Nicotinamide Mononucleotide in the Context of Elevated ...€¦ · Wheeler, and Dr. Pausova for...

Nicotinamide Mononucleotide in the Context

of Elevated Plasma Levels of Free Fatty Acids

Improves Glucose Tolerance by Decreasing

Insulin Clearance

By

Ashraf Nahle

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Physiology

University of Toronto

© Copyright by Ashraf Nahle (2016)

ii

Nicotinamide Mononucleotide in the Context of Elevated

Plasma Levels of Free Fatty Acids Improves Glucose

Tolerance by Decreasing Insulin Clearance

Ashraf Nahle

Master of Science

Department of Physiology

University of Toronto

2016

Abstract

The NAD-dependent deacetylase SIRT1 has been shown to be beneficial to beta cell function.

Nicotinamide Mononucleotide (NMN), the product of the rate-limiting enzyme in NAD synthesis,

has recently been shown to have positive effects on glucose tolerance in mice fed a high fat diet.

This is the first study to examine the effects of NMN on insulin clearance and FFA-induced beta

cell dysfunction. NMN was i.v. infused, with or without oleate, in C57BL/6 mice over 48h in

order to elevate intracellular NAD levels and consequently increase SIRT1 activity. We

demonstrated that administration of NMN in the context of elevated plasma FFA levels results in

a significant decrease in insulin clearance as well as partial protection against FFA-induced beta

cell dysfunction in vivo. This culminated in a large improvement in glucose tolerance. In

summary, NMN may have a therapeutic potential to improve glucose tolerance in conditions of

increased plasma FFA levels, which is typical in patients with type 2 diabetes mellitus.

iii

Acknowledgements

I am very grateful to Dr. Adria Giacca for providing me with the opportunity to contribute

to the front-line of research in type 2 diabetes mellitus, a disease that afflicts many millions

globally. Almost always available, Dr. Giacca has been a beacon of wisdom, guidance, and

patience. I am also incredibly grateful to my supervisory committee, Dr. I. George Fantus, Dr.

Carolyn Cummins, and Dr. Jonathan Rocheleau for their wise guidance, encouragement, and

understanding throughout the years. I would like to thank Dr. Zdenka Pausova for introducing me

to research during my undergraduate degree, as well as Dr. Denise Belsham, Dr. Michael

Wheeler, and Dr. Pausova for their enlightening courses on presentation skills and on the critical

analysis of studies.

In the beginning, I found leading basic science research projects to be challenging;

however, through perseverance, dedication to researching this important illness, and generous

support from friends and colleagues, we have discovered a potentially therapeutic biochemical

pathway. I have also matured both educationally and personally.

I would like to thank my friends and colleagues, Dr. Prasad Dalvi, Dr. Yusaku Mori, Alex

Ivovic, Frankie Poon, Loretta Lam, Lucy Yeung, Alex Orazietti, Tiffany Yu, Tejas Desai, Dr.

Khajag Koulajian, Dr. June Guo, Sammy Cai, and Cynthia Putra for helping make my experience

enjoyable and memorable. I trust that we will stay in touch. I am especially grateful to Loretta

Lam and Frankie Poon for their considerable experimental assistance, and to Dr. Prasad Dalvi,

Dr. Yusaku Mori, and Alex Ivovic for their generous advice, support, and encouragement during

my tough times doing research. Thank you, as well, to Dr. Sonia M. Najjar and her lab, especially

Hilda Ghadieh, for their collaboration in our research and their valuable scientific advice. Of

course, thank you to Ms. Rosalie Pang for her indispensable administrative advice.

I am extremely grateful to my mother, father, and sister for their unwavering support, love,

and frequent phone calls while they were overseas for years. I am very grateful to my close

friends, especially my significant other Lindsay Kuipers and her family, for their kind support,

wise advice, and positivity.

It feels very rewarding to have worked with highly motivated and skilled researchers and to

have contributed to the development of therapeutic and preventive healthcare for patients with

type 2 diabetes. I will always be grateful to everyone involved in our research. Best wishes to

your life and career!

iv

Table of Contents

Abstract………………………………………………………………………………………..ii

Acknowledgements…………………………………………………………………………...iii

Table of Contents……………………………………………………………………………..iv

List of Abbreviations………………………………………………………………………….vi

List of Figures……………………………………………………………………………….viii

List of Tables………………………………………………………………………………….ix

Chapter 1: Introduction ..................................................................................................... 1 1.1 Glucose Homeostasis .......................................................................................................... 1 1.2 Diabetes Mellitus ................................................................................................................ 3 1.3 Complications ..................................................................................................................... 5 1.4 Risk Factors ........................................................................................................................ 7

1.5 Epidemiology ...................................................................................................................... 9 1.6 Insulin Secretion ............................................................................................................... 10

1.7 Insulin Clearance .............................................................................................................. 13 1.8 Fat-Induced Beta Cell Dysfunction .................................................................................. 19

1.8.1 .... Methods of Assessing FFA-Induced Beta Cell Dysfunction ............................................ 20 1.8.2 .... Effects of Different Lipid Treatments on Beta Cell Dysfunction ..................................... 22 1.8.3 .... Genetic Predisposition to Lipid-Induced Beta Cell Dysfunction ..................................... 23 1.8.4 .... Beta Cell Replenishment .................................................................................................. 24 1.8.5 .... Effect of FFAs on Beta Cell Mass .................................................................................... 25

1.9 Glucotoxicity and Glucolipotoxicity ................................................................................ 26 1.10 Mechanisms of FFA-Induced Beta Cell Dysfunction ...................................................... 28

1.10.1 .. The Role of Oxidative Stress ............................................................................................ 28 1.10.2 .. The Role of Endoplasmic Reticulum Stress ..................................................................... 32 1.10.3 .. The Role of Inflammation ................................................................................................ 34 1.10.4 .. The Role of Protein Kinase C ........................................................................................... 35 1.10.5 .. The Role of Ceramides ..................................................................................................... 38

1.11 Sirtuins .............................................................................................................................. 38 1.11.1 .. Sirtuin-1 ............................................................................................................................ 41 1.11.2 .. Role of SIRT1 in Beta Cells ............................................................................................. 42 1.11.3 .. Roles of SIRT1 in the Liver, Skeletal Muscle, and Brain ................................................ 44 1.11.4 .. Roles of FOXO Proteins ................................................................................................... 45 1.11.5 .. Regulation of SIRT1 ......................................................................................................... 46 1.11.6 .. Nicotinamide Mononucleotide ......................................................................................... 50 1.11.7 .. Potential Role of SIRT3.................................................................................................... 54

1.12 Potential Treatments for Lipid-Induced Beta Cell Dysfunction ....................................... 55

Chapter 2: Rationale ........................................................................................................ 59 2.1 Previous Results from the Giacca Lab .............................................................................. 59 2.2 My Experimental Design ................................................................................................... 63

Chapter 3: Materials and Methods ................................................................................. 65 3.1 Animal Models .................................................................................................................. 65 3.2 Mouse Cannulation Surgery .............................................................................................. 65

v

3.3 Treatment Infusion in Mice ............................................................................................... 66 3.4 One-Step Hyperglycemic Clamp ....................................................................................... 66

3.5 Glycemia ............................................................................................................................ 67

3.6 Plasma Insulin Levels ........................................................................................................ 68 3.7 Plasma C-Peptide Levels ................................................................................................... 69 3.8 Plasma FFA Levels ............................................................................................................ 69 3.9 Western Blotting ................................................................................................................ 70 3.10 Insulin Sensitivity Index .................................................................................................... 70

3.11 Disposition Index ............................................................................................................... 71 3.12 Insulin Clearance Index ..................................................................................................... 71 3.13 Statistics ............................................................................................................................. 72

Chapter 4: Results ............................................................................................................ 73 4.1 Glycemia ............................................................................................................................. 74 4.2 Glucose Infusion Rate ......................................................................................................... 75

4.3 Plasma Insulin Levels ......................................................................................................... 76 4.4 Plasma C-Peptide Levels .................................................................................................... 77

4.5 Insulin Clearance Index ...................................................................................................... 78 4.6 Insulin Sensitivity Index ..................................................................................................... 79

4.7 Disposition Index ................................................................................................................ 81

4.8 Plasma Free Fatty Acids Levels ......................................................................................... 82 4.9 CEACAM1 Western Blots ................................................................................................. 84

Chapter 5: Discussion ....................................................................................................... 85 5.1 Potential Mechanisms Behind NMN’s Effects on Insulin Clearance ................................. 87

5.1.1 Synergistic Effects of SIRT1 and FFA on PPARα-Mediated Decrease in CEACAM1

Expression .............................................................................................................................. 87 5.1.2 NMN Accentuates the FFA–Akt–FOXO1–PGC-1α–PPARα–Mediated Decrease in

CEACAM Expression............................................................................................................ 89 5.1.3 Potentially Opposing Effects of FFA and SIRT1 on PKCδ/ε-Mediated Decrease in Insulin

Clearance ............................................................................................................................... 91

5.2 Potential Mechanisms Behind NMN’s Effects on Beta Cell Function ............................... 92 5.2.1 SIRT1-FOXO1/3-Mediated Reduction of Oxidative Stress Ameliorated FFA-Induced Beta

Cell Dysfunction .................................................................................................................... 92 5.2.2 Decreased Plasma FFA Levels Resulted in the Amelioration of FFA-Induced Beta Cell

Dysfunction ............................................................................................................................ 94 5.2.3 Excess Antioxidants During Normal Plasma FFA and Intracellular ROS Levels Resulted in

Sub-Optimal ROS Levels and Decreased GSIS .................................................................... 94 5.2.4 SIRT1-FOXO1-Mediated Nuclear Exclusion of Pdx-1, Resulting in Decreased Insulin

Secretion ................................................................................................................................ 95 5.2.5 NAD-Mediated Increase in PARP Activity Decreases ATP for Insulin Secretion ............... 95

5.3 Limitations ........................................................................................................................... 97 5.4 Future Directions ................................................................................................................. 98 5.5 Conclusions ....................................................................................................................... 103

References ........................................................................................................................ 105

vi

List of Abbreviations

8-OHdG – 8-hydroxy-2’-deoxyguanosine

ACS – Acyl-CoA Synthetase

ANOVA – One-way non-parametric analysis

of Variance

AROS – Active Regulator of SIRT1

ATM – Ataxia Telangiectasia Mutated

ATP - Adenosine triphosphate

BAT – Brown Adipose Tissue

BIM – Bisindolylmaleimide

BMI – Body Mass Index

BSA – Bovine Serum Albumin

CDA – Canadian Diabetes Association

Cdk-1 – Cyclin-dependent kinase-1

CEACAM1 – Carcinoembryonic Antigen-

related Cell Adhesion Molecule-1

ChIP – Chromatin Immunoprecipitation

CHOP – CCAAT-enhancer-binding protein

homologous protein

CPT1B – Carnitine palmitoyltransferase 1B

CV – Coefficient of Variation

DAG – Diacylglycerol

DBC-1 – Deleted in Breast Cancer-1

DCF – Dichlorodihydrofluorescein

DI – Disposition Index

DKA – Diabetic Ketoacidosis

DPP-4 – Dipeptidyl Peptidase-4

ER – Endoplasmic Reticulum

ETC – Electron Transport Chain

FFA – Free Fatty Acid

FPG – Fasting Plasma Glucose

FFAR – Free Fatty Acid Receptor

FOXO – Forkhead box-O

FRD – Fructose-Rich Diet

GAPDH – Glyceraldehyde-3-phosphate

dehydrogenase

GDH – Glucose dehydrogenase

GDM – Gestational Diabetes Mellitus

GINF – Glucose Infusion Rate

GIP – Gastric Inhibitory Polypeptide

GIP-R – Gastric Inhibitory Polypeptide

Receptor

GLP-1 – Glucagon-like Peptide-1

GLP1R – Glucagon-like Peptide 1 Receptor

GLUT1/2 – Glucose transporter 1/2

GPCR – G-protein-coupled receptor

GPR40/41/43/119 – G-protein-coupled

receptor 40/41/43/119

GPx4 – Glutathione Peroxidase-4

GSIS – Glucose-Stimulated Insulin

Secretion

H2DCF-DA - 2',7'-dichlorodihydrofluorescein

diacetate

HFD – High Fat Diet

HLA – Human Leukocyte Antigens

HRP – Horseradish peroxidase

IDE – Insulin Degrading Enzyme

IH – Intralipid + Heparin

IKKB – Inhibitor of nuclear factor kappa-B

kinase subunit beta

IPGTT – Intraperitoneal Glucose Tolerance

Test

IR – Insulin Receptor

IRS – Insulin Receptor Substrate

IκBα – Nuclear factor of kappa light

polypeptide gene enhancer in B-cells

inhibitor, alpha

JNK – c-Jun-N-terminal Kinase

KM – Michaelis constant

LC-CoA – Long-Chain Coenzyme A

LDCV – Large Dense Core Vesicles

LPL – Lipoprotein Lipase

L-SACC1 - Liver-specific dominant-negative

phosphorylation-defective S503A CEACAM1

LXR – Liver X Receptor

M/I – Glucose metabolism value/[insulin]

(Index of insulin sensitivity)

