Mechanism and Regulation of Transport of Water-Soluble Vitamins: Cellular and Molecular Aspects

Hamid M. SaidUniversity of California School of Medicine, Irvine

&VA Medical Center, Long Beach

USA

Hamid Said has nothing to disclose

P Srinivasan, PhDA Ghosal, PhDA Biswas, PhDS Senthilkumar, PhDS Subramanya, PhDT Sekar, PhDF Bukhari, MDL Vogeley, MDL Mee, PhDZ Mohammed, MDA Balasubramanien, PhD S Nabokina, PhD

AcknowledgementsCurrent and Previous Postdocs

W Tipton, MDS Khorchid, MDD Dunning, MDD Dyer, PhDE McCloud, MDK Balamurugan, PhDJ Redling, PhDV Subramanian, PhDC Kumar, PhDN Chatterjee, PhDR Ul-Haq, MDT Nguyen, PhD

THANK YOU!

”We thank the NIH and the DVA for their kind support”

Acknowledgements Collaborators

W Strum (Scripps)D Hollander (UCI)C Wagner (Vanderbilt)D Horne (Vanderbilt)F Ghishan (Vanderbilt)I Parker (UCI)J Marchant (U Minn)T Ma (UNM)P Dudeja (UIC)

”We thank the NIH and the DVA for their kind support”

THANK YOU!

N Vaziri (UCI)N Yanagawa UCLA)C Halsted (UCD)J Sze (Albert Einstein U)K Maedler (U Bremen)G Hecht (UIC)V Subramanian (UCI)S Pandol (UCLA)H Tsukamoto (USC)

Physiology of Vitamin

Transport

NutritionMechanism of Disease

OHHN

HOOC

O

OH

H3C CH 3

NH

HN

S O

H

HH

HOOC

Biotin

Pantothenic acid

Folic acid

ThiaminRiboflavin

N

OCl

NN

NS

ON

O

O

O

OO

N

N

NNN

N

O

O

O

OO

O

N

NN

N

OO

N

O

Cl

Pyridoxine

O

Ascorbic Acid

O

O

O

O

O

O

Ascorbic Acid

O

O

O

O

O

Niacin

N

O

O

Niacin

N

O

O

Niacin

N

O

O

*Water – Soluble Vitamins: An Introduction* 1) Water-soluble vitamins are structurally and functionally unrelated organic compounds that share the common features of being essential for normal cellular function, growth and development.

*Water-soluble vitamins: An introduction* -----continues

2) Humans cannot synthesize water-soluble vitamins (exception: some endogenous synthesis of niacin), and thus, must obtain these micronutrients from exogenous sources.

3) Deficiency of water-soluble vitamins leads to a variety of clinical abnormalities that range from anemia, growth retardation, congenital deformities, and neurological disorders.

-Vitamin deficiency is of two types:

i) Systemic (global) deficiency

ii) Tissue-specific (localized) deficiency (e. g., Thiamin- Responsive Megaloblastic Anemia)

4) Optimizing body homeostasis of water-soluble vitamins, on the other hand, brings about positive health benefits

*Folate: -Prevention of neural-tube defects

-Decrease risk of Alzheimer Disease

-Decrease risk of the development of certain types of cancer (e.g. cervical cancer)

*Thiamin: -Has the potential of preventing diabetic- retinopathy, nephropathy, and vascular damage

Why study transport of water-soluble vitamins?

The case for the intestine

The case for the intestine---continues

1) The human body cannot synthesize these micronutrients nor can it store them in significant quantities; it relies on uninterrupted absorption in the gut.

2) Water-soluble vitamins exist in minute quantities in the diet, and thus, efficient mechanisms are needed for their extraction from the moving food digest in gut lumen.

3) A variety of conditions interfere with normal intestinal absorption of water-soluble vitamins.

• Congenital defect in the specific uptake system (e.g., mutations in PCFT leading to Hereditary Folate Malabsorption Syndrome)

• Congenital defects in the enzyme that hydrolyzes non-absorbable dietary forms of the vitamin into the absorbable form (e.g., Biotinidase - deficiency)

• Chronic alcohol consumption

• Bacterial infection (enteropathogenic E. coli, EPEC)

• Drug-interaction

• G.I. diseases (e.g., IBD); intestinal resection

Why study transport of water-soluble vitamins?The case for the intestine----continues

4) Optimization of water-soluble vitamin bioavailability and body homeostasis via optimized absorption improves health and prevents certain diseases.

