylation level of ACC at
both low (2 mM) and high (20 mM) glucose using Western blotting. The AMPK agonist
AICAR (1 mM, Sigma) was used to establish the maximum pACC readout for use as the
normalizing value. Post 48 hour, islets were stimulated with BMHH media at low glucose, high
25
glucose, and AICAR for 30 minutes. Western immunoblotting was performed immediately after
stimulation. The activity of ACC was quantified using the pACC:ACC ratio.
4.2.2 Palmitate Preparation
Palmitic acid (Sigma) was dissolved to palmitate in 0.1 M NaOH in a 70oC water bath.
Fatty acid free-BSA (BioShop Canada, Inc.) was dissolved in ddH2O by shaking at 4oC.
Palmitate was subsequently conjugated to fatty acid free BSA by mixing at a ratio of 1 mM
palmitate:1% BSA in a 60oC water bath. The final BSA-conjugated palmitate solution was
diluted to 0.4mM in subsequent preparations of either standard islet culture media or buffer-
based stimulation/imaging media (BMHH: 125 mM NaCl, 5.7 mM KCl, 2.5 mM CaCl2, 1.2 mM
MgCl2, 10 mM HEPES, and the indicated glucose concentration, pH 7.4).
4.2.3 Applying the Microfluidic Device Imaging Platform
All experiments in this section followed the islet loading protocol described in Section
3.5. On average ~7 islets were loaded into the channel device in each imaging experiment. An
n=3 was collected on separate days to obtain an average response. A total of above 20 islets is
generally accepted as a normalizing factor to account for islet to islet variations and mouse to
mouse variations.
Islets were cultured in the presence or absence of FGF21 for 48 hours in standard islet
media. Post culture, the islets were loaded into microfluidic devices for redox autofluoresence
imaging. The glucose stimulated NAD(P)H dose response experiments all employed 5 minute
stimulation intervals in each stepwise increase in glucose concentration. The 5 minute
���%��������*����������(��������-cells can rapidly sense and uptake glucose [40]. The glucose
26
dose concentrations used were 2, 4, 8, 12, and 20 mM. These concentrations cover the
physiological glucose range of glucose (~5 mM to ~11 mM) as well as subphysiological and
supraphysiological levels at 2 mM and 20 mM respectively. In contrast, all palmitate stimulation
was done for 30 minutes due to the slow multistep conversion of palmitate to acetyl-CoA [29].
The aggregate NAD(P)H autofluorescence signal from the channel-held islets were tracked in the
dose response experiments. The aggregate NAD(P)H and LipDH autofluorescence signals from
the channel-held islets were tracked in the acute citrate and palmitate experiments.
4.3 Results
4.3.1 FGF21 decreases islet ACC protein levels
To examine the effect of FGF21 on islet ACC, we measured ACC expression using
Western blotting. The expression level of ACC was quantified by dividing the protein band
�������%���+�%������%���������$$�(+��-actin from each blot. Averaging n=5, these data show
a significant decrease in islet ACC expression upon stimulation with FGF21 (Figure 7B).
Figure 7. FGF21-dependent ACC regulation in pancreatic islets. (A) A representative Western immunoblotshowing a reduction in mouse islet ACC protein levels when incubated in the presence of FGF21 (48 hrs; 100 � 1%���� �=�%(������*���� ����� ��(��� ���� �-actin as a loading control. (B) The summarized fold-ACC response ���%���>�������-actin for control and FGF21-treated islets. Data shown represents the mean ± s.e.m. for the islets from 5 independently assayed and treated mice. (*P < 0.05, Student’s t-test done prior to converting to fold over control)
27
4.3.2 FGF21 increases islet pACC:ACC ratio at low glucose
AMPK regulates ACC activity via phosphorylation to deactivate the enzyme [29]. To
examine the effect of FGF21 on islet ACC activity at low (2 mM) and high (20 mM) glucose, we
measured islet pACC and ACC levels using Western blotting. The results show FGF21-treated
islets exhibited higher pACC:ACC ratio at low glucose (Figure 8). At high glucose, the
pACC:ACC ratio decreased, but no difference was observed between control and FGF21-treated
islets.
Figure 8. FGF21 increases islet pACC:ACC ratio at low glucose. The summarized pACC:ACC response normalized to AICAR-treated islets for control and FGF21-treated islets. Data shown represents the mean ± s.e.m. for the islets from 3 independently assayed and treated mice.
28
4.3.3 FGF21-treated islets maintain higher NAD(P)H glucose dose response post 24 hr culture in palmitate
To examine the effect of FGF21 on islet glucose metabolism post 24 hr culture in
palmitate, the glucose-stimulated NAD(P)H response was measured using two-photon
microscopy. The result show FGF21-treated islets exhibited a higher NAD(P)H response to
glucose from 4 mM to 20 mM as indicated by the glucose dose response curve (Figure 9). These
data show significant differences in islet glucose handling in FGF21-treated islets post a 24 hr
culture in palmitate.
Figure 9. Glucose-stimulated NAD(P)H response of islets cultured in palmitate for 24 hrs. Pancreatic islets were cultured in full RPMI media 1640 at 11 mM glucose in the absence (control) and presence (FGF21) of FGF21. At 24hr, 0.4mM palmitate was introduced to islets. At 48hr, the islets were removed from culture and incubated in imaging media containing 2 mM glucose at 37ºC (minimum 30 min) followed by loading into a microfluidic device on the microscope stage for imaging the two-photon glucose-stimulated NAD(P)H response. The summarized mitochondrial NAD(P)H intensities throughout the glucose dose-response. These data represent the pooled response from 20-30 islets harvested on separate days from 3 mice. (*P < 0.05).