MCAD – Medium-chain acyl-CoA

dehydrogenase

MDA – Malondialdehyde

vii

MEHA – 3-methyl-N-ethyl-N-(B-

hydroxyethyl)-aniline

MnSOD – Manganese superoxide dismutase

MUFA – Monounsaturated Fatty Acid

NAC – N-acetylcysteine

NAD – Nicotinamide Adenosine

Dinucleotide

NADPH oxidase – Nicotinamide Adenine

Dinucleotide Phosphate oxidase

NAMPT – Nicotinamide

phosphoribosyltransferase

NCLX – Na+/Ca2+ exchanger

NCoR – Nuclear receptor co-repressor

NFκB – Nuclear factor kappa-light-chain-

enhancer of activated B cells

NMN – Nicotinamide mononucleotide

NMNAT – Nicotinamide mononucleotide

adenylyltransferase-1

OGTT – Oral Glucose Tolerance Test

OLE – Oleate

PARP – Poly (ADP-Ribose) Polymerase

PBA – Phenylbutyrate

PC – Proprotein Convertase

PCOS – Polycystic Ovary Syndrome

PDK4 – Pyruvate Dehydrogenase Kinase 4

Pdx-1 – Pancreas duodenum homeobox-1

PGC-1α - Peroxisome proliferator-activated

receptor gamma coactivator 1-alpha

PI3K – Phosphoinositide 3-kinase

PKA – Protein Kinase-C Activator

PKC – Protein Kinase C

PML - Promyelocytic leukemia

POD – Peroxidase

POMC – Proopiomelanocortin

PP Cells – Pancreatic Polypeptide Cells

PTP1B – Protein-tyrosine phosphatase 1B

PUFA – Polyunsaturated Fatty Acid

PVDF – Polyvinylidene fluoride

RER – Rough Endoplasmic Reticulum

ROS – Reactive Oxygen Species

RPMI media – Roswell Park Memorial

Institute media

RPTPs – Receptor-like Protein Tyrosine

Phosphatases

RPTPs – Receptor-like Protein Tyrosine

Phosphatases

SAL – Saline

SAS – Statistical Analysis System

SDS-PAGE – Sodium dodecyl sulfate

polyacrylamide gel electrophoresis

SE – Standard Error

SF-1 – Steroidogenic Factor-1

SFA – Saturated Fatty Acid

SIR genes – Silent Information Regulator

genes

siRNA – Small interfering ribonucleic acid

SIRT – Silent mating type information

regulation 2 homolog

SMRT – Silencing Mediator of Retinoid and

Thyroid hormone receptors

SPT – Serine palmitoyltransferase

STZ – Streptozotocin

T1DM – Type 1 Diabetes Mellitus

T2DM – Type 2 Diabetes Mellitus

TCA Cycle – Tricarboxylic Acid Cycle

TGN – Trans-Golgi Network

TLR-4 – Toll-like receptor-4

TMB – 3,3',5,5'-tetramethylbenzidine

TZDs – Thiazolidinediones

UCP1/2 – Uncoupling protein 1/2

UPR – Unfolded Protein Response

VDCC – Voltage-Dependent Ca2+ Channels

WAT – White Adipose Tissue

WHO – World Health Organization

ZDF rats – Zucker Diabetic Fatty rats

viii

List of Figures

Figure 1. Anatomy of the Islets of Langerhans in the Human Pancreas. ................................................. 2

Figure 2. Mechanisms of glucose-stimulated insulin secretion (GSIS) in the beta cell. ....................... 12

Figure 3. Summary of the process of insulin clearance in the hepatocyte. ............................................ 16

Figure 4. The Disposition Index (DI) is the product constant of insulin secretion and insulin

sensitivity ............................................................................................................................................... 21

Figure 5. Illustrations of the three-dimensional protein structure and catalytic activity of the NAD-

dependent protein deacetylase Sirtuin-1 (SIRT1). ................................................................................. 41

Figure 6. Chemical structure of nicotinamide mononucleotide (NMN). ............................................... 50

Figure 7. Summary of the Nampt-mediated NAD synthesis. ................................................................ 54

Figure 8. Resveratrol, a SIRT1 activator, partially protects against FFA-induced beta cell

dysfunction in rats in vivo ...................................................................................................................... 60

Figure 9. Beta cell-specific SIRT1 overexpressing (BESTO) mice were partially protected against

FFA-induced beta cell dysfunction in vivo ............................................................................................ 60

Figure 10. Islets of Wistar rats i.v. infused with oleate did not demonstrate decreased NAD

bioavailability, compared with the saline-infused control ..................................................................... 62

Figure 11. Glucose tolerance and plasma levels of insulin, cholesterol, triglycerides, and free fatty

acids of mice fed a high-fat-diet and treated with nicotinamide mononucleotide.. ............................... 63

Figure 12. Gradual elevation in glycemia of wildtype mice i.v. infused with 37.5% glucose during

the hyperglycemic clamp.. ..................................................................................................................... 74

Figure 13. Glucose infusion rate (GINF) required for obtaining and maintaining hyperglycemia in

mice. ....................................................................................................................................................... 75

Figure 14. Plasma insulin levels prior to i.v. glucose infusion (basal glycemia) and after the

maintenance of hyperglycemia (clamp) for ~30 minutes in C57BL/6 mice.. ........................................ 76

Figure 15. Plasma C-peptide levels prior to i.v. glucose infusion (basal glycemia) and after the

maintenance of hyperglycemia (clamp) for ~30 minutes in C57BL/6 mice. ......................................... 77

Figure 16. Insulin Clearance Indices prior to i.v. glucose infusion (basal glycemia) and after the

maintenance of hyperglycemia (clamp) for ~30 minutes in C57BL/6 mice.. ........................................ 78

Figure 17. Insulin Sensitivity Indices (M/I) obtained after the maintenance of hyperglycemia

(clamp) in C57BL/6 mice for ~30 minutes ............................................................................................ 79

Figure 18. The Disposition Indices obtained after the maintenance of hyperglycemia (clamp) in

C57BL/6 mice for ~30 minutes ............................................................................................................. 81

Figure 19. Plasma FFA levels of mice during basal glycemia (after 4 h of fasting and prior to

glucose infusion) and during hyperglycemia (after ~30 minutes at steady-state) ................................. 82

Figure 20. Western blots of CEACAM1 (Cc1) performed on livers of mice infused with SAL,

OLE, NMN+OLE, or NMN for 48h ...................................................................................................... 84

ix

List of Tables

Table 1. Summary of the seven mammalian sirtuins SIRT1-7 ....................................................... 40

1

Chapter 1: Introduction

1.1 Glucose Homeostasis

Glucose homeostasis is mainly regulated by the secretion of hormones from the islets of

Langerhans (hereinafter referred to as “islets”), which are sphere-like tissues that make up the

endocrine portion of the pancreas1,2. These islets were discovered in 1869 by Paul Langerhans in

Germany3. The islets contain hormone-secreting cells, which include alpha, beta, delta, epsilon,

and pancreatic polypeptide (PP) cells (Figure 1).

While the islets are crucial for glucose homeostasis, they only make up ~2% (1-2 g) of the

total pancreatic mass (60-100 g) in adult humans1,2. There is an estimated one million islets

scattered throughout the pancreas, together containing about one billion beta cells. In humans, the

islets consist of approximately 54.6% beta cells, 33.6% alpha cells, 12% delta cells, 1.6% PP

cells, and < 1% epsilon cells. The proportion of cells in islets is known to slightly vary between

different species1,2. In addition, islets are highly vascularized (10 - 15% of pancreatic blood flow)

and are innervated by sympathetic and parasympathetic nervous fibres. These allow the islets to

be quick and precise at regulating glucose homeostasis1,2.

2

Figure 1. Anatomy of the Islets of Langerhans in the Human Pancreas. Reprinted courtesy of

Encyclopaedia Britannica, Inc., copyright 2003; used with permission.

In response to elevated blood glucose levels, insulin is secreted from beta cells located in

the islets. Insulin travels through the blood stream and binds to insulin receptors, which are

located mainly on hepatocytes, skeletal myocytes, and white adipocytes2. Via an insulin-signaling

cascade, hepatocytes increase their uptake of glucose from the blood and decrease their output of

glucose via gluconeogenesis and glycogenolysis. In response to insulin, skeletal myocytes and

adipocytes increase their rate of glucose transport, resulting in increased glucose uptake2. Overall,

these effects result in decreased blood glucose levels.

In the case of hypoglycemia, glucagon is secreted from alpha cells, located in the islets.

Glucagon is transported in the blood and mainly binds to the glucagon receptors (GR) on

hepatocytes. Via a signaling cascade, this results in an increased output of glucose from the liver

to the blood via glycogenolysis and gluconeogenesis, consequently increasing circulating glucose

levels2.

3

As beta cells and alpha cells are crucial in glucose homeostasis, it is no surprise that they

comprise the majority of cells in the islets, at ~54.6% and ~33.6%, respectively. Delta cells

secrete the hormone somatostatin, which inhibits the secretion of both insulin and glucagon1. PP

cells and epsilon cells secrete the hormones pancreatic polypeptide, and ghrelin, respectively1.

1.2 Diabetes Mellitus

Diabetes mellitus, commonly known as “diabetes”, is characterized by chronically elevated

blood glucose levels due to insufficient insulin production in relation to the body’s need2.

Diabetes seems to have been first described in Egyptian manuscripts dating back to 1500 BC4.

The term “diabetes” is originally Greek for “siphon” or “to pass through”. It was probably coined

by Apollonius of Memphis approximately 250 BC. The word “mellitus” originates from Latin,

meaning “honey” or “sweet”. This term was added to form “diabetes mellitus” by the English

physician Thomas Willis in 16755. The disease was named as such because of the copious amount

of sweet urine produced from its afflicted individuals5,6.

According to the International Diabetes Federation, the global prevalence of diabetes is

estimated to be at least 415 million adults (~9% of adults)7,8. Diabetes has increased in incidence

by ~50% over the past decade. It is expected that, by 2040, the incidence of diabetes will have

risen to 642 million7–9. Although diabetes occurs worldwide, a majority of people with diabetes

live in developing countries. Furthermore, the number of cases of diabetes in developing countries

is increasing, and unfortunately, due to inferior healthcare systems, sufferers of diabetes tend to

have worse outcomes in developing countries, compared with developed countries10–12.

There are three main types of diabetes: type 1 diabetes mellitus, type 2 diabetes mellitus,

and gestational diabetes mellitus. Type 1 Diabetes Mellitus (T1DM) is a disease caused by the

autoimmune-mediated destruction of the insulin-producing beta cells in the pancreas. This

4

eventually results in the inability to produce insulin, causing hyperglycemia to ensue. Although

T1DM can occur at any age, it is typically manifested before the age of 30. As such, it is also

termed juvenile-onset diabetes. T1DM accounts for ~5-10% of all diabetes cases13.

Type 2 Diabetes Mellitus (T2DM) makes up ~85-90% of diabetes cases. T2DM occurs

mainly due to decreased insulin sensitivity (termed “insulin resistance”) in skeletal muscle, liver,

and adipose tissue, in combination with decreased beta cell function (the ability of beta cells to

secrete insulin). The decrease in insulin sensitivity is typically caused by relatively high levels of

free fatty acids (FFA; also called nonesterified fatty acids) and cytokines in plasma. This is

common in overweight and obese individuals, who happen to be ~90% of T2DM patients14. In

response to insulin resistance, beta cells increase their insulin secretion in order to maintain

normal glycemia1. This is termed compensatory hyperinsulinemia since insulin levels must be

elevated above normal levels.

Although hyperinsulinemia can prevent hyperglycemia in the context of moderate insulin

resistance, this does not remain the case after prolonged periods of high plasma FFA levels. This

is because prolonged periods of elevated FFA decrease beta cell function (termed “beta cell

dysfunction”), consequently impairing their ability to compensate for insulin resistance. When

compensation for insulin resistance is no longer sufficient, hyperglycemia ensues. The diagnostic

criteria for prediabetes are: 1) fasting plasma glucose (FPG) levels between 6.1 – 6.9 mmol/L; 2)

plasma glucose between 7.8 – 11.0 mmol/L two hours following a 75g oral glucose tolerance test

(OGTT); and 3) Hb A1c levels between 6.0% to 6.4%15. If hyperglycemia is above these

diagnostic criteria, it is termed T2DM. About one third of cases of prediabetes eventually

progress to T2DM16–18. In T2DM, approximately 4% of the capacity of the beta cell to secrete

insulin is decreased per year19.

5

The third type of diabetes is Gestational Diabetes Mellitus (GDM). Over the past 20 years,

the prevalence of GDM has increased by ~10-100% in several race/ethnicity groups in USA20,21.

GDM occurs to varied degrees in ~7-14% of pregnancies in USA, depending on demographics

and ethnicities21. It is characterized by hyperglycemia, which is caused by increased insulin

resistance during pregnancy, typically during the third trimester. Women who experience GDM

have an elevated risk of acquiring T2DM later in life21–23.

All three types of diabetes share hyperglycemia as an outcome. Accordingly, the most

common symptoms of diabetes are polyuria (frequent urination), polydipsia (frequent thirst), and

polyphagia (excessive hunger)13,14,21. Polyuria and polydipsia are caused by the excessive loss of

water and salts in the form of urine (due to the osmotic effect of glycosuria), and polyphagia is

caused by the calorie loss caused by glycosuria21.