Sources of water- soluble vitamins

Two sources are available to the human gut:1)Dietary2)Bacterial (i. e., the normal microflora of the large intestine)

Sources of water- soluble vitamins----dietary source-

-Early studies on the mechanisms involved in the intestinal absorption of (many) water-soluble vitamins have concluded that these events occur via simple diffusion.

-This came out as a result of the use of non-physiological/non-optimal experimental conditions. Further, when evidence for the existence of a specific mechanism was observed, e. g., saturability, it was attributed to intracellular metabolism, limited solubility of the vitamin (due to existence of an acidic microclimate at the surface of the small intestine), etc.

-The above erroneous belief has significantly delayed progress in the field. Thus, very limited studies were attempted to investigate if and how these absorptive events are regulated, whether they are affected by certain disease conditions or by drug interaction, and whether certain human genetic diseases are due to defects in vitamin transporters.

This is where we were in the early 1980s

This is where we are in 2012

Involvement of specific, carrier-mediated and highly regulated transport systems in the absorption of dietary water-soluble vitamins in the human small intestine

SVCT 1

SVCT 2 RFT1THTR-1

??MDR-3

RFT2?? THTR1

THTR2

PCFTSMVT

Ascorbic Acid Riboflavin Pyridoxine Thiamine Biotin/Pantothanate

Folate Lumen

Blood

??

Niacin

Sources of water- soluble vitamins----dietary source Absorption of certain dietary water-soluble vitamins in the small intestine is site-

specific; However adaptation (induction) occur following resection

Resected

Sham-operated

Am J Clin Nutr 47:75-9, 1988

Sources of water- soluble vitamins

1)Dietary source

1)Bacterial source, i. e., the normal microflora of the large intestine.

Humans have more bacteria in their GI tract than they have cells

1012

Cells

1014

Bacteria

Human Body

GI Tract

* There are over 1,800 different species of bacteria in our GI tract identified so far.

Microbial distribution in the GI tract

Aerobes

AnaerobesAnaerobes

Stomach<102 cfu/mL

pH, 1-2

Colon<1010-12 cfu/mL

pH, 5-7

Colon<1010-12 cfu/mL

pH, 5-7

Duodenum101-3 cfu/mL

pH, 6-7

Duodenum101-3 cfu/mL

pH, 6-7

Jejunum103-4 cfu/mL

pH, 6-7

Jejunum103-4 cfu/mL

pH, 6-7

Ileum107-9 cfu/mL

pH, 6-7

Ileum107-9 cfu/mL

pH, 6-7

According to Mayo Clin Proc 2008; 83:460-469

Happy VS unhappy intestinal microflora

Adopted from Gastroenterology 2009;136:2015-2031

vitamin

vitamin

vitamin

vitamin

vitamin

vitamin

vita

min

vitamin

vitamin

vitam

in

vitamin

-Bacterial source-----continues

Enterotypes of the human gut microbiome Arumugam et al, Nature 473:174-180, May 2011

*Human microbiota can be functionally classified into three distinct enterotypes:

-Enterotype A: Has an over-represented Biotin (vitamin B7; vitamin H) biosynthetic pathway

-Enterotype B: Has an over-represented Thiamin (vitamin B1) biosynthetic pathway

-Enterotype C: Has an over-represented Haem biosynthetic pathway

Functional differences between enterotypes (Nature 473:174-80, 2011)

Arumugam M, Raes J, Pelletier E, Ehrlich DS , Bork P et. al. “Enterotypes of the human nature in microbiome.” Nature. 2011 A473(7346):174-80pr 20. Doi: 10.1038.

Bacteria Divide People Into 3 Bacteria Divide People Into 3 Types, Scientists Say Types, Scientists Say

By Carl ZimmerPublished: April 20, 2011

“The ability of the microbiota to synthesize vitamins has been known for many years, but this was considered unimportant for nutritional health, because it was assumed they were malabsorbed and lost in stools. However, the recent demonstration of specific transporters for folate (24), biotin (25), thiamine (26), riboflavin (27), and pyridoxine (28) in the colonic mucosa has forced us to reconsider our view.”