4.3.4 FGF21-treated islets exhibit lower NADPH levels at high glucose
To examine the effect of FGF21 on normal islet glucose metabolism, the glucose-
stimulated NAD(P)H response was measured using two-photon microscopy. The result shows
29
no difference in the glucose stimulated NAD(P)H response between FGF21-treated and control
islets from 2 mM glucose to 12 mM glucose. In contrast, the response of islets at 20 mM
glucose was significantly lower in FGF21-treated islets compared to the control islets (Figure
10). These results demonstrate that FGF21 changed the islet NAD(P)H response at supra-
physiological levels of glucose.
Figure 10. Glucose-stimulated NAD(P)H response. Pancreatic islets were cultured for 48 hrs in full RPMI media 1640 at 11 mM glucose in the absence (control) and presence (FGF21) of FGF21. The islets were subsequently incubated in imaging media containing 2 mM glucose at 37ºC (minimum 30 min) followed by loading into a microfluidic device on the microscope stage for imaging the two-photon glucose-stimulated NAD(P)H response. (A) Images of a representative device-immobilized islets’ NAD(P)H autofluorescence at 2 and 20 mM glucose. (B)The summarized mitochondrial NAD(P)H intensities throughout the glucose dose-response. These data represent the pooled response from 20-30 islets harvested on separate days from 3 mice. (*P < 0.05).
Since two photon NAD(P)H imaging cannot spectrally distinguish NADH and NADPH,
the aggregate NAD(P)H response and the LipDH(mNADH) response were tracked at 2 mM, 10
mM and 20 mM glucose concentrations to gather insight into the individual NADH and NADPH
responses. At 20 mM glucose, FGF21-treated islets exhibited a significantly smaller NAD(P)H
30
response (Figure 11A) coupled with a comparable LipDH(mNADH) response (Figure 11B)
compared to control islets. Therefore, there was a smaller NADPH response in FGF21-treated
islets at 20 mM glucose.
Figure 11. Glucose-stimulated NAD(P)H and LipDH(mNADH) responses. Pancreatic islets were cultured for 48 hr in full RPMI media 1640 at 11 mM glucose in the absence (control) and presence (FGF21) of FGF21. The islets were subsequently incubated in imaging media containing 2 mM glucose at 37ºC (minimum of 30 min) followed by loading into a microfluidic device on the microscope stage for imaging of the glucose-stimulated NAD(P)H and LipDH (mNADH) redox index responses. (A) The summarized fold NAD(P)H response of mitochondrial regions from islets exposed sequentially to 2, 10, and 20 mM glucose. (B) The summarized LipDH(mNADH) redox index for the same islets shown in (A). The LipDH response (458:488 nm intensity ratio) is indexed to pharmacological treatments that minimize and maximize mitochondrial NADH reduction. The data shown was collected from control (n=24) and FGF21-treated (n=28) islets harvested from 3 separate mice. (*P < 0.05).
4.3.5 FGF21-treated islets exhibit decreased NADPH with citrate stimulation
To examine the FAS pathway upstream of ACC, citrate was used to probe differences
between control and FGF21-treated islets. At 2mM glucose, control islets stimulated with citrate
showed a slight decrease in the aggregate NAD(P)H coupled with a rise in the LipDH(mNADH)
(Figure 12). This result indicates a decrease in NADPH or a lowered NADPH response to citrate
in control islets. In contrast, FGF21-treated islets show both increases in the aggregate
NAD(P)H and lipDH(mNADH) when stimulated with citrate at 2mM glucose (Figure 12)
31
indicating a higher NADPH response. At 20 mM glucose, no differences were observed between
control and FGF21-treated islets as both group show increases in the NAD(P)H and
lipDH(mNADH) signals (Figure 12).
Figure 12. Citrate metabolism in control and FGF21-treated islets. Pancreatic islets were cultured for 48 hr in full RPMI media 1640 at 11 mM glucose in the absence (control) and presence (FGF21) of FGF21. The islets were subsequently incubated in imaging media containing 2 mM glucose at 37ºC (minimum of 30 min) followed by loading into a microfluidic device on the microscope stage for imaging of the glucose-stimulated NAD(P)H and LipDH (mNADH) redox index responses. (A) The summarized fold NAD(P)H response from mitochondrial regions of islets treated with 2 mM glucose (5 min), 2 mM glucose + 10 mM citrate (30 min), and 20 mM glucose + 10 mM citrate (5 min). (B) The summarized LipDH(mNADH) redox index for the same islets shown in (A). The data shown was collected from control (n=21) and FGF21-treated (n=22) islets with islets harvested from three separate mice. (*P < 0.05).
4.3.6 FGF21-treated islets exhibit altered processing of mitochondrialNADH during high fat and high glucose challenge
To examine the effect of the FAS pathway downstream of citrate, the metabolic response
to acute palmitate stimulation was measured at both low and high glucose concentrations. After
addition of 0.4mM palmitate at 2 mM glucose, both control and FGF21-treated islets showed a
decrease in the aggregate NAD(P)H signal (Figure 13A). Addition of 0.4 mM palmitate also
caused a similar concurrent rise in the LipDH(mNADH) signal in both control and FGF21-
treated islets (Figure 13B). These results demonstrate that there is a decrease in NADPH in both
32
control and FGF21-treated islets with acute stimulation with palmitate. Subsequent treatment in
0.4 mM palmitate with 20 mM glucose showed a significantly lower LipDH(mNADH) signal in
the FGF21-treated islets (Figure 13B). Overall, these results indicate FGF21 stimulation induced
altered islet metabolism at high glucose and high palmitate.
