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.

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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.

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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

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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

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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

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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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