1.3 Complications

Diabetes is associated with a variety of complications. Acute life-threatening complications

include coma, as a result of hypoglycemia (caused by an overdose of exogenous insulin), and

diabetic ketoacidosis (DKA), which occurs during severe hyperglycemia24.

Besides the potentially fatal acute complications, diabetes can have devastating and life-

threatening chronic complications. These typically involve blood vessels, which are damaged

over time by high blood glucose levels (angiopathy)24. Depending on the size of the vessels

damaged, angiopathy is categorized as “microvascular disease” (damaged small blood vessels) or

“macrovascular disease” (damaged arteries)24.

As blood vessels are vital to the function of all organs, angiopathy can result in a variety of

complications. For example, microvascular disease can result in damage to the retina of the eye

(retinopathy), possibly causing blindness after many years. In fact, diabetic retinopathy is the

6

leading cause of blindness among adults aged 20 – 74 years25,26. Diabetic retinopathy can include

capillary microaneurysms and/or degeneration (causing hypoxia, ischemia, and/or macular

edema), altered vascular permeability, and excessive blood vessel growth on the retina

(neovascularization-- which can cause retinal detachment or vitreal hemorrhage)25. Microvascular

disease can also result in kidney damage (nephropathy) and contribute to neural damage

(neuropathy) and limb amputations24.

Diabetic nephropathy can lead to end-stage renal failure. This often occurs gradually over a

period of 10 – 20 years27, and can result in fatal uremia if left untreated28. Furthermore,

nephropathy can amplify the risk for cardiovascular disease due to the resulting increase in

hypertension29, 30.

More than half of all people with diabetes will eventually develop a form of neuropathy31.

Diabetic neuropathy is a progressive disease known to affect the nervous system at both its

somatic and autonomic divisions, in particular the parasympathetic division24. As a result of

microvascular damage, blood flow to nerve fibers can be compromised. After prolonged periods,

hypoxia or ischemia would cause nerve fiber deterioration32. Far more common, however, is a

condition known as “metabolic neuropathy” whereby nerve cells slowly degenerate due to

hyperglycemia. Symptoms may vary depending on which nerves are damaged and the severity of

the damage. Typically, sensitivities to vibrations and/or thermal thresholds are significantly

reduced or lost. However, some individuals may suffer from paresthesia (tingling), hyperalgesia

(increased sensitivity to pain), and/or neuropathic pain32. The lack of pain can be very dangerous

as it places patients at a high risk of unknowingly injuring themselves. Combined with impaired

wound healing due to angiopathy and susceptibility to infection because of hyperglycemia,

neuropathy often leads to amputations24.

7

Due to the acceleration of atherosclerosis in T1DM and T2DM33, people with diabetes have

a six-fold increased risk for myocardial infarction (heart attack), compared with the general

population34. This risk is as high as that of an individual who has previously experienced a

myocardial infarction35. Cardiovascular disease, including myocardial infarction and stroke,

accounts for more than half of the mortality of diabetic populations35,36. Associated with diabetes

is myocardial dysfunction. However, the etiology of myocardial dysfunction is currently unclear

and controversial. It seems to occur independently of atherosclerosis and have a metabolic

causation24,37,38. Lastly, many of the previously mentioned complications tend to be reported as

the cause of death of many diabetic patients. This results in the under-reporting of the mortality of

diabetes per se.

1.4 Risk Factors

T1DM, T2DM, and GDM are caused by different mechanisms. As previously described,

T1DM is caused by the autoimmune-mediated destruction of the insulin-producing beta cells. On

the other hand, T2DM is mainly caused by insulin resistance and beta cell dysfunction, both

typically induced by high plasma levels of FFA. The mechanisms of GDM, though very unclear,

likely involve human placental lactogen39,40. As such, the risk factors for these main forms of

diabetes are different. According to the World Health Organization (WHO), a “risk factor” is

defined as “any attribute, characteristic, or exposure of an individual that increases the likelihood

of developing a disease or injury”41.

The risk factors for T1DM include family history, race, the expression patterns of T1DM-

associated genes (the most important of which are certain Human Leukocyte Antigens (HLA)),

and some viral infections42–45. There is currently no cure for T1DM; however, it can be relatively

well-managed mainly by insulin therapy (exogenous insulin injections due to lack of endogenous

8

insulin production), healthy diet, and exercise43. The risk factors for GDM include age, family

history of GDM, prediabetes, or T2DM, adiposity, and race. While there is also no cure for GDM,

patients tend to recover within weeks after the end of their pregnancy22,46,47. Management of

GDM is focused on the mother and child. It includes monitoring blood glucose levels and the

development of the child, adopting a healthy diet, exercise, and, in some cases, insulin therapy22.

The risk factors for T2DM are many. Similar to the other forms of diabetes, T2DM seems to

be caused by the combination of genetic susceptibility and environmental trigger(s). As such, the

risk factors for T2DM include: overweight/obesity; visceral adiposity (abdominal fat); a family

history of T2DM; inactivity; age; race; prediabetes; GDM; and polycystic ovary syndrome

(PCOS)48. PCOS is a common endocrine disorder among women, where the ovaries are enlarged

and contain small collections of fluid, and features insulin resistance49.

The major predisposing factor for T2DM is obesity, particularly the presence of a large

amount of visceral adipose tissue50–52. Obesity is clinically defined as having a body mass index

(BMI) of at least 30 kg/m2. Visceral adipose tissue has a significantly higher lipolytic activity

than subcutaneous adipose tissue does53. Consequently, visceral adipose tissue is a more potent

elevator of plasma FFA levels, which are the main inducers of insulin resistance and beta cell

dysfunction, compared with subcutaneous adipose tissue16,54–56.

Similar to the other forms of diabetes, there is currently no cure for T2DM. However,

T2DM can be reversible in many cases, particularly in cases caused by obesity57,58. T2DM is

usually reversed by weight loss. This can be achieved through a healthy diet and exercise57,58. In

some cases of severe obesity, bariatric surgery may be performed to induce long-term weight

loss59–64. Nevertheless, it should be noted that ~10% of patients with T2DM are not obese and

require treatments besides weight loss65.

9

Bariatric surgery is considered to be the most effective treatment for obesity59. It can be

performed by: 1) reducing the size of the stomach by using a gastric band; 2) dividing the

stomach into a small “pouch” and a larger part, then resecting and re-routing the small intestine to

the small “pouch” in order to bypass a large portion of the stomach (roux-en-Y gastric bypass); or

3) removing a portion of the stomach (sleeve gastrectomy)59–64. Roux-en-Y gastric bypass, which

is the most common form of bariatric surgery in the United States of America, has a T2DM-

reversal rate of 83%. Gastric band surgery confers a reversal rate of 62%64.

In the cases where weight loss fails to reverse fat-induced T2DM, weight loss remains

beneficial in that it improves insulin sensitivity and beta cell function61,62. This decreases the

severity of the patient’s T2DM, consequently improving blood glucose control. Building on that

point, weight loss, whether by healthy diet and exercise or by bariatric surgery, is a treatment for

T2DM. Other treatments include adopting a healthy diet, exercise (independent of weight loss),

insulin-sensitizing medication (such as metformin, which is the first-line antidiabetic medication,

and thiazolidinediones (TZDs)), insulin secretagogues (such as sulphonylureas and analogues),

incretin-based therapies (such as glucagon-like peptide-1 (GLP-1) and dipeptidyl peptidase-4

(DPP-4)-inhibitors), and insulin therapy in severe cases of T2DM66–68. Some advances in current

research of potential treatments for T2DM are described in the section “Potential Treatments for

Lipid-Induced Beta Cell Dysfunction”.

1.5 Epidemiology

As mentioned earlier, over 415 million adults, from both developed and developing

countries, suffer from diabetes7. In Canada, over 9 million people (~31% of the population) are

estimated to have diabetes or pre-diabetes (Canadian Diabetes Association (CDA), May 201569).

In 2012, over 29 million people in USA were estimated to have diabetes (~9% of the US

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population)70. While these figures are already staggering, we must remember that the actual

prevalence of diabetes is probably greater than estimated due to the numerous cases of diabetes

that go undiagnosed71–73. Lastly, the incidence of diabetes, especially T2DM, has been increasing

dramatically and steadily over the past decades. As stated previously, it is expected that the

number of adults with diabetes worldwide will have will have risen to 642 million (an increase of

over 50%) by 20407.

Since obesity is the major risk factor for T2DM, it is not surprising that the incidence of

T2DM has been following the substantial increase in incidence of obesity65. Besides the obvious

negative effects of T2DM on health, this rise in prevalence of T2DM will put huge strains on

healthcare systems and economies. T2DM already costs the Canadian healthcare system ~$13

billion annually; however, this is expected to rise to ~$17 billion by 2020 (CDA, December

200974). This is due to the high cost of treating chronic diseases, including the expenses for

hospitals, drugs, and treatments for T2DM-associated complications, such as amputations. In

addition, T2DM can cause personal disability, potentially weakening the workforce and economy.

Thus, T2DM is significantly increasing in prevalence and represents a serious and considerable

threat to the health and economies of Canada and the rest of the world. As such, it must be

researched vigorously in order to arrive at potential ways of preventing and treating T2DM with

reliable effectiveness.

1.6 Insulin Secretion

Insulin is a peptide hormone consisting of two polypeptide chains: the A- and B-chains,

which are linked by disulfide bonds75. It is produced and secreted by beta cells in response to

elevated blood glucose levels. Insulin is secreted in a biphasic pattern consisting of an acute

(lasting ~10 min) and prolonged secretion of insulin76. Insulin that had already been synthesized

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is stored in large dense core vesicles (LDCV; termed secretory granules) near the membrane in

the beta cells. When glucose enters the beta cell via the glucose transporters GLUT1 (in

humans77,78) and GLUT2, it is metabolized, ultimately causing a signaling cascade which induces

the exocytosis of the insulin granules79,80,77,81. This is the acute phase of insulin secretion, which

responds quickly to increases in glycemia76.

In detail, the mechanism by which glucose metabolism induces insulin secretion is as

follows (Figure 2): after entering the beta cell, glucose is metabolized to produce adenosine

triphosphate (ATP) via glycolysis and the tricarboxylic acid (TCA) cycle. The rise in cellular

ATP levels causes the closure of KATP channels, which results in the build-up of K+ and

depolarization of the cell membrane. This depolarization causes voltage-dependent Ca2+ channels

(VDCC) to open, allowing an influx of Ca2+ into the beta cell. The rise in cytosolic Ca2+ levels

then induces the exocytosis of insulin granules76. While glucose is the primary stimulant of

insulin secretion, insulin secretion can also be induced by other means. These include a number of

amino acids, acetylcholine (secreted by the peripheral nervous system), gastric inhibitory

polypeptide (GIP), and glucagon-like peptide (GLP), the latter two being incretin hormones

secreted by the gut following food ingestion82.

Acetylcholine binds to the M3 muscarinic acetylcholine receptor, and GIP and GLP bind to

the gastric inhibitory polypeptide receptor (GIP-R) and glucagon-like peptide 1 receptor

(GLP1R), respectively. The M3 muscarinic acetylcholine receptor, GIP-R, and GLP1R, the three

of which are expressed on beta cells, enhance insulin secretion82,83. Interestingly, FFAs, such as

oleate, have specific receptors on a wide variety of cells in the body, including beta cells. These

receptors, known as Free Fatty Acid Receptors (FFARs), are G-protein-coupled receptors

(GPCRs) which act as signaling molecules in many physiological processes related to energy

metabolism84–87. FFARs are classified according to the chain-length of their FFA ligands. With

12

regards to FFARs found on beta cells, medium- and long-chain FFAs, such as oleate, are known

to activate G-protein-coupled receptor 40 (GPR40), whereas short-chain FFAs activate GPR41

and GPR4385,88. Derivatives of FFAs have a specific FFAR on beta cells, namely GPR11989.

GPR40 and GPR119 have positive effects on insulin secretion, and thus may explain the acute

stimulatory effect of FFAs on insulin secretion84,87,89. Although the roles of GPR41 and GPR43 in

beta cells are at present not completely clarified, GPR41 appears to be mainly inhibitory and

GPR43 appears to be mainly stimulatory90.

Figure 2. Summary of the mechanism of glucose-stimulated insulin secretion (GSIS) in the beta cell.

Glucose enters the cell via GLUT2 transporters. Glucose is then metabolized via glycolysis and the

tricarboxylic acid (TCA) cycle, resulting in the production of ATP. The rise in intracellular ATP levels

results in the closure of KATP channels, which leads to the depolarization of the cell membrane. This

depolarization then stimulates voltage-dependent Ca2+ channels (VDCC), resulting in an influx of Ca2+ into

the cytosol. The increase in intracellular Ca+ concentration induces the exocytosis of insulin secretory

granules located at the membrane. Figure adapted from Desai T., M.Sc. Thesis, Dept. of Physiology,

University of Toronto, 2013.

Once the store of insulin granules is secreted, the beta cells must synthesize and secrete

additional insulin. This is a relatively slow process which is rate-limited by the synthesis of

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insulin91. This is the second phase of insulin secretion. Insulin is first translated in the form of a

single polypeptide termed preproinsulin. Preproinsulin contains a 24-residue signal peptide that

directs the polypeptide to the rough endoplasmic reticulum (RER). In the RER, preproinsulin is

cleaved to form proinsulin, which is then folded into the correct conformation. This includes the

formation of disulfide bonds. Subsequently, proinsulin is transported to the Trans-Golgi Network

(TGN), where it is cleaved by Proprotein Convertase (PC) 1 and 2 to release the two chains of

insulin along with C-peptide. At this point, insulin and C-peptide are ready to be secreted from

the beta cells upon proper stimulation91. As C-peptide is always co-secreted with insulin, the

concentration of C-peptide in plasma is correlated with that of insulin. As such, it is often

assessed as an index of insulin secretion92–95.