Figure 13. Palmitate metabolism in control and FGF21-treated islets. Pancreatic islets were cultured for 48 hr in full RPMI media 1640 at 11 mM glucose in the absence (control) and presence (FGF21) of FGF21. The islets were subsequently incubated in imaging media containing 2 mM glucose at 37ºC (minimum of 30 min) followed by loading into a microfluidic device on the microscope stage for imaging of the glucose-stimulated NAD(P)H and LipDH (mNADH) redox index responses. (A) The summarized fold NAD(P)H response from mitochondrial regions of islets treated to 2 mM glucose (5 min), 2 mM glucose + 0.4 mM palmitate (25 min), and 20 mM glucose + 0.4 mM palmitate (5 min). (B) The summarized LipDH(mNADH) redox index for the same islets shown in (A). The data shown was collected from control (n=14) and FGF21-treated (n=14) islets with islets harvested from three separate mice. (*P < 0.05).
4.4 Discussion
The FGF21-induced decrease in islet ACC expression is consistent with changes in the
metabolism of islets by potentially modifying fatty acid partitioning. Therefore a follow-up
experiment was conducted to examine the ACC phoshorylation levels. AMPK phosphorylates
ACC to deactivate the enzyme [29]. The pACC:ACC ratio was used to determine the level of
ACC activation. A ratio of 1 indicates 100% ACC phosphorylation by AMPK and this value
was established by treating with the AMPK-agonist AICAR. A high pACC:ACC ratio indicates
33
low ACC activity and low ratio indicates high ACC activity. Examining the trends, there is a
decrease in pACC:ACC ratio going from low to high glucose (Figure 8). This response is
consistent with an activation of ACC at high glucose to partition towards glucose metabolism by
activating the conversion of citrate to malonyl-CoA to inhibit FAO. Comparing control and
FGF21-treated islets, a difference was observed at low glucose with FGF21-treated islets
exhibiting a higher pACC:ACC ratio (Figure 8). Although not statistically significant, this result
is consistent with the observed decrease in total ACC induced by FGF21. A significant
reduction in total ACC potentially allows active AMPK to more rapidly phosphorylate a majority
of the ACC pool at low glucose and partition towards FAO in FGF21-treated islets. Overall, the
FGF21-induced decrease in islet ACC and increase in pACC:ACC ratio at low glucose may
indicate increased ability to metabolise fatty acids at low glucose. The increase of FAO in
FGF21-treated islets may provide a release valve to prevent the build up of long chain fatty
acids. Therefore, it was valuable to examine the nutrient stimulated metabolism in live islets
using autofluorescence imaging and compare potential differences between control and FGF-21
treated islets.
The first imaging experiment examined the NAD(P)H response of islets to increasing
glucose challenge post culture in chronic (24 hr) palmitate. Stimulation with glucose will cause
���� ���������������&$���+�����������+���������������-cells causing rising levels of both NADH and
NADPH. Therefore, measuring the change in aggregate NAD(P)H was used as a measurement
of islet function. The result demonstrated that FGF21-treated islets have a higher glucose
stimulated NAD(P)H response post chronic culture in palmitate (Figure 9). This may be an
indication of protection of function in glucose sensing by FGF21 and is consistent with data in
previous literature measuring islet function post chronic culture in palmitate [12]. However, I
34
cannot make a claim that FGF21 treatment maintained the glucose induced NAD(P)H response
because another set of control islets without the 24 hour palmitate culture was not assayed on the
same day. Moreover, this experiment was done as a preliminary test for imaging the glucose
stimulated NAD(P)H response of islets in our microfluidic device.
The second imaging experiment examined the response of healthy islets to glucose
stimulation. The results showed that FGF21-treated islets have a lower NADPH response at 20
mM glucose (Figure 11). FGF21-treated islets have lowered ACC expression and should build
up TCA cycle citrate faster than control islets at high glucose concentrations. This model would
indicate that FGF21-treated islets should turn on the pentose phosphate pathway (PPP) sooner by
TCA-exported citrate inhibition of glycolysis and in turn generate more NADPH. As well,
FGF21-treated islets should have a slower conversion of citrate to malonyl-CoA and consume
less NADPH in the FAS pathway. However, instead of an expected increase in NADPH
response at high glucose, FGF21-treated islets demonstrated lower NADPH response at high
glucose. A possible explanation for the observed decrease in NADPH in FGF21-treated islets at
high glucose may be the consumption of NADPH in the scavenging of reactive oxygen species
(ROS). At the supra-physiological level of glucose, TCA cycle activity will generate a high
level of NADH to further increase electron transport chain (ETC) activity. High levels of NADH
may lead to increased leakage of electrons in the ETC to create ROS in the form of superoxides
or hydrogen peroxide [41]. A cellular mechanism to soak up excessive ROS is the reduction of
ROS by conjugation with glutathione [42]. The reduction of ROS such as superoxides oxidizes
glutathione and reactivation by glutathione reductase requires the consumption of NADPH [42].
Therefore, the lower level of NADPH exhibited by FGF21-treated islets at 20 mM glucose may
suggest a higher level of consumption in ROS scavenging.
35
The third imaging experiment examined the response of islets to citrate stimulation at 2
mM and 20 mM glucose. The metabolic pathway model (Figure 4) suggests that a decrease in
ACC protein level will lead to a decrease in the conversion of citrate to malonyl-CoA. In turn,
this may lead to a decrease in the FAS pathway due to a reduction in the substrate for palmitate
synthesis. At 2 mM glucose, stimulation upstream of ACC with citrate resulted in a decrease in
NADPH in control islets at 2 mM glucose (Figure 12A, 12B). The decrease in NADPH suggests
initiation of FAS in control islets. In contrast, the FGF21-treated islets showed citrate-stimulated
rises in both the aggregate NAD(P)H and LipDH(mNADH), indicating either minimal or no
initiation of FAS at low glucose. These results suggest that FGF21-treated islets undergo lower
levels of FAS when challenged with citrate. A reduction in FAS is expected to be protective in
FGF21-treated islets by limiting the build up of toxic long chain fatty acids [22].