O”Keefe et al, J. Nutr. 139: 2044-2048, 2009

Identification of carrier-mediated transport systems for water-soluble vitamins in the human colon

Studies with colonic tissue from human organ donors and with cultured colonocytes

??

RFT1

RFT2?? THTR1

THTR2

RFCSMVT

Niacin Riboflavin Pyridoxine Thiamine Biotin/Pantothanic acid

Folate

Lumen

Blood

MDR-3THTR-1

TPP

Said et al 1995- present

Implications for the identification of efficient mechanisms for absorption of water-soluble vitamins in the human colon are:

1) They indicate that the bacterially synthesized vitamins are nutritionally available to the host and contribute toward overall vitamin homeostasis, especially toward the cellular nutrition and health of the local colonocytes.

2) Provides physiological basis for important nutritional and clinical observations.

Intestinal absorption of water-soluble vitamins

Biotin-

Biotin-

Brush Border Membrane

Basolateral Membrane

hSMVT

2. Energetics of intestinal transport processes of water-soluble vitamins:

i) The case for biotin

Na+ Biotin-Na+

Folate-

Folate-

Brush Border Membrane

Basolateral Membrane

PCFT

2. Energetics of intestinal transport processes of water-soluble vitamins:

ii) The case with folate

H+ Folate-H+

12 11

Cytosol

N-glycosylation sites PKC phosphorylation sitesPKA phosphorylation site

II. Molecular aspects 1) Molecular identity of many of the transporters of water-soluble vitamins have been determined

by cloning.Predicted topology of hTHTR-1

II. Molecular aspects 1) Molecular identity of many of the transporters of water-soluble vitamins have been determined

by cloning.Predicted topology of hTHTR-1

NHNH22

1 2 3 5 7 8 9 10

COOHCOOH

4 6

Asn63

Asn414

Thr17

Thr22 Ser357

Thr158

Ser186

Thr360Ser222

Thr223

Ser292

Ser49

Thr477

Asn314

12

SLC19A2 (hTHTR-1)-Promoter Luciferase

SLC19A3 (hTHTR-2)-Promoter Luciferase

GK

LF/S

P1

AP1

SP1NF1

SP1

SP1 N

F1

OCT1

ERG

2/3

2. The 5’ regulatory region (promoters) of a number of the genes that encode transporters of water-soluble vitamins have also been cloned and

characterized both in vitro and in vivo.

Reidling and Said, AJP, 285:C633-41, 2003Nabokina and Said, AJP 287:G822-9, 2004.

SLC19A3 Promoter Luciferase

In vivo validation of promoter activity in transgenic mice.

SLC19A2 Promoter Luciferase

In vivo validation of promoter activity of the C. elegans folate transporter cfolt-1 (fused to GFP) at the integrative whole animal level

AJP 293:C670-681, 2007

3) Relative contribution of water-soluble vitamin transporters toward carrier-mediated uptake processes

i) Gene-specific knock down

ii) Gene-specific knockout

-

i) Gene-specific knock down (siRNA)

Relative contributions of hTHTR-1 and hTHTR-2 toward carrier-mediated thiamin uptake by human intestinal epithelial Caco-2 cells

AJP 286:G491-G498, 2003

ii) Gene-specific knockout: the mouse model

Intestinal thiamin absorption in THTR-2 KO mouse model: In vivo perfusion

Gastroenterology 138:1802-1809, 2010

P < 0.01

Intestinal thiamin absorption in THTR-1 KO mouse model: In vivo perfusion

Relative expression of THTR-2 in THTR-1-/- mouse intestine

II. Cell Biology Aspects

1) Live cell confocal imaging showed expression of hTHTR-1 at both the apical and basolateral membrane domains (A, B), while that of hTHTR-2 (C, D) only at the apical membrane domain of polarized human epithelial cells.

JBC 278:3976-84, 2003; JBC 281:5233-45, 2006

2. Targeting of water-soluble vitamin transporters to the apical membrane domain of polarized intestinal/renal epithelia involves specific motifs (signals) that are embodied in the individual polypeptide: The story with the human folate transporter, PCFT.