The autocrine effects of insulin on beta cells have been a controversial topic for over a

decade96,97. Although insulin signaling in beta cells was first suggested to negatively regulate

insulin secretion98–100, recent evidence depicts a positive effect on insulin transcription,

translation, and secretion, as well as beta cell survival101–104.

1.7 Insulin Clearance

While insulin action is vital for glucose homeostasis, excess insulin action can result in

hypoglycemia and possibly death. As such, besides regulating the secretion of insulin, the body

modulates insulin levels by removing insulin from the blood plasma. This is termed insulin

clearance. It is mainly performed by the liver and to a lesser extent by the kidneys105.

While increases in insulin secretion elevate circulating insulin levels, decreases in insulin

clearance can also produce this effect106. This is evident in certain liver diseases where impaired

insulin clearance results in hyperinsulinemia, which, after chronic periods, can induce peripheral

insulin resistance107. Insulin resistance can be induced by chronic hyperinsulinemia via the

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downregulation of insulin receptors in the cell membrane as a result of the internalization of the

insulin-insulin receptor complex as well as the impairment of synthesis of the insulin receptor.

Hence, insulin clearance plays an important role in the regulation of insulin action and, in turn,

glucose homeostasis108.

Insulin clearance occurs through receptor-mediated insulin endocytosis followed by

degradation108–110. Approximately 50% of circulating insulin is removed during its first passage

through the liver111. This takes place via the endocytosis and degradation of the insulin-insulin

receptor complex in hepatocytes. While insulin clearance mainly occurs via the liver, insulin in

the systemic circulation is removed to a significant extent by the kidneys.

The insulin clearance rate by the liver is known to be proportional to portal insulin

concentrations112,113 and inversely proportional to portal FFA concentrations in rats114–116.

Elevated levels of plasma FFA are well-known to decrease insulin clearance114–117. FFAs are

known to impair insulin clearance at two main levels. First, by inducing hepatic insulin resistance,

insulin signaling is decreased, resulting in decreased activation of carcinoembryonic antigen-

related cell adhesion molecule 1 (CEACAM1; formerly known as pp120) by the insulin-insulin

receptor complex activity118. CEACAM1 is a transmembrane glycoprotein that is responsible for

the endocytosis of the insulin-insulin receptor complex (described below)118–120. Second, as

insulin action is known to increase CEACAM1 transcription, decreased insulin signaling may

reduce CEACAM1 levels121. In addition, FFAs taken up by the liver are mainly esterified to form

triglycerides. Afterwards, this pool of triglycerides is used in several metabolic processes,

including fatty acid oxidation122. Hepatic triglyceride levels have been shown to be inversely

proportional to insulin clearance123.

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1.7.1 Mechanisms of Insulin Clearance

Hepatic insulin clearance occurs via the internalization of the insulin-insulin receptor

complex, which is mediated by the tyrosine autophosphorylation of the insulin receptor upon

insulin binding. The tyrosine-phosphorylated insulin receptor subsequently phosphorylates

CEACAM1124,125. Via clathrin-coated pits, the insulin-insulin receptor complex is internalized

into a clathrin-coated vesicle and subsequently an endosome. Insulin degradation begins in the

acidic environment of the endosome by the action of Insulin Degrading Enzyme (IDE; also

known as insulysin). The endosome is then processed into the late endosome where insulin is

further degraded. Eventually, the late endosome returns to the plasma membrane by diacytosis in

order to recycle the insulin receptor and exocytose the degradation products of insulin126.

Alternatively, the late endosome may transition to a lysosome, where insulin and the insulin

receptor are both degraded127. A basic illustration of the process of insulin clearance by the

hepatocyte is summarized below in Figure 3.

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Figure 3. Summary of the process of insulin clearance in the hepatocyte. After insulin binds

to the insulin receptor, it triggers the tyrosine autophosphorylation of the insulin receptor. The

insulin receptor, in turn, tyrosine phosphorylates carcinoembryonic antigen-related cell adhesion

molecule-1 (CEACAM1; also known as pp120), resulting in its activation. CEACAM1 then

induces the internalization of the insulin-insulin receptor complex into an endosome. In the acidic

endosome, IDE begins degrading insulin. The endosome is then processed into a lysosome, where

insulin and the insulin receptor are completely degraded. Alternatively, the late endosome fuses

with the plasma membrane, resulting in the recycling of the insulin receptor and the exocytosis of

the degradation products of insulin. Figure modified from Duckworth W.C. et al. 1998. Endocr

Rev. 19(5), 608–624.

In kidneys, insulin clearance occurs via two mechanisms: 1) glomerular filtration followed

by proximal tubular reabsorption and degradation, and 2) diffusion of insulin from peritubular

capillaries followed by the binding of insulin to contraluminal membranes of tubular cells and the

endocytosis of insulin128–131. Glomerular filtration of insulin occurs by nonspecific diffusion and

by specific-receptor-mediated transport. More than 99% of insulin that enters the renal tubule is

reabsorbed by proximal tubule cells, mainly via endocytosis132. Hence, almost no insulin is

excreted in the urine of healthy individuals. However, insulin that is endocytosed by the proximal

tubule cells is processed for degradation in a manner similar to that in the hepatocyte, which is

described above129. Nevertheless, while insulin in the proximal tubule cell proceeds through the

17

endosomal complex, it is transferred to lysosomes much earlier than in hepatocytes128. In

addition, some insulin is released intact into the circulation via retroendocytosis131.

CEACAM1 was originally discovered as a substrate of the insulin receptor tyrosine kinase

in rat hepatocytes124. While CEACAM1 protein is ubiquitously produced, it is predominantly

expressed in the liver and has limited expression in skeletal muscle and white adipose tissue

(WAT)108,133. CEACAM2, a less expressed isoform of CEACAM1, plays a role in the regulation

of metabolic rate and insulin sensitivity and is mainly expressed in the brain, spleen, kidneys, and

testes134,135. CEACAM2 is not expressed in peripheral insulin-targeted tissues, such as the liver,

skeletal muscles, and WAT134,135. In addition, only CEACAM1 is expressed in humans and

rats134. Although CEACAM1 is significantly expressed in beta cells, CEACAM1-null mice

exhibited normal beta cell area and insulin secretion in response to glucose in vivo and in isolated

islets111.

CEACAM1 is known to be highly conserved among different species108. It has been

suggested that tyrosine-phosphorylated CEACAM1 reduces the mitogenic effects of insulin by

binding with Shc, which is another substrate of the insulin receptor. This results in the

sequestration of Shc, preventing it from coupling Grb2 to the insulin receptor and thus results in

the downregulation of the Ras/MAP kinase mitogenesis pathway136,137. In addition, CEACAM1 is

known to act as a cell adhesion molecule, mediating Ca2+-dependent and Ca2+-independent cell

adhesion138. CEACAM1 has also been reported to suppress tumor growth139 and to mediate

angiogenesis140. Phosphorylation of CEACAM1 is necessary for its activation, with the exception

of its role in cell adhesion16,141–143.

IDE is a highly conserved Zn2+-dependent endopeptidase that plays a key role in the

degradation of insulin in endosomes144,145. IDE was first described as “insulinase” by Mirsky and

Broh-Kahn in 1949146. The expression of IDE is highest in the cytosol followed by the

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peroxisomes of cells144,147–149. Yet, in regards to concentration, one study, using immunoelectron

microscopy, reports that IDE is ~3.5 times more concentrated in peroxisomes than it is in the

cytosol of rat hepatocytes (due to the relatively large volume of the cytosol), suggesting that

peroxisomes contain 10-20% of total IDE147. However, the precise difference of IDE

concentration between the two subcellular compartments is unclear because, using immunoblot

analysis, the same study found similar concentrations of IDE in the peroxisomal and cytosolic

fractions147. This difference may have been in part due to leakage of protein from the fragile

peroxisomes during homogenization147,150.

Overexpression of IDE has been shown to increase the rate of insulin degradation by cells,

supporting the significance of IDE in insulin degradation151. However, while IDE is important for

insulin proteolysis, a 2015 study published in the Journal of Biological Chemistry suggests that

IDE may not be necessary for the clearance of insulin from blood plasma152. Utilizing novel IDE

inhibitors which do not bind with IDE’s catalytic Zn domain (and hence do not need to compete

with IDE’s substrates), the authors of this study performed insulin tolerance tests and euglycemic

clamps on High Fat Diet (HFD)-fed obese mice152. The authors found that plasma insulin levels

and insulin action were not significantly altered, compared with control mice in vivo.

It is possible that insulin clearance during inhibited IDE activity was not affected because of

the internalization of insulin by CEACAM1, which effectively removes insulin from the blood

plasma even if the downstream IDE action is impaired. Alternatively, it is possible that, in the

HFD model, insulin clearance was already impaired by the elevated plasma FFA levels. A study

by the lab of Dr. Malcolm A. Leissring (University of California, Irvine) reported that whole-

body IDE-null mice exhibited ~3-fold higher fasting levels of insulin in plasma and improved

glycemic control at 2 months of age, compared with wildtype mice; however, at 6 months of age,

IDE-null mice exhibited severe insulin resistance and glucose intolerance due to their chronic

19

exposure to extremely high plasma levels of insulin (caused by the complete genetic deletion of

IDE)153.

Unpublished data from Dr. Leissring’s lab show that liver-specific IDE-KO mice do not

exhibit a significant difference in insulin clearance, compared with wildtype mice. This may have

been due to a compensatory increase in insulin clearance by the kidneys or due to the extent of

IDE knockout in the liver. Thus, the magnitude of the effect of IDE on insulin clearance is

currently controversial.

In addition to degrading insulin, studies based on pharmacological inhibition of IDE show

that IDE degrades amylin (also known as islet amyloid polypeptide)154–157. Amylin is a 37-residue

peptide hormone that is co-secreted with insulin by beta cells. Pharmacological levels of amylin

help suppress glucagon secretion, delay gastric emptying, and induce satiety158–160. Physiological

levels of amylin have been shown to contribute to the regulation of glucagon release and gastric

emptying161–164.

In conclusion, insulin clearance is an important process in the regulation of the levels and

action of insulin in the body. It occurs mainly by the liver and kidneys, primarily by CEACAM1-

mediated endocytosis followed by degradation in peroxisomes by the action of IDE.

1.8 Fat-Induced Beta Cell Dysfunction

Many studies have shown that acute exposure of beta cells to elevated FFA levels results in

increased glucose and non-glucose-stimulated insulin secretion in vitro165,166 and in vivo in

animals167 and humans168–171. The responsible mechanisms (which include the interactions of

FFAs with FFARs) have been extensively reviewed84,86,87,89,172,173. In contrast, prolonged

exposure of beta cells to elevated FFA levels impairs glucose-stimulated insulin secretion (GSIS)

in vitro. There are comprehensive reviews outlining the molecular mechanisms of fat-induced

20

beta cell dysfunction172,173. In vivo studies assessing absolute GSIS after prolonged i.v. infusion of

fat showed inconsistent results, namely increased174,175, unchanged168,176–184, or decreased170,185

absolute GSIS in humans and rats.

There are several explanations for the differences observed in these in vivo studies. First, the

assessment of absolute GSIS does not account for insulin resistance induced by elevated plasma

levels of FFA. Second, the compositions of triglyceride or fatty acid infusates administered in

each study were not necessarily similar. Third, a number of human studies have demonstrated that

genetic predispositions to diabetes greatly influence the effect of FFA on beta cell function. The

detrimental effects of FFA on beta cell function and/or viability fall under the category of beta

cell lipotoxicity186–188. In addition, chronic hyperglycemia is also known to cause beta cell

dysfunction. This is termed glucotoxicity189–191.

1.8.1 Methods of Assessing FFA-Induced Beta Cell Dysfunction

As explained earlier, beta cells increase insulin secretion during insulin resistance

(compensatory hyperinsulinemia) in order to maintain proper glucose homeostasis1. As insulin

sensitivity decreases, insulin secretion increases, and as insulin sensitivity increases, lower plasma

levels of insulin are required, resulting in decreased insulin secretion. Since chronically high FFA

levels induce insulin resistance as well as beta cell dysfunction, absolute GSIS is not a reliable

assessment of beta cell function. This is because compensatory hyperinsulinemia induced in

response to insulin resistance could mask a decrease in beta cell function.

As the relationship between insulin secretion and insulin sensitivity is hyperbolic, their

product is a constant. This constant, first published by Toffolo G., Bergman R.N., and Cobelli C.

in Diabetes192, is termed the Disposition Index (DI; Figure 4). The DI is the current reliable index

of beta cell function in vivo193. This is because the DI expresses beta cell function while

controlling for potential changes in insulin sensitivity.

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Figure 4. The Disposition Index (DI) is the product constant of insulin secretion and insulin

sensitivity. The arrow depicts a decrease in the DI as beta cell function is decreased. This is demonstrated

by the fact that for the same insulin sensitivity, insulin secretion is decreased. Figure modified from

Stumvoll M. et al.194.