The fourth imaging experiment examined the response of islets to palmitate stimulation at
2 and 20 mM glucose. At 2mM glucose, stimulation with palmitate caused an increase in the
LipDH(mNADH) signal. This suggests an initiation of FAO and entry of fatty acyl-CoA into the
mitochondria to increase TCA cycle NADH. However, the concurrent drop in the aggregate
NAD(P)H response in both control and FGF21-treated islets indicate a net decrease in NADPH.
Two possible processes that can account for a decrease in NADPH include the activation of fatty
acid elongation downstream of palmitate and the initiation of ROS scavenging. Although
palmitate stimulation induced an increase in the LipDH(mNADH) signal suggesting initiation of
FAO, the high concentration of palmitate may also force the cell to undergo long chain fatty acid
synthesis. The elongation of palmitate to stearate requires the consumption of NADPH via
catalysis by fatty acid synthase [43]. As well, the metabolism of palmitate can increase TCA
cycle activity to yield a buildup of high mitochondrial membrane potential, which can in turn
36
induce the generation of ROS. It has been shown in related studies that palmitate stimulation
causes an increase in ROS in pancreatic islets [41]. Therefore, the palmitate-induced decrease in
NADPH may also be a result of depletion of cellular NADPH in the scavenging of ROS.
Furthermore, stimulation with 20 mM glucose after the acute palmitate treatment caused a
significant drop in the LipDH(mNADH) signal in FGF21-treated islets (Figure 12B). Since the
mitochondrial NADH is used to establish the mitochondrial membrane potential for subsequent
ATP synthesis, this result reflects a change in mitochondrial energetics in FGF21-treated islets.
Changes in membrane potential may also alter the ATP dependent insulin secretion. Therefore,
it was of interest to examine whether the decrease in mitochondrial NADH in FGF21-treated
islets is also reflected in the mitochondrial membrane potential and its effects on insulin
secretion.
37
CHAPTER 5 – THE EFFECT OF FGF21 ON ISLET MITOCHONDRIAL ENERGETICS AND INSULIN SECRETION
5.1 Introduction
In the last chapter, FGF21-treated islets exhibited a decrease in mitochondrial NADH
when treated with 20 mM glucose after the 30 minute palmitate stimulation. This section will
examine whether the decrease in mitochondrial NADH is also reflected in the mitochondrial
membrane potential. As well, it was of interest to examine mechanisms involved in regulating
mitochondrial membrane potential. One mechanism to account for changes in the mitochondrial
membrane potential may be an altered expression of mitochondrial uncoupling proteins by
FGF21. When an excessively high mitochondrial membrane potential is created during high
levels of oxidative respiration, the generation of ROS from electron leakage occurs. Uncoupling
proteins such as uncoupling protein 2 (UCP2) are ubiquitously expressed in various tissues to
prevent the build up of ROS during high respiration levels by leaking protons across the
mitochondrial membrane to reduce the gradient [44]. This proposed mechanism was examined
in this chapter using Western blotting to determine examine whether the treatment of FGF21
induces changes in UCP2 protein expression in islets. Lastly, to relate the change in
mitochondrial NADH observed in FGF21-treated islets to physiological function, the glucose-
and palmitate-induced insulin secretion was examined. The insulin secretion profile of control
and FGF21-treated islets at 2 mM glucose, 2 mM glucose with 0.4 mM palmitate, and 20 mM
glucose with 0.4 mM palmitate were quantified using a sandwich ELISA.
38
5.2 Chapter Specific Methods
General methods were described previously in Chapter 3. Methods specific to this
chapter are described below.
5.2.1 Rhodamine123 Imaging
Rhoadmine123 (Rh123) fluorescent dye (Invitrogen) was used to examine islet
mitochondrial membrane potential. Rh123 is a fluorescent dye commonly used to track islet
mitochondria membrane potential [45]. The dye is normally distributed across all cellular
compartments, but collects to the mitochondrial membrane when the membrane potential is high
(ie. when NADH builds up in high nutrient states). Sequestration of the dye in the mitochondria
leads to a decrease in fluorescence intensity due to collisional quenching. This effect makes the
dye bright during low islet metabolism and dim during high islet metabolism. Overall, the
Rh123 intensity is inversely related to the mitochondrial membrane potential.
��<��!�7�0 1%���*����������������������< mM glucose-imaging buffer and incubated for
30 minutes at 37oC. Islets were subsequently loaded into microfluidic devices and the 514-nm
laser line was used to excite the loaded dye. Fluorescent emission signals were detected using a
525-655-nm bandpass filter. Pinhole size was set to 1.73 AU and images were taken with pixel
dwell time of 12.6 μs. The Rh123 signal was tracked at 2 mM glucose, 2 mM glucose with 0.4
mM palmitate and 20 mM glucose with 0.4 mM palmitate for both control and FGF21-treated
islets.