AJP 294:C233-240, 2008

3. A predicted β-turn motif between TMD 2 and 3 of PCFT was found using a β-Turn Predictive Algorithm Program: Progressive mutagenesis of amino acids within this region is predicted to disrupt formation of the β-turn.

hPCFT-YFPhPCFT[GRR112-

114AAA]-YFP

xyxz

A

3H-F

olic

aci

d u

pta

ke(

pm

ol/m

g p

rote

in/3

min

)

Control hPCFT-YFP hPCFT[GRR112-

114AAA]-YFP

B

4. Disrupting the β-turn between TMD 2 and 3 of the PCFT polypeptide leads to: A) intracellular retention of the mutant protein in polarized epithelial cells, and 3) inhibition in the induction of folate uptake.

a

5. Different mutants of hTHTR-1 found in patients with TRMA display different cellular expression phenotypes.

Clin Sci (Lond). 113: 93-102, 2007

6. Intracellular movement of hTHTR -1 & 2 (fused to GFP) in living human epithelial cells involves trafficking vesicles (A), whose movement depends on an intact microtubule network (B).

A. Control B. Nocodazol

JBC 278:3976-84, 2003; JBC 281:5233-45, 2006

Tspan1 Dynein light chain road block

hRFChSMVT

PDZD11

hTHTR1

7. Specific accessory proteins interact with membrane transporters of water-soluble vitamins and modulate their function, stability, and membrane expression: Studies using bacterial and yeast two-hybrid system to screen human intestinal cDNA libraries.

AJP 301:G808-13, 2011 AJP 300:G561-7, 2010 AJP 297:G480-7, 2009

III. Regulatory aspects of the intestinal absorption processes of water-soluble vitamins

A) Adaptive - regulation by dietary (extracellular) substrate levels.

B) Developmental - regulation during early stages of life.

C) Differentiation - dependent regulation.

D) Regulation by specific intracellular protein kinase-mediated pathways.

AJP 256:G306-311, 1989

Adaptive - regulation of the intestinal biotin absorption process

1)Dietary-induced biotin deficiency in rats leads to a specific induction in intestinal biotin uptake, while dietary over-supplementation of biotin leads to suppression of the uptake.

2) Similarly, maintaining the human-derived intestinal epithelial Caco-2 and HuTu-80 cells under biotin-deficient conditions leads to a specific induction in biotin uptake and in the level of expression of the hSMVT protein and mRNA.

AJP 292:G275-281, 2007*p < 0.01

3) The effect of biotin deficiency on biotin uptake by Caco-2 and HuTu-80 cells was not mediated via changes in mRNA stability of hSMVT.

RNA decay rate assay (i.e., RNA stability assay)

4) The induction in hSMVT mRNA levels in Caco-2 and HuTu-80 cells in biotin deficiency is mediated, at least in part, via transcriptional mechanism(s).

hSMVT (SLC5A6)- promoter Luciferase

*p < 0.01

5) The biotin-deficiency responsive region of the hSMVT P1 is located in a specific (103 bp) region between -130 and -233

6) A KLF4 cis-regulatory element in the biotin deficiency-responsive region of the hSMVT P1 is responsible for mediating the biotin deficiency effect on the

hSMVT promoter.

Integrative aspects of the adaptive - regulation of vitamin transport by substrate level:

Whole animal studies utilizing living C. elegans

1) Folate over-supplementation inhibits folate uptake (A), as well as the level of expression of the cfolt-1 mRNA (B) in adult C. elegans

0

1

2

-

Deficie

ntRel

ativ

e fol

t -1m

RN

A e

xpre

ssio

nov

er -a

ctin

(in

fol

ds)

0

1

2

overOve

r-sup

plemen

ted

Rel

ativ

e fol

t -1m

RN

A e

xpre

ssio

nov

er -a

ctin

(in

fol

ds)

0

0.5

1.0

Over-s

uppl

emen

ted

Defici

ent

[3 H] -f

olic

aci

d up

take

(fm

ol/5

ani

mal

s/5

min

)

0

0.5

1.0

[3 H] -f

olic

aci

d up

take

(fm

ol/5

ani

mal

s/5

min

)

A B

AJP 293:C670-681, 2007*p < 0.01 for both

2) Folate over-supplementation suppresses the activity of the cfolt-1 promoter (fused to GFP) in living C. elegans.