In all of Dr. Giacca’s collaborative studies on humans, aside from one185, 48-h intravenous

fat infusions did not affect absolute GSIS. However, lipid infusions for 16- to 48-h impaired DI,

indicating that the ability of beta cells to compensate for insulin resistance had been

diminished168,176,177,180,181,183–185. During hyperglycemic clamp studies, these collaborative studies

found that the glucose infusion rate (GINF) required for maintaining hyperglycemia (an index of

glucose tolerance) was lower in humans infused with lipids for 48h, compared with saline

controls. In another study, a graded i.v. glucose infusion, rather than a hyperglycemic clamp, was

performed following prolonged fat infusion. This resulted in slightly elevated blood glucose

levels, further demonstrating that prolonged fat infusion causes beta cell dysfunction16. Beta cell

dysfunction usually increases in severity with the duration that beta cells are exposed to high

levels of FFA187. After very long periods of beta cell lipotoxicity, such as high-fat feeding in

rodents or T2DM, beta cell death via apoptosis can occur195,196.

In summary, acute elevation of plasma FFA levels results in increased beta cell function,

whereas chronic elevation of plasma FFA levels results in decreased beta cell function. If beta cell

22

dysfunction is severe enough to prevent insulin secretion from compensating for FFA-induced

insulin resistance, T2DM would manifest.

1.8.2 Effects of Different Lipid Treatments on Beta Cell Dysfunction

Several FFA and lipids are used to model FFA-induced beta cell dysfunction. In humans

and rodents, the two most abundant fatty acids present in blood plasma are oleate and

palmitate197. Oleate is a monounsaturated fatty acid (MUFA) whereas palmitate is a saturated

fatty acid (SFA). Compared with non-obese individuals, obese individuals have 1.5 – 2 fold

greater levels of both fatty acids and a higher ratio of SFA to MUFA198,199. The chronic elevation

of either of those fatty acids has been shown to result in beta cell

dysfunction168,176,177,180,181,183,185,200.

Our studies suggest that the in vivo elevation of MUFA in rats is most effective in causing

beta cell dysfunction, compared with SFA and polyunsaturated fatty acid (PUFA) infusion201,202.

There is currently no convincing evidence that this is true in humans203.

Models of FFA-induced beta cell dysfunction have included the elevation of oleate and/or

palmitate plasma levels in animals in vivo. This is often done by i.v. infusion of albumin-bound

oleate, as has been done in a number of our studies56,201,204,205. This is not feasible with palmitate

due to its toxicity and low solubility. More recently, it has been possible to non-toxically elevate

plasma palmitate concentrations in vivo. This method consists of i.v. infusing ethyl palmitate

instead of palmitate200. The ethyl moiety which is esterified to the carboxylic end of the fatty acid

abolishes the fatty acid’s detergent action, and thus its toxicity. Plasma esterase present in the

blood plasma act on ethyl palmitate to release palmitate and ethanol into the circulation.

Another method of elevating plasma FFA levels is by i.v. infusion of a fat emulsion with

heparin. Conventional fat emulsions that can be infused in humans include Intralipid and Liposyn,

23

which mainly contain unsaturated triglycerides206–208. Heparin is co-infused in order to release

lipoprotein lipase (LPL) into the blood stream in order to hydrolyze the triglycerides into FFA.

A third common model of FFA-induced beta cell dysfunction is the chronic administration

of a HFD (typically > 45% of energy by fat) to rodents, as opposed to a standard diet (typically <

20% fat209–211. Although this model of diet-induced obesity is common and convenient, it has

several limitations. A HFD model is non-selective in regards to circulating FFA and involves the

expansion of cytokine-releasing adipose tissue and the secretion of gut hormones, both of which

affect glucose metabolism82.

Lastly, numerous studies have assessed beta cell function in vitro by exposing beta cell

models or isolated islets to cell culture media containing specific fatty acids205,212–217. Fatty acids,

such as oleate or palmitate, are complexed with Bovine Serum Albumin (BSA) in the culture

media in order to simulate in vivo conditions and to prevent the toxic effect of excess unbound

FFA. These in vitro models facilitate mechanistic studies.

1.8.3 Genetic Predisposition to Lipid-Induced Beta Cell Dysfunction

A study found that having a positive family history of T2DM was correlated with increased

susceptibility to lipid-induced beta cell dysfunction218. This was also found to be the case for

glucose-intolerant first-degree relatives of patients with T2DM182.

A collaborative study by our lab examined genetic susceptibility to lipid-induced beta cell

dysfunction in the Sandy Lake Oji-Cree community of Ontario, Canada176, which has the third

highest prevalence of T2DM in the world219. Counter-intuitively, our lab found that nondiabetic

subjects with a family history of T2DM experienced a lower decrease in beta cell function

following a 2-day lipid infusion, compared with non-Oji-Cree subjects. This was assessed using

the DI. Although the Oji-Cree subjects exhibited a lower decrease in beta cell function, their

overall beta cell function was less than that of non-Oji-Cree subjects. Thus, Oji-Cree subjects

24

seemed to have had impaired beta cell function prior to the infusion of lipid. Some of the

genetically susceptible individuals may normally have relatively high plasma levels of FFA, as

decreasing FFA levels improved insulin secretion in the Oji-Cree population220–222.

Further collaborative work by our lab demonstrated that Caucasians with a family history of

T2DM tend to have higher levels of FFA during i.v. Intralipid + Heparin (IH) infusion due to

enhanced FFA spillover into the plasma223. Physiologically, triglycerides infused into blood

plasma are broken down by LPL primarily in the capillaries of muscles and adipose tissue,

releasing FFA and glycerol224. The released FFA are normally taken up and esterified

immediately within the organs, resulting in very little amounts of FFA remaining in the plasma. In

conditions where FFA esterification is impaired, plasma FFA levels are elevated above

physiological levels. This is termed spillover225. FFA spillover is considered a measure of

inefficiency in dietary fat storage.

Given the recent discovery of many genetic variants associated with T2DM226, it may be

worth investigating which genetic variants increase susceptibility to lipotoxicity. For instance, it

has been shown that some genetic variants of the KATP channel increase susceptibility to T2DM

by having a greater sensitivity to opening in response to FFA in vitro227. The opening of the KATP

channel allows K+ to flow out of the beta cell, causing a shift towards hyperpolarization of the cell

membrane. This increases the barrier that must be overcome for the cell membrane to depolarize

and induce insulin secretion227.

1.8.4 Beta Cell Replenishment

Beta cell populations are mainly regulated by cell proliferation and cell death. In adult

humans, the rate of proliferation of beta cells is very low228,142. Beta cells arise from pre-existing

beta cells, pancreatic cells, and extra-pancreatic cells. In rodents, the main source of new beta

cells seems to be replication from pre-existing beta cells. Secondary sources of new beta cells in

25

rodents are pancreatic cells (mainly duct cells), whereas extra-pancreatic cells including

stem/progenitor cells, although capable of generating beta cells, do not normally contribute to

beta cell replenishment. In humans, beta cells appear to mainly originate from non-beta cells in

the pancreatic duct due to limited beta cell replication, particularly in adulthood229.

In metabolic states of increased insulin demand, beta cells may adapt by increasing in size

and/or number1. However, after chronic periods of T2DM, beta cell mass is decreased, further

exacerbating hyperglycemia. Beta cell mass is reduced mainly through apoptosis, although

necrosis, autophagy, and ferroptosis may also cause cell death1.

The discovery that lipid-induced apoptosis occurs in vivo is important since in vitro

experiments involving beta cell incubation in media containing albumin-bound FFA can simulate

in vivo FFA effects to a limited extent. This is because beta cell death in vitro may be due to the

toxic effect of excess unbound fatty acids, which does not occur in vivo. Fatty acids released from

adipose tissues are immediately buffered by the great amount of albumin in plasma.

1.8.5 Effect of FFAs on Beta Cell Mass

Although a 16-20 week HFD diet administration has been shown to increase insulin

resistance and absolute GSIS in rodents230,231, a chronic HFD ultimately results in glucose

intolerance. The increased GSIS is attributed to increased beta cell function and/or mass230,231.

Nevertheless, the final outcome of glucose intolerance demonstrates the failure of beta cells to

compensate for insulin resistance following a chronic HFD.

Fat-induced beta cell failure is typically due to beta cell dysfunction and/or beta cell

death231,232. Beta cell dysfunction typically precedes the decrease in beta cell mass174,233. This

decrease in beta cell mass tends to occur because of beta cell apoptosis230,231,234–237 and beta cell

senescence237. Thus, although absolute GSIS and beta cell mass are increased after a chronic

26

HFD, the effects of lipotoxicity on beta cells result in beta cell failure. Consequently, glucose

intolerance ensues.

1.9 Glucotoxicity and Glucolipotoxicity

In addition to having elevated plasma levels of FFA, individuals with prediabetes or T2DM

have high levels of blood glucose. Chronic hyperglycemia is known to lead to beta cell

dysfunction through a process termed glucotoxicity190,238. A recent study in our lab suggests that

high-glucose-induced beta cell dysfunction in vivo mainly occurs through oxidative stress,

endoplasmic reticulum (ER) stress, and the stress-activated enzyme c-Jun-N-terminal kinase

(JNK)239. This study utilized a pharmacological model and a genetic model: 1) inhibition of JNK

by administering SP600125 to rats, and 2) JNK1-null mice i.v. infused with glucose or saline.

SP600125 protected against high-glucose-induced beta cell dysfunction without significantly

decreasing total and mitochondrial superoxide levels. The administration of the antioxidant

tempol with the chemical chaperone sodium phenylbutyrate (which reduces ER stress) prevented

the activation of JNK by high glucose.

There are many studies, including one done by our lab240, which implicate oxidative stress

in high-glucose-induced beta cell dysfunction both in vitro241–244 and in vivo240,245,246. According

to one study, high-glucose-induced beta cell dysfunction stems from decreased insulin gene

expression caused by a defect in the mRNA maturation of pancreas duodenum homeobox-1 (Pdx-

1), which is an important insulin transcription factor238. The study discovered an absence of Pdx-1

in glucotoxic beta cells, and demonstrated an increase in insulin promoter activity after Pdx-1

transfection into glucotoxic beta cells238.

The combined toxicity of elevated plasma fatty acid and glucose levels on beta cells is

termed glucolipotoxicity. Glucolipotoxicity has been demonstrated in many in vitro studies and is

27

thought to be more detrimental to beta cell function than lipotoxicity and glucotoxicity are

alone247,248. Glucolipotoxicity has been extensively documented in vitro173; however, in vivo

studies of glucolipotoxicity are far less common.

In one in vivo study, 48h i.v. co-infusion of Intralipid + Heparin (IH) and glucose in

dexamethasone-treated rats induced greater beta cell dysfunction than when IH or glucose were

each infused alone248. In another study, the co-infusion of IH and glucose in normal rats resulted

in a synergistic effect, i.e., a significant decrease in insulin gene transcription which was not

observed after infusion of IH or glucose alone249. This supports the theory that glucolipotoxicity

tends to be a more potent cause of beta cell dysfunction, compared with glucotoxicity or

lipotoxicity.

In humans, prolonged hyperglycemia has been shown to result in beta cell dysfunction250.

However, experimentally inducing prolonged hyperglycemia is usually not performed on humans

due to inflammation of the i.v. site, swelling, nausea, and electrolyte imbalance. Nevertheless,

there have been a few studies investigating glucolipotoxicity in humans. In a collaborative study

by our lab, IH was co-infused with glucose (achieving 7.5 mM glycemia) in humans for 24h180.

Although this resulted in beta cell dysfunction, its severity was not greater than that induced by

IH infusion alone. Thus, this did not manifest the effects of true glucolipotoxicity. Interestingly,

the 24h infusion of glucose alone (achieving 7.5 mM glycemia) enhanced beta cell function.

However, it is important to note that 7.5 mM glycemia is a mild elevation of blood glucose, and

that a greater degree of hyperglycemia, when prolonged, is known to cause beta cell

dysfunction251,252,190.

In summary, although glucolipotoxicity has been clearly demonstrated in vitro173 and in

animal models in vivo249, it has not been sufficiently demonstrated in humans likely due to the

limitations in experimentally elevating glycemia.

28

1.10 Mechanisms of FFA-Induced Beta Cell

Dysfunction

FFA-induced beta cell dysfunction has been widely studied in vitro; however, it has been

far less studied in vivo173. As a result, many of the mechanisms discovered in in vitro studies have

not been tested in vivo, especially in humans. Mechanisms for beta cell dysfunction and death

which are supported by in vitro and in vivo studies include FFA-induced oxidative stress177,212,253,

ER stress184, and inflammation230,254,255.

1.10.1 The Role of Oxidative Stress

In beta cells, Reactive Oxygen Species (ROS) are mainly produced in mitochondria via

the oxidation of glucose and, to a lesser extent, via oxidation of FFA. More specifically, ROS are

produced downstream of glucose and fatty acid oxidation as a product of the electron transport

chain (ETC). This form of ROS is mainly superoxide (O2•-)256,257. FFA lead to increased ROS

production by directly disrupting the mitochondrial membrane composition, resulting in

decreased efficiency of oxidative phosphorylation and an increase in ROS production258.