5.2.2 UCP2 Western Immunoblot
Due to the low abundance of UCP2 protein in the mouse islet, it was difficult to obtain a
high protein concentration sample using the Triton-X based lysis buffer method. Instead, the hot
39
sample buffer method was used to minimize the loss of protein. The SDS-based sample buffer
was boiled at 100oC in a water filled heat block and added directly to the islet pellet in a
microtube. The lysis and protein denaturation process was allowed to continue in the heat block
for 5 minutes. Forty-eighty islets were used per lane in this method. Proteins were separated by
10% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked by
incubating with 5% non-fat dry milk powder, and 1% BSA in Tris-buffered saline-Tween20
(TBS-T) for 1hr at RT. Proteins of interest were detected by overnight incubation at 4°C in
block solution containing goat anti-mouse UCP2 antibody [(Novus Biologics), 0.5ug/ml]. Blots
were subsequently incubated with anti-goat peroxidase-linked antibody [(Roche), 1:1000] diluted
in block solution for 1 hr at RT, and proteins were detected by enhanced chemiluminescence
(Pierce, Thermo Scientific Inc.).
5.2.3 Insulin ELISA
Isolated islets were cultured in the presence or absence of FGF21 (100 ng/mL). Post-
culture, ~20 islets were picked from each of control and FGF21-treated dishes into microfuge
tubes containing BMHH buffer supplemented with 2 mM glucose and equilibrated for 30
minutes. Islets were stimulated at 37°C in 2 mM glucose followed 20 mM glucose. As well,
another set of islets were stimulated with 2 mM glucose followed by 2 mM glucose with 0.4 mM
palmitate and finally 20 mM glucose with 0.4 mM palmitate (40 min for each stimulation step).
All stimulation solutions were prepared in BMHH based buffer, and supernatant was collected
from microtubes prior to adding the succeeding stimulation media. After collection of the final
set of supernatants, total islet insulin content was released by treatment with 1% Triton X-100
with immediate freeze storing at -20oC to permeate the islet cells’ membrane. Fractional total
40
insulin was quantified using a sandwich insulin ELISA assay (Millipore), where the measured
secreted insulin in the supernatant in each sample was normalized to the total insulin measured in
the final Triton X-100 treated sample.
5.3 Results
5.3.1 FGF21-treated islets exhibit lower mitochondrial membrane potential during high fat and high glucose challenge
To examine the effect of FGF21 on islet mitochondrial membrane potential, Rh123
imaging was performed at different nutrient states. The addition of 0.4 mM palmitate caused a
similar drop in Rh123 fluorescence intensity in both control and FGF21-treated islets (Figure
14B). However, when islets were subjected to 20 mM glucose with 0.4 mM palmitate, FGF21-
treated islets showed a significantly lower decrease in Rh123 intensity compared to control islets
(Figure 14B). The lower decrease in Rh123 intensity indicates a smaller build up of
mitochondrial membrane potential in FGF21-treated islets.
41
Figure 14. Palmitate and glucose-stimulated changes in mitochondrial membrane potential. Pancreatic islets were cultured for 48 hr in full RPMI media 1640 supplemented with 11 mM glucose in the absence (control) and presence (FGF21) of FGF21. The islets were subsequently incubated at 37 ºC in imaging media containing 2 mM glucose for 1 hr followed by 2 mM glucose with Rh123 (10 μg/ml). The islets were then loaded into a microfluidic device on the microscope stage for imaging. (A) Representative images of a Rh123-labelled control islet stimulated with 2 mM glucose (5 min), 2 mM glucose + 0.4 mM palmitate (25 min) and 20 mM glucose + 0.4 mM palmitate (5 min). (B) The summarized data from 13 and 11, control and FGF21-treated islets, respectively. The data are plotted as the fold Rh123 intensity relative to 2 mM glucose. (*P < 0.05).
5.3.2 FGF21 decreases islet UCP2 protein levels
To examine the effect of FGF21 on islet UCP2, Western immunoblot was used to
measure UCP2 expression levels. Protein band densitometry measurements were taken for both
?$�<������-actin, and the UCP2 content in each sample was quantified with normalization to
�����-actin. FGF21-treated islets exhibited a significant decrease in UCP2 levels (Figure 15B;
n=4). This result suggests a down-regulation of UCP2 expression with FGF21 stimulus.
42
Figure 15. FGF21-dependent UCP2 regulation in pancreatic islets. (A) A representative Western immunoblot showing a reduction in mouse islet UCP2 protein levels when incubated in the presence of FGF21 (48 hrs; 100 � 1%���� �=�%(������*��������� ��(��� �����-actin as a loading control. (B) The summarized fold-UCP2 response ���%���>�������-actin for control and FGF21-treated islets. Data shown represents the mean ± s.e.m. for the islets from mice assayed on 4 independent days.
5.3.3 FGF21 induces detection of UCP2 of higher than expected molecular mass
The UCP2 Western immunoblot studies also yielded an unexpected larger UCP2 band at
~80 kDa in FGF21-treated islets (Figure 16). The expected molecular weight of UCP2 is ~30
kDa.
43
Figure 16. Potential FGF21-dependent UCP2 activation in pancreatic islets. Representative Western immunoblot revealing expression of a high molecular mass UCP2 species in islets when incubated in the presence of FGF21 (48 hrs; 100 ng/ml).
5.3.4 FGF21-treated islets secrete less insulin during high fat and high glucose challenge
To study the functional consequence of FGF21 stimulation, the glucose- and palmitate-
induced insulin secretion was investigated using a sandwich ELISA assay. The result showed no
significant difference in insulin secretion between control and FGF21-treated islets at 2 mM
glucose or 20 mM glucose. However, FGF21-treated islets exhibited a significantly lower
fractional insulin response compared to control islets at 20 mM glucose following the addition of
0.4 mM palmitate (Figure 17B).