0

50

100

Def

icie

nt

over

-sup

plem

ente

d

GF

P f

luor

esce

nce

inte

nsi

ty (

%)

Level of GFP fluorescence in the C. elegans intestineDeficient

Over-supplemented

(p < 0.01)

1. Carrier-mediated uptake of [3H]-Thiamine by mouse intestinal BBMV during development was found to decrease

with maturation (suckling > weanling > adult).

Developmental - regulation of intestinal and renal thiamin uptake process

2. A decrease in levels of the mouse endogenous mTHTR-1 and mTHTR-2 protein and mRNA (suckling > weanling > adult) was observed in the intestine during developmental maturtion using Western analysis and qPCR

3. Luciferase activity in the intestine of transgenic mice carrying the human SLC19A2 or SLC19A3 promoters showed

a decrease in activity with maturation (suckling > weanling > adult).

4. Carrier-mediated uptake of [3H]-thiamine by mouse kidney BBMV during development was found to decrease with

maturation (suckling > weanling > adult).

5. A decrease in levels of the mouse endogenous mTHTR-1 and mTHTR-2 protein and mRNA (suckling > weanling > adult) was observed in the kidney during developmental maturation using Western analysis and

qPCR

6. Luciferase activity in the kidney of transgenic mice carrying the human SLC19A2 or SLC19A3 promoters showed

a decrease in activity with maturation (suckling > weanling > adult).

IV. Mechanism of Disease

A) Effect of chronic alcohol use on intestinal (and renal) absorption of water-soluble vitamins.

A) Effect of Enteropathogenic Escherichia coli (EPEC) on intestinal absorption of water-soluble vitamins.

A) Thiamin-Responsive Megaloblastic Anemia (TRMA):

-Why only certain tissues are affected?

-How mutations in hTHTR-1 in patients with TRMA impair thiamin uptake.

Effect of chronic alcohol feeding/exposure on cellular and molecular parameters of the

intestinal (and renal) thiamin absorption process

- Thiamin deficiency and sub-optimal levels are highly prevalent in chronic alcoholics (up to 75%) and leads to serious clinical consequences (e.g., Wernicke-Korsakoff syndrome).

- We examined the physiological and molecular parameters of the intestinal (and renal) thiamin absorption process that are affected by chronic alcohol consumption.

Mechanism of Disease-----continues

1. Chronic alcohol feeding of rats leads to a significant inhibition in thiamin uptake by jejunal BBMV.

AJP 299:G23-31, 2010

*p < 0.01 for all

2. Chronic alcohol feeding of rats also leads to a significant inhibition in thiamin uptake by jejunal BLMV.

*p < 0.01

3. Chronic alcohol feeding of rats also inhibits thiamin uptake in the colon, suggesting possible impairment in uptake of the bacterially

synthesized biotin.

*p < 0.01

4. Chronic alcohol feeding of rats inhibits the expression of THTR-1 (but not THTR-2) protein at the intestinal BBM.

*p < 0.01

5. Chronic alcohol feeding of rats inhibits mRNA expression of THTR-1 (but not THTR-2) in the jejunum.

*p < 0.01

7. Chronic alcohol exposure (72 hr) of cultured human-derived intestinal epithelial HuTu-80 cells leads to significant inhibition in thiamin uptake.

*p < 0.01

8. Chronic alcohol exposure (72 hr) of HuTu-80 cells leads to a significant inhibition in the level of expression of the hTHTR-1 and

hTHTR-2 proteins.

*p < 0.01 for both

9. Chronic alcohol exposure (72 hr) of HuTu-80 cells also leads to significant inhibition in the expression of the hTHTR-1 and hTHTR-2

mRNA.

*p < 0.01 for both

10. Effect of chronic alcohol exposure (72 hr) of human intestinal epithelial HuTu-80 cells on the activity of hTHTR-1 (SLC19A2) and hTHTR-2 (SLC19A3) promoters.

hTHTR-1 (SLC19A2)-Promoter) Luciferase hTHTR-2 (SLC19A3)-Promoter) Luciferase

*p < 0.01 for both

11. Chronic alcohol feeding of transgenic mice carrying the SLC19A2 (hTHTR-1)- and SLC19A3 (hTHTR-2)- promoters leads to a significant inhibition in activity of the human promoters in the jejunum.