In addition, oxidation of FFA in the peroxisome plays a role in ROS production via the

generation of H2O2256. While mitochondria primarily oxidize small- to long-chain fatty acids,

peroxisomes tend to oxidize very long-chain fatty acids187,256. High levels of plasma FFA are

known to induce the shifting of the energy pathway towards the oxidation of FFA and away from

glucose oxidation and glycolysis259.

Recently, ROS has been shown to be produced in beta cells via plasma membrane

Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase187,260. NADPH oxidase is a

membrane-bound enzyme complex that is found on the plasma membrane and on membranes of

phagosomes in neutrophils. It is responsible for the generation of superoxide during phagocytosis

of microorganisms187. A study done by our lab demonstrated that NADPH-oxidase-derived

29

cytosolic superoxide is increased following 48h of oleate i.v. infusion in mice56. This study also

made the valuable finding that pharmacological and genetic whole-body inhibition of NADPH

oxidase decreases superoxide levels and prevents oleate-induced beta cell dysfunction. In

addition, studies have shown increased concentrations of components of NADPH oxidase in islets

of animal models of T2DM126 and in beta cell lines treated with palmitate for 24h261. As NADPH

oxidase is activated by elevated levels of Protein Kinase C (PKC), cytokines, and increased

activity of toll-like receptor-4 (TLR-4; which is activated by saturated FFA200,262), NADPH

oxidase can be a significant source of ROS production in beta cells exposed to high levels of

FFA56,263.

Oxidative stress is defined as an elevation in levels of intracellular ROS to a level that

begins to cause damage to the cell242,264,265. This typically occurs when the production of ROS is

beyond the cell’s ability to decrease ROS levels. Being highly reactive, ROS can cause damage to

DNA, enzymes, and membranes in cells, resulting in dysfunction of the cells, and in some cases,

cell death266. Patients with T2DM have been shown to exhibit 5-fold higher levels of markers of

ROS in their beta cells, compared with healthy controls267. Markers of oxidative stress include

malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), which are indicators of

lipid peroxidation and oxidative DNA damage, respectively268.

Beta cells are particularly susceptible to dysfunction via oxidative stress. Normally, cells

use antioxidants in order to control their levels of ROS. These include manganese superoxide

dismutase (MnSOD), glutathione, and catalase, to name a few257. However, beta cells have

significantly lower levels of antioxidants, compared with most cells, and are therefore very

susceptible to damage caused by increased ROS141,269.

Since beta cells have a limited capacity to store triglycerides189,143,188, exposure to high

levels of FFA tends to result in the metabolism of excess FFA in beta cells including their

30

conversion into toxic FFA esterification products, such as diacylglycerol (DAG) and ceramides.

This leads to increased ROS production188. As the very low rate of beta cell proliferation renders

beta cells susceptible to significant loss of beta cell mass, chronic oxidative stress could lead to

the inability of beta cells to compensate for insulin resistance, thus resulting in T2DM. Overall, it

is clear that the intrinsic physiology of beta cells renders them very vulnerable to FFA-induced

oxidative stress.

In beta cells, oxidative stress has been consistently shown to be causal in beta cell

dysfunction, which is the impairment of the ability of the beta cell to synthesize or secrete

insulin242,264,265. In collaboration with Dr. Michael B. Wheeler’s lab, our studies have shown that

the antioxidant taurine protects GSIS of both rat islets and MIN6 beta cells exposed to FFA for

48h270. Our lab found that the antioxidants N-acetylcysteine (NAC), taurine, and tempol

prevented beta cell dysfunction in oleate-infused rats in vivo, in vitro, and ex vivo212. The in vivo

assessments of beta cell function utilized hyperglycemic clamps, whereas the ex vivo experiments

measured insulin secretion from isolated islets of i.v. infused rats. Our lab’s results are consistent

with another study, which showed that NAC administration prevented beta cell dysfunction in rats

infused with IH over 96h253.

As mentioned earlier, inhibition of NADPH oxidase with apocynin as well as its deletion

in NADPH oxidase-null mice prevented oleate-induced beta cell dysfunction56. In addition, a

recent study in our lab has shown that elevated levels of lipid peroxides are causal to FFA-

induced beta cell dysfunction in vivo and ex vivo (isolated islets obtained from mice i.v. infused

with treatments)205. Our study utilized mice overexpressing Glutathione Peroxidase-4 (GPx4),

which is an enzyme that specifically reduces lipid peroxides, and wildtype control mice. After i.v.

infusion with saline or oleate for 48h, dichlorodihydrofluorescein diacetate was used to image

levels of ROS in isolated islets. Alternatively, beta cell function was assessed in vivo using the

31

hyperglycemic clamp method or ex vivo via an insulin secretion assay (measurement of insulin

secretion after incubating islets in glucose-containing media for 2h).

A potential mechanism by which ROS can decrease insulin synthesis is by decreasing the

levels and activities of the transcription factors Pdx-1 and MafA264,271. This was suggested by

studies which showed that the antioxidant NAC prevented the ROS-induced decrease of Pdx-1

and MafA expression in HIT-T15 cells (beta cell line) or human islets incubated in high glucose

concentrations in vitro244–246,264. One of these studies also found that NAC protected against a

ROS-induced decrease in insulin expression and secretion in vivo246. Pdx-1 is important for the

transcription of insulin, whereas MafA plays a role in beta cell growth and maturation as well as

insulin transcription272. Oxidative stress can decrease insulin transcription by activating the

general stress-related enzymes JNK and NFκB (nuclear factor kappa-light-chain-enhancer of

activated B cells)273. This is known to disrupt the process of insulin secretion. The responsible

mechanisms will be discussed later in my thesis. This can occur via inflammatory pathways,

eventually implicating forkhead box-O (FOXO) proteins in insulin signaling265.

In pancreatic beta cells, ROS can also decrease GSIS via mitochondrial dysfunction, which

can consist of the inhibition of the Na+/Ca2+ exchanger (NCLX) directly by ROS in vitro274,275.

This prevents the transport of Ca2+ in mitochondria, causing a decrease in ATP production. As

insulin secretion depends on ATP, it may also be decreased as a consequence.

A decrease in ATP levels can also occur via the ROS-mediated activation of uncoupling

protein 2 (UCP2). Uncoupling protein 1 (UCP1; also known as thermogenin) has been clearly

shown to be responsible for the uncoupling of the ETC and ATP synthesis, resulting in decreased

ATP synthesis and increased thermogenesis257,276,276,277. However, the role of UCP2 in beta cells

is not clear. Studies regarding the role of UCP2 in beta cells will be described in section “Role of

SIRT1 in Beta Cells”.

32

Severe ROS damage can result in the death of the beta cell via apoptosis. ROS-induced

apoptosis is usually the result of mitochondrial damage that allows the release of proapoptotic

factors such as cytochrome c278. In sufficiently high levels, these proapoptotic factors activate a

signaling cascade that leads to apoptosis256,278.

While high levels of ROS have been shown to cause beta cell dysfunction and beta cell

death in some cases, it is important to keep in mind that a certain level of ROS is necessary for the

proper functioning of all cells, including beta cells266. Depending on the type of cell, there is a

level of ROS that is optimal to its function. It has been shown that exposure of beta cells to high

levels of antioxidants results in a decrease in GSIS266. Furthermore, some studies have found that

ROS can serve as important signaling molecules in insulin secretion257,266. For instance, a

transient increase in intracellular ROS levels has been shown to promote insulin signaling via the

oxidation and inhibition of Receptor-like Protein Tyrosine Phosphatases (RPTPs), which would

normally impair the tyrosine-phosphorylation-mediated activation of the insulin receptor279,280.

While the mechanisms behind the beneficial effects of ROS are currently not clear, the

aforementioned findings certainly shed a new light on the role of ROS in the beta cell.

1.10.2 The Role of Endoplasmic Reticulum Stress

As mentioned earlier, the synthesis of insulin involves the cleavage and folding of

preproinsulin within the RER. When the beta cell must produce excessive amounts of insulin,

such as during compensatory hyperinsulinemia in response to FFA-induced insulin resistance, the

RER can become congested with preproinsulin272,281,282. This leads to disruption of the RER,

which manifests itself as ER stress. This has been demonstrated by elevated ER stress markers in

islets and beta cell lines exposed to elevated FFA levels272,281,282. As 50% of the protein

synthesized by beta cells is insulin283, an increase in insulin synthesis can cause significant ER

stress. This can result in beta cell dysfunction, and in some cases, beta cell apoptosis284.

33

Prolonged ER stress is known to activate the unfolded protein response (UPR), which is a

common cellular response that reduces ER stress. This occurs by degrading misfolded proteins,

halting protein translation, and increasing the levels of molecular chaperones involved in protein

folding. However, if the UPR is prolonged or is initiated too late, it progresses towards

apoptosis281,282. Studies have shown that markers of apoptosis, such as CHOP (CCAAT-enhancer-

binding protein homologous protein), are increased in the beta cells of T2DM patients, and in beta

cells exposed to FFA in vitro272,283.

In addition to increasing the load of insulin synthesis on beta cells, there is evidence that

FFA can decrease the capacity of the ER to process proteins284. This is in part due to the FFA-

induced depletion of ER Ca2+ stores, which are necessary for ER function282. By decreasing ER

capacity, FFA also increase the susceptibility to ER stress.

In contrast to in vitro studies, few in vivo studies have investigated the role of ER stress in

FFA-induced beta cell dysfunction. A recent study done by our lab found that ER stress plays a

causal role in high-glucose-induced beta cell dysfunction in vivo239. This was shown to occur via

the positive feedback relationship between ER stress and oxidative stress, particularly

mitochondrial superoxide. In the context of FFA-induced beta cell dysfunction, recent data in our

lab suggest that neither the administration of IH nor oleate affect ER stress markers in rodent beta

cells in vivo (unpublished data). As i.v. infusion of palmitate in vivo has very recently become

feasible, our lab aims to investigate the role of ER stress in palmitate-induced beta cell

dysfunction in vivo in the future. Nevertheless, a collaborative study by our lab has shown that

oral administration of sodium phenylbutyrate (PBA), which reduces ER stress, in humans

ameliorates beta cell dysfunction and insulin resistance induced by IH i.v. infusion184. Hence, it is

also possible that the positive effect of PBA on FFA-induced beta cell dysfunction is not mediated

34

by ER stress reduction due to the multiple effects of PBA. Thus, additional studies are needed in

order to elucidate the role of ER stress in FFA-induced beta cell dysfunction in vivo.

1.10.3 The Role of Inflammation

There is a reciprocal relationship between oxidative stress, ER stress, and inflammation as

they are known to induce each other. Oxidative stress is known to activate IKKB (inhibitor of

nuclear factor kappa-B kinase subunit beta)285, which subsequently phosphorylates the inhibitor

IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha),

resulting in the activation of the proinflammatory NFκB. In addition, IKKB has been shown to

prevent the tyrosine phosphorylation of insulin receptor substrate (IRS) by serine phosphorylating

IRS286,287. This prevents the activation of IRS, consequently impairing insulin signaling.

A study performed by our lab suggests that IKKB is causal to FFA-induced beta cell

dysfunction in vitro, ex vivo, and in vivo (unpublished data). This study utilized the IKKB-

inhibitor salicylate as well as beta cell-specific IKKB-null mice. Nevertheless, as with ROS, some

studies have found that a certain degree of activation of the IKKB/NFκB pathway may be

beneficial to beta cell function288–290. A collaborative study by our lab and the lab of Dr. Gary F.

Lewis found that oral administration of the anti-inflammatory sodium salicylate at a high dose in

men did not prevent insulin resistance and beta cell dysfunction induced by i.v. infusion of IH183.

However, the majority of studies have reported positive results in the treatment of FFA-induced

beta cell dysfunction using salicylate in humans291–293.

IKKB can be activated by FFA through multiple mechanisms. These include oxidative

stress induced by FFA, ER stress294, inflammatory cytokines, PKC295–298, and TLRs which are

directly activated by saturated FFA299.

Another mechanism by which inflammation can cause beta cell dysfunction is the

activation of JNK. Similar to IKKB, JNK is known to serine phosphorylate IRS, consequently

35

impairing insulin signaling300. JNK has also been implicated in FFA-induced beta cell

dysfunction300. In addition, JNK is at least partially responsible for retention of FOXO1 in the

nucleus, which results in the exclusion of Pdx-1 from the nucleus and the consequent decrease in

insulin transcription301,302. Based on in vivo and in vitro experiments, a recent study suggests that

nuclear translocation of FOXO1 results in decreased transcription of peroxisome proliferator-

activated receptor-γ (PPARγ), which is an important transcription factor for Pdx-1303. As a stress-

activated enzyme, JNK can also be activated by inflammatory cytokines, ceramides, ER stress,

and PKC16.

Recent results in our lab have shown that FFA-induced beta cell dysfunction is mediated

by JNK in the context of palmitate, but not in the context of oleate, in vitro. Isolated islets

obtained from JNK1-null mice and wildtype controls were cultured for 48h in Roswell Park

Memorial Institute (RPMI)-based media with or without palmitate or oleate (0.4 mM in 0.5%

BSA). Afterwards, the islets underwent an insulin secretion assay followed by the measuring of

their intracellular insulin content. As mentioned above, other data in our lab have demonstrated

that JNK plays a causal role in high-glucose-induced beta cell dysfunction downstream of

superoxide generation and ER stress in vivo.

1.10.4 The Role of Protein Kinase C

PKC is a family of protein kinase enzymes that play a significant role in regulating

signaling cascades by phosphorylating other proteins. There are many isoforms of PKC304.