44
Figure 17. Palmitate and glucose-stimulated insulin secretion. Pancreatic islets were cultured in media containing FGF21 for 48 hrs prior to measuring the glucose- and palmitate-stimulated insulin responses. The treated islets were subsequently incubated at 37ºC for 30 min in imaging media containing 2 mM glucose followed by sequential effluent collection. Effluent collections were done after 1 hr incubations with the indicated nutrient stimulants by careful collection of the sample supernatant. (A) The normalized insulin response from control and FGF21-treated islets at 2 and 20 mM glucose. (B) The normalized insulin response from islets exposed to sequential 40 minutes treatments of 2 mM glucose, 2 mM glucose + 0.4 mM palmitate, and 20 mM glucose + 0.4 mM palmitate. The data are normalized to total insulin content collected post-islet permeabilization with 1% triton-X 100. The data shown are summarized from the islets harvested and cultured from 5 mice on independent days. (*P< 0.05).
5.4 Discussion
The decrease in mitochondrial membrane potential exhibited by FGF21-treated islets
stimulated with 20 mM glucose and 0.4 mM palmitate correlates to the drop in the
LipDH(mNADH) signal observed in the autofluorscence microscopy studies in Section 4.3.6.
Stimulation with 20 mM glucose in the presence of 0.4 mM palmitate should generate a high
level of NADH to in turn drive higher mitochondrial membrane potential. Therefore, the
decrease in mitochondrial NADH and lowered membrane potential may indicate an altered
handling of high TCA cycle flux.
An increase in UCP2 expression in FGF21-treated islets would be consistent with
increased uncoupling of the mitochondrial membrane under high glucose and high palmitate
45
stimulation. Therefore, the subsequent experiment examined whether islet UCP2 expression was
increased with FGF21 treatment. However, we found that UCP2 expression was decreased in
FGF21-treated islets and this result was consistent with a FGF21-induced reduction in UCP2
mRNA shown by another student in the Rocheleau group. Overall, the decrease in mitochondrial
membrane potential cannot be accounted for simply by UCP2 protein and mRNA expression.
Another possibility is post-translational regulated activation of UCP2 in FGF21-treated
islets. Interestingly, a larger than expected UCP2 band was detected in FGF21-treated islets only
(Figure 16). It is known that UCP2 is an inner mitochondrial transmembrane protein that
dimerizes upon activation [46], and thus the higher band may be a product of an activated
population of UCP2. However, this band still has a larger size than predicted for a UCP2 dimer.
A recent study suggested that UCP2 activity is dynamically regulated by rapid proteolysis
following its activation in proposed mechanisms that involves poly-ubiquitination and
subsequent degradation by 26S proteosome binding [47]. This novel mechanism of UCP2
regulation may resolve the larger UCP2 band detected in FGF21-treated islets as a post-activated
product of poly-ubiquitinated UCP2 or 26S proteosome-bound UCP2. Therefore, the decrease in
mitochondrial membrane potential observed in FGF21-treated islets may be explained using a
model based on post-translational increase in UCP2 protein activity.
The functional consequence of lowered mitochondrial NADH and mitochondrial
membrane potential in FGF21-treated islets was reflected in the insulin secretion studies.
Mitochondrial NADH is used to establish the mitochondrial membrane potential for subsequent
ATP generation, and the rise in ATP/ADP ratio leads to insulin secretion. Lowering the
mitochondrial NADH levels during high glucose and high fat challenge in FGF21-treated islets
was reflected in a lower fractional insulin secretion (Figure 17). However, the glucose-
46
stimulated insulin response was not abolished in FGF21-treated islets after high palmitate
stimulation and remained comparable to the level induced by glucose stimulation alone (Figure
17A, 17B). Therefore, a decrease in insulin secretion under high nutrient challenge may be a
protective mechanism to prevent the over-secretion of insulin and in the long term preserve the
�����������������������-cells.
47
CHAPTER 6 – GENERAL DISCUSSION
6.1 Discussion
The goal of this thesis was to examine potential mechanism of FGF21 in the regulation of
������ �-cell metabolism at the early stages of high glucose and high fat loading. Based on
previous literature demonstrating FGF21-regulation of the key metabolic regulator ACC in the
liver and the adipose [35], I confirmed that FGF21 induced a decrease in mouse islet ACC
protein expression (Figure 7). A model was subsequently formulated based on the decrease in
ACC expression to test potential changes in islet metabolism and the associated mechanisms.
The first experiment aimed to dissect potential differences in glucose handling. Using a
microfluidic device as an imaging platform, the NAD(P)H and LipDH(mNADH) of islets were
tracked as a readout of metabolism at different concentrations of glucose challenge. It was
revealed that FGF21-treated islets exhibit lower NADPH at supra-physiological glucose
concentration (20 mM) (Figure 10,11). If analysis of this result was based only on the decreased
ACC expression, an opposite trend would be expected where FGF21-treated islets should exhibit
increased NADPH from theoretically higher PPP generation and lower FAS pathway
consumption. However, at supra-physiological concentrations of glucose, the available ACC
pool in control and FGF21-treated islets should both be highly activated to handle the high TCA
cycle flux. Therefore, although there was a decrease in total ACC protein level, the difference
observed in this experiment may not be ACC dependent as both control and FGF21-treated islets
have a sufficient pool of activated ACC to handle the high TCA cycle flux. In addition, an
interesting observation made was that the difference in NADPH only occurs after a prolonged
exposure to 20mM glucose. Therefore, a different mechanism was discussed in Section 4.4
hypothesizing that FGF21-treated islets may start the NADPH dependent ROS scavenging
48
earlier than control islets as the prolonged glucose stimulus begins to introduce excess electron
leakage to create superoxides. However, this alternative mechanism cannot be confirmed
without further experimentation. Overall, the divergence of FGF21-treated islets in its NAD(P)H
response to high glucose indicated potential differences in the handling of high nutrient loading,
and subsequent experiments aimed to further probe the metabolic pathway centered around
ACC.