SLC19A2 Promoter LuciferaseSLC19A2 Promoter Luciferase

0

20

40

60

80

100

120

Control Alcohol

Relati

ve lu

cifer

ase a

ctivit

y

(Pair-fed)

0

20

40

60

80

100

120

Control Alcohol

Relati

ve Lu

cifer

ase a

ctivit

y

(Pair-fed)

*p < 0.01 for both

Effect of chronic alcohol feeding on physiological and molecular parameters of renal thiamin transport

1) Effect on [3H]thiamin uptake by rat renal BBMV

2) Effect of chronic alcohol feeding on [3H]thiamin uptake by rat renal BLMV

3) Effect of chronic alcohol feeding on level of expression of THTR-1 and THTR-2 in kidney cortex of rats fed alcohol chronically

4) Effect of chronic alcohol feeding of rats on level of expression of heterogeneous nuclear RNA (hnRNA) of the Slc19a2 & Slc19a3 genes

Control(Pair-fed) Alcohol

Rel

ativ

e SL

C19

A2

luci

fera

se a

ctiv

ity(%

of

cont

rol)

Control(Pair-fed)

Alcohol

Rel

ativ

e SL

C19

A3

luci

fera

se a

ctiv

ity(%

of

cont

rol)

_

**

* p < 0.01

SLC19A2 Promoter Luciferase SLC19A2 Promoter Luciferase

5) Chronic alcohol feeding of transgenic mice carrying the SLC19A2 and SLC19A3 promoters leads to a significant inhibition

in activity of the human promoters in the renal cortex.

1) EPEC, a gram-negative food-borne pathogen, infects human intestine leading to significant morbidity and mortality, especially in infants. Infection with EPEC is common in developing countries; it also occurs in developed countries as a result of food contamination and improper hygiene.

2) Whereas diarrhea is a major consequence of EPEC infection, malnutrition also occurs especially in severe and prolonged cases.

Mechanism of Disease-------continues

Effect of Enteropathogenic Escherichia coli (EPEC) infection and intestinal thiamin uptake

3) Pathogenicity of EPEC involves attachment of the bacteria to enterocytes, effacement in the microvilli, and delivery of effector molecules via a syringe-like type III secretion system (TTSS).

4) Nothing is known about the effect of EPEC infection on intestinal

absorption of thiamin. Addressing this issue of physiological and nutritional importance since humans have limited capability to store thiamin, and thus, prolonged and severe infection with EPEC may negatively impact normal body homeostasis leading to further aggravation of the health status of the infected hosts (many of whom are already nutritionally compromised).

Results

1) EPEC treatment of Caco-2 cells leads to a significant inhibition in thiamine uptake. Uptake was performed immediately after treatment (A) or 6 hr later (B).

A B

AJP 297: G825-833. 2009*p < 0.01 for both

2) The inhibition in thiamin uptake by Caco-2 cells caused by EPEC (100 MOI) increases as a function of the pretreatment (contact) time.

3) The inhibition in thiamin uptake by Caco-2 cells requires live EPEC.

*p < 0.01

4) EPEC inhibits thiamin uptake at both the nanomolar range (mediated by hTHTR-2) and the micromolar range (mediated by hTHTR-1).

5) EPEC reduces the level of expression of the hTHTR-1 and hTHTR-2 proteins at the apical membrane domain of confluent Caco-2 monolayers.

Biotinylation assay*p < 0.01 for both

6) EPEC inhibits the level of mRNA expression of hTHTR-1 (A), and hTHTR-2 (B).

A B

*p < 0.01 for both

7) EPEC, but not the non pathogenic E. coli, suppresses the activity of the human SLC19A2 and SLC19A3 promoters in Caco-2 cells.

*p < 0.01 for both

8) Functional type III secretion system (TTSS) of EPEC is required for the bacteria to inhibit thiamin uptake by Caco-2 cells.

*p < 0.01

“Few Take Home Messages”

1) Absorption of dietary water-soluble vitamins in the small intestine involves specific, and regulated carrier-mediated systems.

2) The water-soluble vitamins synthesized by the normal microflora of the large intestine can be absorbed via efficient carrier-mediated systems, and thus, contribute to the overall body homeostasis of these micronutrients, and especially toward the cellular nutrition and health of the local colonocytes.

3) A variety of conditions/factors interfere with the intestinal absorption of water-soluble vitamins; this may lead to a compromise in their normal body homeostasis.

Thank you