Activated PKC is thought to induce beta cell dysfunction and, in some cases, apoptosis partly by

impeding insulin signaling in beta cells305. During excess energy conditions, lipid-derived by-

products can begin to accumulate. These include Long-Chain Coenzyme A (LC-CoA), DAG, and

ceramides306. LC-CoA and DAG are known to activate classical and novel isoforms of PKC,

36

which subsequently lead to beta cell dysfunction307–309. Ceramides are also known to induce beta

cell dysfunction310,311, as described below.

Activated PKC, particularly its novel isoforms (δ, ε, η, and θ isoforms), serine/threonine

phosphorylate the insulin receptor (IR) and IRS. This prevents the tyrosine-phosphorylation-

mediated activation of the IR and IRS, and consequently impairs insulin signaling. This

mechanism of preventing tyrosine-phosphorylation of IRS was evidenced using human IRS-1 in

vitro312. Another study found that HFD-fed mice overexpressing dominant-negative Akt exhibit

decreased GSIS, suggesting that impaired insulin signaling may be involved in FFA-induced beta

cell dysfunction313. Thus it is possible that PKC mediates FFA-induced beta cell dysfunction by

impairing insulin signaling.

FFA-induced activation of PKCε results in decreased transcription of insulin249, 314. It has

been shown that PKCε whole-body knockout mice are protected against glucose intolerance

induced by a HFD, compared with wildtype controls314. In addition, deletion of PKCε in mice

treated with high levels of FFA resulted in improved GSIS and modestly decreased whole-body

and hepatic insulin clearance314. The most likely mechanism by which PKCε inhibition improves

GSIS is the restoration of the efficiency of FFA esterification via glucose-stimulated flux through

the TCA cycle314,315. The study reported that deletion of PKCε did not alter hepatic insulin

signaling or the number of insulin receptors on hepatocytes or their affinity to insulin, but rather

resulted in decreased insulin internalization by hepatocytes (assessed using [125I]insulin). The

mechanism responsible for the decrease in hepatic insulin internalization following PKCε-

deletion was not investigated in that study. The article concluded that a modest reduction in

insulin internalization by hepatocytes can be accomplished without necessarily impairing insulin

signaling.

37

In a study done by our lab using the PKC inhibitor bisindolylmaleimide (BIM), oleate was

found to induce the translocation of PKCδ from the cytosol to membrane fractions, mediating the

FFA-induced decrease in hepatocyte insulin binding316. Although the mechanism by which PKCδ

in membrane fractions affects hepatocyte insulin binding requires further research, it has been

reported that PKC increases the internalization of membrane receptors, including the IR, via a

non-specific and ligand-independent process of coated-pits internalization317. This may be

mediated by the phosphorylation of internalization motifs on the cytoplasmic juxtamembrane

domain of the membrane receptors317,318. Thus, PKCδ may mediate the oleate-induced decrease in

hepatocyte insulin binding by increasing the internalization of the IR, resulting in a deficiency of

IRs on the plasma membrane.

As CEACAM1 is a key mediator in the internalization of the insulin receptor, it is possible

that it is implicated in the PKC-mediated internalization of the insulin receptor. CEACAM1 can

be activated via serine/threonine phosphorylation by PKC120,319. Indeed, a study found that

Protein Kinase-C Activator (PKA) increased the Ser503 phosphorylation of CEACAM1 in

hepatocytes, resulting in its activation319. However, since the administration of a PKC inhibitor

(calphostin C) in colon carcinoma cells did not alter the phosphorylation of CEACAM1319, it is

currently unclear whether or not PKC had mediated the PKA-induced Ser503 phosphorylation of

CEACAM1 in hepatocytes.

Acute overexpression of kinase-negative PKCδ specifically in beta cells of mice on a HFD

was found to improve glucose tolerance and increase plasma insulin levels, compared with

wildtype controls235. Although the mechanism is unknown, this seems to have been caused by

PKCδ’s effect of preventing the translocation of FOXO1 into the nucleus where it would cause

the nuclear exclusion of Pdx-1235. Thus, Pdx-1 was uninhibited and insulin transcription was not

impaired. In addition, kinase-negative PKCδ overexpression in beta cells was found to prevent

38

apoptosis and mitochondrial dysfunction235. Thus, PKC, in particular its novel isoforms, are

known to be causal to FFA-induced beta cell dysfunction. Nevertheless, the responsible

mechanisms are currently unclear.

1.10.5 The Role of Ceramides

Ceramides are a family of waxy lipids that are found in cell membranes and can act as

signaling molecules in apoptosis. Ceramides are endogenously formed in highest amounts by the

metabolism of saturated fatty acids, such as palmitate. Studies have shown that ceramides can

decrease insulin transcription by impairing the nuclear translocation of Pdx-1 and by preventing

the glucose-induced expression of MafA, though the mechanisms are unknown320,138. Many

studies have shown that ceramides can cause apoptosis in beta cells196,321,195. Furthermore, other

studies have demonstrated that inhibition of serine palmitoyltransferase (SPT), which catalyzes

the first step in ceramide production, prevents apoptosis in human and rat islets exposed to high

levels of palmitate in vitro322. Inhibition of ceramide production also protected beta cells from

apoptosis in Zucker Diabetic Fatty (ZDF) rats311. In summary, oxidative stress, ER stress,

inflammation, PKC, and ceramides all play important roles in the development of beta cell

dysfunction in the context of exposure to elevated FFA levels.

1.11 Sirtuins

In the past decade, sirtuins were discovered in yeast and were named Silent Information

Regulator (SIR) genes323. Members of the SIR gene family were later discovered in other species,

eventually indicating that the SIR genes are expressed in all kingdoms of life. The proteins

encoded by the SIR genes were found to be Nicotinamide Adenosine Dinucleotide (NAD)-

dependent deacetylases324,325. Importantly, SIR genes were found to extend the lifetime of

yeast326,327. As a result, research into the role of SIR genes in human aging skyrocketed. Many

39

years of research in this area have revealed that animals contain seven members of the SIR

family. They are named SIRT (silent mating type information regulation 2 homolog) 1 - 7. While

most mammalian SIRT enzymes are deacetylases, their tissue-specific expression, intracellular

localization, and specific roles in physiological processes vary328. SIRT1 is the most studied

sirtuin in mammals. It is also the closest mammalian homologue to the first yeast sirtuin. The

similarities and differences between the mammalian sirtuins are briefly summarized in Table 1.

40

Table 1. Summary of the seven mammalian sirtuins SIRT1-7.

Sirtuin Associated

Diseases Main Processes

Enzymatic

Activity

Main

Substrates

Subcellular

Localization

SIRT1

Metabolic,

neurological,

renal,

cardiovascular,

cancer,

mitochondrial

Glucose

production, β-

oxidation,

insulin

secretion,

neuroprotection,

cell stress

response

Deacetylase

FOXO1/3/4,

p53, NF-κB,

PGC-1α,

eNOS,

Histone

H1/4, LXR,

DBC-1

AROS

Nuclear,

cytoplasmic

SIRT2

Neurological,

metabolic,

cancer

Cell cycle

control, tubulin

deacetylation

Deacetylase

Tubulin,

FOXO,

Histone H4

Nuclear,

cytoplasmic

SIRT3 Metabolic,

mitochondrial

ATP production,

mitochondrial

protein

deacetylation, β-

oxidation

Deacetylase ACS2 Mitochondrial

SIRT4 Metabolic,

mitochondrial

Amino acid-

stimulated

insulin secretion

ADP-

ribosyltransferase

GDH, IDE,

ANT2/3 Mitochondrial

SIRT5 Neurological Urea cycle

regulation Deacetylase CPS1 Mitochondrial

SIRT6 Cancer

NF-κB

regulation, base

excision repair

Deacetylase,

ADP-

ribosyltransferase

Histone H3 Nuclear

SIRT7 Cardiovascular RNA Pol I

transcription Deacetylase

RNA Pol I,

p53 Nucleolar

Abbreviations and definitions (in alphabetical order): ACS2, acetyl-CoA-synthetase 2; ANT2/3, adenine

nucleotide translocator – transports ATP into mitochondria; AROS, active regulator of SIRT1 – an

activator of SIRT1 and a suppressor of p53 activity; CPS1, carbamoyl phosphate synthetase 1 - a

mitochondrial ligase involved in the production of urea; DBC-1, deleted in breast cancer 1 - an inhibitor of

SIRT1; eNOS, endothelial nitric oxide synthase; FOXO, forkhead box O - a family of transcription factors

that regulate cell growth and cell differentiation; GDH, glutamate dehydrogenase - a mitochondrial enzyme

involved in urea synthesis; Histone H1/4, two of five main histone components of chromatin in eukaryotes;

IDE, insulin degrading enzyme; LXR, liver X receptor - a member of a family of transcription factors that

regulate cholesterol levels, fatty acids and glucose homeostasis; NF-κB, nuclear factor kappa B; p53, also

known as TP53 is a protein that suppresses tumor growth; PGC-1α, peroxisome proliferator-activated

receptor gamma co-activator 1 alpha; RNA Pol I, RNA polymerase I; Tubulin, a major component of the

eukaryotic cytoskeleton. Table adapted from references 323,329–332.

41

1.11.1 Sirtuin-1

Figure 5. Illustrations of the three-dimensional protein structure and catalytic activity of the NAD-

dependent protein deacetylase Sirtuin-1 (SIRT1). Protein structure adapted from J. Mol. Biol. 426: 526-

541, 2014.

SIRT1 is an NAD-dependent protein deacetylase that has been shown to have beneficial

effects, such as improving beta cell function by increasing insulin secretion333. The three-

dimensional structure of the SIRT1 protein is depicted above in Figure 5. Modest transgenic

overexpression of SIRT1 in C57BL/6 mice was reported to protect against HFD-induced glucose

intolerance in vivo334. Although SIRT1 has been found in almost all tissues of the body, it plays a

different role depending on the tissue in which it is expressed. In most tissues, SIRT1 has been

shown to be important in the regulation of nutrient metabolism. SIRT1 has been involved in

various diseases, including diabetes, neurodegenerative diseases, and cancer. As was shown in

Table 1, SIRT1 affects many molecules and physiological processes. Significant regulators of

energy metabolism which are influenced by SIRT1 activity include PGC-1α (peroxisome

proliferator-activated receptor gamma coactivator 1-alpha), PPARγ332, and UCP2335,336. PGC-1α

is activated by direct deacetylation by SIRT1 at specific lysine residues333,337. In adipocytes,

SIRT1 was shown to repress PPARγ by binding to PPARγ’s cofactors NCoR (nuclear receptor

42

co-repressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptors)332.

SIRT1 represses the transcription of UCP2 by directly binding to its promoter336.

According to the aforementioned study, unpublished results obtained by its authors

demonstrate that the inhibition of SIRT1 by nicotinamide (a SIRT1 inhibitor) restores UCP2

transcription, suggesting that deacetylation may be involved in the suppression of UCP2

expression by SIRT1336. Nevertheless, although deacetylation has been shown to be involved in

most of the effects of SIRT1, it is not clear whether deacetylation is involved in all of SIRT1’s

effects338–340. In summary, deacetylation can increase or decrease function depending on the target

protein.

1.11.2 The Role of SIRT1 in Beta Cells

While SIRT1 has been most studied in the liver, there are relatively few studies focused on

SIRT1 in beta cells. In beta cells, SIRT1 is primarily found in the nucleus341, although it is also

present in the cytosol342,343. Overall, these studies support the role of SIRT1 as an enhancer or

protector of beta cell function in response to glucose, as described below.

One of the studies utilized a potent SIRT1-activator, resveratrol, to activate SIRT1 in INS-

1E insulinoma cells in vitro344. Resveratrol is a natural polyphenol that has been widely used as an

activator of SIRT1. Resveratrol is typically found in grapes and red wine. The study found that

exposure to 1 µM resveratrol significantly increased insulin secretion by the insulinoma cells

acutely and over 24h of exposure344. Furthermore, this improvement in beta cell function

remained after removing the cells from the resveratrol-containing culture following 24h of

resveratrol exposure. The same study also assessed the effects of resveratrol on islets obtained

from a type 2 diabetic human donor. It found that resveratrol partially enhanced insulin secretion

in response to glucose.

43

Another study found that Beta Cell-Specific Sirtuin 1-Overexpressing (BESTO) transgenic

mice exhibited improved glucose tolerance and glucose-stimulated insulin secretion in vivo,

compared with wildtype mice335. SIRT1 has been shown to positively regulate insulin secretion

in beta cells335. As described earlier, SIRT1 accomplishes this effect by suppressing the

transcription of UCP2 via directly binding with the UCP2 promoter336. As UCP2 is involved in

dissipating the H+ electrochemical gradient in mitochondria, its decreased expression increases

the efficiency of oxidative phosphorylation, leading to greater ATP production345. As insulin

secretion depends on ATP, it is also increased346. The lack of dissipation of the electrochemical

gradient also results in increased ROS production257,347. However, net ROS levels do not increase

since SIRT1 also activates FOXO proteins, which increase the transcription of antioxidant

enzymes. This ultimately leads to decreased oxidative stress348, as will be explained in the section

“Role of FOXO Proteins”.