The metabolism of islets was further examined by stimulating upstream and downstream
of ACC with citrate and palmitate respectively. By tracking the aggregate NAD(P)H and
LipDH(mNADH) signals, I observed lower NADPH response in FGF21-treated islets when
stimulating with citrate at 2 mM glucose (Figure 12). This result is consistent with FGF21-
treated islets undergoing lower levels of FAS when challenged with citrate. A reduction in FAS
is expected to be protective in FGF21-treated islets by limiting the build up of long chain fatty
acids [22]. However, a more concrete quantification such as an absorbance based triglyceride
assay would be required to correlate the decreasing trend in NADPH to actual FAS.
Furthermore, stimulation with palmitate at 2mM glucose caused similar increases in the
LipDH(mNADH) in both control and FGF21-treated islets (Figure 13B). This was a surprising
result as FGF21-treated islets were expected to undergo a higher level of FAO. By combining
the aggregate NAD(P)H and LipDH(mNADH), the data suggests a net decrease in NADPH. The
possible mechanisms contributing to the decrease in NADPH were discussed in Section 4.4
including the consumption of NADPH in the production of long chain fatty acids downstream of
palmitate [43] and the generation and subsequent scavenging of ROS during prolonged fatty acid
metabolism. However, future studies are necessary to confirm these mechanisms. Finally,
stimulation with 20 mM glucose after acute stimulation with palmitate showed a significantly
49
lower build up of mitochondrial NADH in FGF21-treated islets (Figure 13). One possible
mechanism to account for a reduction in mitochondrial NADH is feedback inhibition of
metabolism. A buildup of TCA cycle intermediates inhibits multiple steps of the TCA cycle.
For example, citrate inhibits glycolysis at phosphofructokinase and acetyl-CoA inhibits pyruvate
dehydrogenase [29]. A reduction in ACC in FGF21-treated islets may allow a more rapid
buildup of intermediates during high glucose and high fat stimulation leading to a reduction in
TCA cycle flux and NADH generation. Furthermore, another mechanism that may account for
the reduction in NADH may be FGF21-modified anaplerosis and metabolite cycling. Normally,
pyruvate can enter the TCA cycle through conversion by pyruvate carboxylase (PC) to
oxaloacetate [29]. This process triggers the replenishing of TCA cycle intermediates. As well,
anaplerosis enables intermediate cycling such as conversion of isocitrate to alpha-ketoglutarate
to generate NADPH [29]. These cycling pathways can contribute to a reduction in NADH by
unloading intermediates early in the TCA cycle and shifting metabolism towards NADPH
production. It is possible that FGF21-treated islets compile an acetyl-CoA pool faster due to
lowered ACC expression and the acetyl-CoA in turn allosterically activates PC to induce the
anaplerotic cycling pathways.
To understand the decrease in mitochondrial NADH in FGF21-treated islets during high
glucose and high fat loading, subsequent studies were conducted to examine the mitochondrial
membrane potential and insulin secretion of FGF21-treated islets. The difference in
mitochondrial NADH in FGF21-treated islets was correlated with a lower buildup of
mitochondrial membrane potential measured by Rh123 (Figure 14) during high glucose and high
fat stimulation. NADH generated in the TCA cycle is used by the electron transport chain to
establish mitochondrial membrane potential for ATP generation, and a rise in the ATP/ADP ratio
50
leads to insulin secretion. Moreover, any source of mitochondrial membrane uncoupling such as
increased ATP synthase activity can contribute to a decrease in NADH. Consequently, I
examined the glucose- and palmitate-induced insulin response of FGF21-treated islets. We
found lower secretion of insulin during high glucose and high fat stimulus (Figure 17). This
result indicates that the decrease in mitochondrial NADH is not due to an increased consumption
by ATP synthase. Another mechanism potentially responsible for reducing the mitochondrial
membrane potential in FGF21-treated islets is membrane uncoupling by UCP2. Although UCP2
protein expression was found to be decreased by FGF21, post-translational regulation of UCP2
by 26S protesome degradation is possible. Lastly, an interesting and less known candidate that
can lead to reduced mitochondrial membrane potential and contribute to a drop in mitochondrial
NADH is the mitochondrial protein nicotinamide nucleotide transhydrogenase (NNT). When the
mitochondrial membrane potential is high, NNT catalyzes the conversion of NADH to NADPH
using energy from a proton translocation event across the inner membrane [48]. It has been
shown that mice carrying mutations in NNT have impaired glucose tolerance, reduction in GSIS
���� ��*����� �&�� ����������� � ��� *���)� %������ 88&� �-cells show enhanced glucose usage
coupled with increased ROS production [48]. Since NADPH is consumed by glutathione
reductase in the process of replenishing glutathione stores for defence against oxidative stress
[48], it would be beneficial to have a large store of NADPH during high nutrient challenge.
Therefore, FGF21-treated islets may have an increased expression of NNT to raise the NADPH
pool during high glucose and high fat challenge to protect against the mitochondrial-generated
ROS.
Although mechanistically unclear, the lowered insulin secretion of FGF21-treated islets
during high glucose and high fat stimulus is an important result. Since �-cell exhaustion is a
51
hallmark of type 2 diabetes disease pathogenesis, evidence showing lowered insulin response
during high nutrient loading may indicate protection from over-secretion of insulin by FGF21. It
is also of value to consider the physiological environment in FGF21-treated islets under high
glucose and high fat stress. Results from this thesis showed a net increase in NADPH response
in FGF21-treated islets when challenged with high glucose and high fat. Since glutathione
handling of ROS requires NADPH for reactivation, it is beneficial to have a large store of
NADPH during high nutrient challenge. As well, it is important to note that the glucose-
stimulated insulin response was decreased, but not abolished during high glucose and high fat
loading, and actually remained comparable to the glucose-only response (Figure 17A, 17B).
Overall, the decrease in insulin secretion provides evidence that FGF21 may protect islets from
over-secretion of insulin during acute high glucose and high fat stress.
52
6.2 Future Directions
Future studies should continue investigating the mechanisms of FGF21-induced changes
in the metabolic response during high nutrient challenge.
Reactive oxygen species (ROS) is an important measure of cellular toxicity. In the
discussion, I postulated that FGF21-treated islets shifts metabolism towards NADPH to provide
better protection against ROS generation during high nutrient loading. Therefore, it would be
valuable to measure the level of ROS generation in FGF21-treated islets during high glucose and
high fat loading with comparison to non-treated control islets. Dichlorofluorescein (DCF) and
MitoSox Red are commonly used fluorescent dyes to measure changes in ROS levels. These
dyes are chemically deactivated and only become fluorescent via interaction with ROS such as
hydrogen peroxide. My preliminary attempts to measure ROS using DCF and Mitosox Red
failed due to difficulties loading the dye. The data point of interest is the quantification of ROS
levels at high glucose and high fat. However live islets under high metabolic activity readily
pumps out both of these dyes. As a result, it was difficult to track significant ROS induced
changes in fluorescence intensity due to the low levels of dye accumulation in the live islet tissue
during high glucose and high fat loading. Future experiment should examine potential methods
to overcome the dye loading issue. One possible experiment would be loading the dye at low
glucose first prior to loading the islets into the microfluidic device. Subsequently, the high
glucose and high fat stimulus can be flown into the channel. Time series imaging should be
immediately started to track changes in dye intensity and the tissue with more ROS generation
should retain a higher level of fluorescence. Overall, a quantification of ROS levels would
provide valuable information on the level of oxidative stress in the islet tissue during high
nutrient challenge.
53
Another potential candidate involved in mitochondrial membrane uncoupling is NNT.
The ability of NNT to reversibly catalyze the conversion of NADH and NADPH is a powerful
mechanism for the cell to regulate metabolism. It was discussed that NNT expression may be
altered by FGF21 stimulation to increase the capacity to generate NADPH during high nutrient
stimulus. Therefore, a future experiment should investigate possible changes in islet NNT
mRNA and protein levels post FGF21 stimulation.
Lastly, another valuable experiment would be to examine the metabolic kinetics in
FGF21-treated islets. Since FGF21-treated islets have a lower expression of ACC, it is possible
that the toggling between fatty acid oxidation and fatty acid synthesis occurs at different rates.
Interesting experiments would include examination of the glucose-induced NAD(P)H and
NADH response of islets in a high fat environment. The microfluidic device we used allows
consistent imaging of islets while the desired stimulation media is continuously provided by
controlled laminar flow. Preliminary experiments were not successful due to focal drift. The
difficulties occurred when the 37oC temperature chamber was opened during the addition of
stimulation media and the cooling of the chamber by room temperature air caused the objective
to drift out of focus. An attempt to solve this issue was applying a stoppage in flow during the
addition of stimulation media and allowing time for the chamber temperature to re-equilibrate to
37oC before simultaneous initiation of flow and the time series imaging sequence. This method
allowed successful collection of time series images. However, islets reorient in the channel after
prolonged incubation in the absence of flow. Specifically, the re-initiation of flow after stoppage
causes significant movement of the islet in the channel. Therefore, data obtained using this
method proved difficult to analyse because the early images in the time series set had large
inconsistencies in islet position. To improve this experiment, a new microfluidic device should
54
be designed to allow switching of stimulation media without accessing the temperature control
chamber. Overall, further development of the microfluidic device can provide valuable insight
into metabolic kinetics of islets.
6.3 Concluding Remarks
This thesis aimed to address the hypothesis that (1) FGF21 stimulation will decrease islet
ACC protein expression and modulate islet metabolism during high glucose and high fat loading,
and (2) FGF21 stimulation will modulate islet mitochondrial energetics and insulin secretion.
To address the first hypothesis, biochemistry studies confirmed a FGF21-induced
decrease in islet ACC expression. Subsequently, a microfluidic device was developed to study
the metabolism of live islets by measuring the NADH and NADPH responses using
autofluorescence microscopy. It was determined that FGF21-treated islets exhibit a lower
NADPH response when stimulated with citrate which correlates to a decrease in FAS. As well,
addition of high glucose in the presence of palmitate caused a decrease in mitochondrial NADH
in FGF21-treated islets only. Overall, these results confirmed FGF21 regulates islet ACC
expression and alters the metabolic response of islets to nutrient stimulus as measured by NADH
and NADPH.
To address the second hypothesis, imaging of Rh123 labelled islets during high glucose
and high palmitate challenge confirmed a decrease in mitochondrial membrane potential in the
presence of FGF21. Subsequent ELISA assay of islet insulin secretion showed FGF21-treated
islets secrete a lower fraction of total insulin under high glucose and high palmitate stimulation.
To establish a mechanistic explanation to these results, the expression of UCP2 protein was
examined. FGF21-treated islets exhibited lower expression level of UCP2 protein. Overall,
55
these results confirmed altered handling of high glucose and high palmitate challenge in FGF21-
treated islets with reduced mitochondrial energetics and diminished fractional insulin secretion.
In summary, this thesis provided evidence of FGF21-stimulated regulation of islet
metabolism based on a model centered on the key metabolic regulator ACC. The FGF21-
induced changes in mitochondrial energetics and insulin secretion provides motivation for
continued investigation of the mechanisms of FGF21 action in the protection of pancreatic islets
during high nutrient loading.
56
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