Levels of UCP2 have been shown to decrease when SIRT1 levels are increased, and to

increase when SIRT1 levels are decreased335,336. As mentioned earlier in my thesis, studies

regarding the effects of UCP2 on beta cell function have been controversial. However, it is

possible that this is due to the dual effect of UCP2 resulting in decreased levels of ATP as well as

ROS. Thus, in the absence of an increase in ROS (because SIRT1 decreases ROS levels), the

decrease in UCP2 levels by SIRT1 results in increased beta cell function.

Some studies have demonstrated that knockdown or knockout of UCP2 expression

improves GSIS due to elevated ATP levels within beta cells231,336,347,349. While some studies state

that activation of UCP2 in beta cells decreases ATP levels and insulin secretion231,345,350, other

studies report contradicting results276,350,351. Nevertheless, UCP2 seems to be involved in some

way in ROS-induced beta cell dysfunction345.

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Some studies showed that increased UCP2 levels decrease ATP production and insulin

secretion346,352. There is evidence that decreased UCP2 expression protects mice against a HFD-

induced decrease in beta cell function, although the responsible mechanism is currently

unclear231,334,353. On the other hand, a number of studies did not find a link between UCP2 and

FFA-induced beta cell dysfunction. For instance, a study utilizing UCP2-null mice have reported

decreased GSIS and no changes in islet insulin content, compared with controls350. Other studies

which involved the overexpression of UCP2 did not find any significant changes in GSIS or ATP

levels within beta cells, compared with wildtype controls277,354. Thus, although most studies

support the hypothesis that UCP2 plays a role in FFA-induced beta cell dysfunction, some

confounding studies render the evidence unclear. It is possible that the contradictory studies are

due to the dual effects of UCP2 in increasing the production of ATP as well as ROS.

1.11.3 Roles of SIRT1 in the Liver, Skeletal Muscle, and Brain

In hepatocytes, SIRT1 deacetylates FOXO1, PGC-1α, and Liver X Receptor (LXR),

resulting in their activation. Although FOXO1, PGC-1α, and LXR have different roles in the

liver, they collectively regulate beta oxidation and gluconeogenesis340,355,356. Although the

upregulation of gluconeogenesis seems to portray SIRT1 as an antagonist to insulin action in the

liver, SIRT1’s effect of decreasing intrahepatic fat content ultimately results in increased hepatic

insulin sensitivity357. It has been shown that although liver-specific SIRT1-knockdown mice had

improved glucose tolerance, they had greater hepatic steatosis and steatohepatitis after being fed a

HFD, compared with controls358. Since SIRT1 activates PGC-1α, and PGC-1α upregulates

mitochondrial biogenesis, it is thought that SIRT1 increases energy expenditure in hepatocytes358.

In WAT, SIRT1 has been shown to exert significant effects via PPARγ and FOXO1.

SIRT1’s direct repression of PPARγ was shown to result in an increase in FFA mobilization355.

SIRT1’s deacetylation and activation of FOXO1 results in increased transcription and production

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of adiponectin since FOXO1 is a transcription factor for adiponectin359. In a study, mice

overexpressing SIRT1 exhibited an ~30% increase in adiponectin concentration in blood

plasma360.

In skeletal muscle, one of SIRT1’s main roles is the upregulation of mitochondrial

biogenesis, resulting in an increase in beta oxidation and energy production358. As explained

above, this occurs via the direct deacetylation and activation of PGC-1α by SIRT1. In addition,

SIRT1 in skeletal muscle is known to increase insulin sensitivity via histone H3 deacetylation,

resulting in the repression of the expression of PTP1B (protein-tyrosine phosphatase 1B), which

is a negative regulator of insulin signaling361,362. This was evident from two studies: the first

utilized muscle-specific PTP1B-null mice363, and the second used whole-body PTP1B-null

mice364. Thus, SIRT1 increases insulin sensitivity in skeletal muscle.

In the brain, SIRT1 is found in the hypothalamus, particularly in the proopiomelanocortin

(POMC) and steroidogenic factor-1 (SF-1) neurons365,366. The effects of SIRT1 in the brain are

less clear, compared with its effects in the liver, skeletal muscles, and WAT. Studies involving

transgenic mouse models have shown that SIRT1 in the brain may increase energy expenditure

and prevent obesity367,368. In addition, neuronal SIRT1 has been found to protect against oxidative

stress, ameliorate inflammation and apoptosis, and regulate neuronal and stem cell

differentiation369,370. Studies suggest that the protective effects of SIRT1 on neurons are based on

the SIRT1-mediated decrease in inflammation and apoptosis. These are likely caused by SIRT1’s

deacetylation of NFκB, resulting in the suppression of the inflammatory pathway and

apoptosis323,371.

1.11.4 Roles of FOXO Proteins

FOXO proteins are transcription factors, and consist of FOXO 1, 3, 4, and 6 in mammals.

These transcription factors are inhibited by the insulin/PI3K (phosphoinositide 3-kinase)/Akt

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pathway and are involved in oxidative stress, metabolism, and organismal longevity372,373.

FOXO2 is a pseudogene of FOXO3, and FOXO5 is the fish orthologue of FOXO3373. Acetylated

FOXO4 has been shown to promote podocyte apoptosis374. FOXO6, mainly found in the brain

and liver, has been recently shown to have negative effects on glucose metabolism, where

overexpressing FOXO6 in livers of mice gradually resulted in glucose intolerance, fasting

hyperglycemia, and insulin resistance375,376.

FOXO1 is the most common FOXO protein in beta cells377. FOXO1 and 3 are known to

decrease oxidative stress by upregulating antioxidant genes, such as MnSOD348,378. In addition, a

study has reported that FOXO1 may form a complex with the promyelocytic leukemia protein

(Pml) and SIRT1. This complex subsequently increases the expression of the transcription factors

NeuroD and MafA379. In addition to Pdx-1, MafA and NeuroD are transcription factors that

regulate insulin. Moreover, NeuroD and MafA are involved in the regulation of beta cell

maturation380. FOXO1 has also been shown to directly activate the SIRT1 promoter, increasing

SIRT1 mRNA and protein levels381. Similarly, FOXO3 is a transcription factor for SIRT1372,382.

Thus, FOXO1 or FOXO3 activation may upregulate SIRT1 via a feed-forward mechanism.

With that said about the protective role of FOXOs against oxidative stress and the effects

of FOXOs on NeuroD and MafA, it must be noted that FOXO1 can also indirectly decrease

insulin transcription. Active FOXO1 is localized in the nucleus, where it induces the nuclear

exclusion of Pdx-1, which is a major transcription factor for insulin300. In summary, although

activation of FOXO proteins seems to ultimately attenuate ROS, the overall effect of FOXO1 on

beta cell function in particular is not very clear.

1.11.5 Regulation of SIRT1

As mentioned earlier, SIRT1 is a deacetylase that is NAD-dependent. Hence, one of the

main regulators of SIRT1 activity is the availability of intracellular NAD. During fasting, reduced

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energy production results in an increase in the NAD/NADH ratio in cells323. Thus, provided that

the sum of NAD and NADH levels are not significantly altered, this results in a relative increase

in intracellular NAD levels. As a result, SIRT1 activity is upregulated383,384. Consistently, the

activity of SIRT1 is known to be increased during fasting385–387. A decrease in NAD levels

reduces SIRT1 activity388–390. This can be caused by a number of factors, ranging from decreased

NAD/NADH ratio due to energy excess to depletion of NAD levels by enzymes other than

SIRT1391.

Calorie restriction has been shown to increase lifespan in several organisms, including

primates, via increased SIRT1 activity391–393 and levels394–396. While this may be caused by

increased NAD availability, a 2004 study published in Science suggests that p53 can also

upregulate SIRT1 activity382. A transcription factor, p53, can function as either an activator or a

repressor. Normally, p53 directly decreases SIRT1 transcription; however, under caloric

restriction, p53 has been shown to play a complex role in which it is required for the FOXO3-

mediated increase in SIRT1 expression382. Activated FOXO3a subsequently increases SIRT1

transcription by directly interacting with p53 and two p53-binding sites at the SIRT1 promoter.

The same study found that FOXO3a-knockdown mice did not exhibit an increase in SIRT1

expression after chronic caloric restriction, compared with wildtype controls. Hence, FOXO3a

seems to be important in the caloric restriction-mediated increase in SIRT1 expression.

While caloric restriction increases SIRT1 activity in many tissues, such as skeletal muscle

and WAT, caloric restriction was found to decrease SIRT1 activity in the liver by reducing the

NAD/NADH ratio in hepatocytes397. Interestingly, a high-caloric diet was shown to increase

SIRT1 activity in hepatocytes. The reason for the opposite effect of caloric restriction on SIRT1

in the liver, compared with the other tissues, is thought to be because the liver plays a large role in

the synthesis of triglycerides and cholesterol. As these processes are usually in proportion to

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caloric intake and are highly reductive, the highly-fed liver should have an oxidized redox state

(increased NAD/NADH ratio) in order to perform these processes397,398. Hence, the effects of

caloric restriction on SIRT1 may depend on the tissues in which SIRT1 is expressed.

Incubation of rat islets in palmitate-containing media or intravenous infusion of IH in rats

decreased SIRT1 mRNA and protein levels in beta cells399, 400. The mechanisms responsible for

the FFA-induced decrease in SIRT1 are unclear. However, one known mechanism is that FFA-

induced ROS damages DNA, leading to the activation of the DNA repair enzyme Poly (ADP-

Ribose) Polymerase (PARP). PARP consumes NAD to repair DNA, resulting in decreased NAD

availability401. As SIRT1 is dependent on the bioavailability of NAD, a decrease in SIRT1

activity may ensue384,402,403. Accordingly, studies using whole-body PARP-knockout mice (PARP

decreases NAD availability) have demonstrated increased SIRT1 activity404,405; however, this

effect has not been assessed in beta cells. Decreased SIRT1 activity may reduce the activity of the

SIRT1-transcription factors FOXO1 and FOXO3 via a feed-forward mechanism372, and thus

result in decreased SIRT1 expression.

As explained above, FFA-induced ROS can attenuate SIRT1 activity by decreasing NAD

bioavailability. Nevertheless, ROS can alter SIRT1 activity independently of NAD. For instance,

the oxidative stress-activated enzyme JNK1 has been shown to serine-phosphorylate SIRT1406,

transiently activating it. However, this serine-phosphorylation of SIRT1 has additional effects on

SIRT1. It induces SIRT1’s ubiquitination, resulting in SIRT1’s gradual degradation and decreased

activity over time. This form of SIRT1 ubiquitination was demonstrated using a model of HFD-

fed mice in vivo407. ROS have also been shown to directly induce the alkylation of cysteine

residues on SIRT1. This results in increased SIRT1 proteosomal degradation, ultimately leading

to decreased SIRT1 protein levels408.

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In addition to being serine-phosphorylated by JNK1, SIRT1 can be serine- and threonine-

phosphorylated by cyclin-dependent kinase-1 (Cdk-1), independently of JNK, resulting in

decreased SIRT1 activity409. Cdk-1 is a highly conserved kinase that plays a regulatory role in the

G2/M phase of mitosis. SIRT1 activity can be positively regulated by the nuclear protein AROS

(Active Regulator of SIRT1). This takes place by AROS directly binding to SIRT1, inducing a

conformational change in SIRT1410. As a result of this conformational change, SIRT1’s

deacetylation activity is enhanced.

A negative regulator of SIRT1 is the nuclear protein Deleted in Breast Cancer-1 (DBC-

1)411,412. DBC-1 directly binds to the catalytic site of SIRT1, consequently inhibiting SIRT1’s

activity412. It has recently been shown that DBC-1 is phosphorylated and activated by the kinase

Ataxia Telangiectasia Mutated (ATM) following DNA damage and oxidative stress413,414.

Interestingly, DBC-1 can also act as a switch for SIRT1 activity. For example, activated AMPK

induces the dissociation of the DBC-1-SIRT1 complex, resulting in activated SIRT1415. As the

regulation of SIRT1 by DBC-1 seems to occur independently of intracellular changes in NAD

levels415–417, DBC-1 might have been involved in the decrease of SIRT1 activity in our lab’s

previous experiments since we did not observe decreased NAD levels in rat islets treated with

FFA. While AROS and DBC-1 do regulate SIRT1 function, their mechanisms are not quite clear

yet since their effects on SIRT1 have only recently been discovered.

Lastly, it should be mentioned that over 3,500 activators of SIRT1 have been

developed329. These small-molecule compounds are structurally unrelated to resveratrol, yet are

1,000 fold more potent than resveratrol at activating SIRT1. These activators bind to the SIRT1

enzyme-peptide substrate complex at an allosteric site near the catalytic domain, lowering the

Michaelis constant (KM) for SIRT1’s deacetylation activity418. The KM is a widely used index of

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enzyme activity. It is defined as the concentration of substrate required for a chemical reaction to

occur at half of its maximum velocity419.

1.11.6 Nicotinamide Mononucleotide

As described earlier, SIRT1 being an NAD-dependent enzyme is affected by NAD

availability. As such, altering intracellular levels of NAD is a viable method of regulating SIRT1

activity. A number of studies have demonstrated that altering NAD levels by altering NAD

synthesis affects SIRT1 activity420–424. NAD is derived from nicotinamide by the action of several

enzymes. A key intermediate of NAD synthesis is nicotinamide mononucleotide (NMN), which is

produced from nicotinamide and 5-phosphoribosyl-pyrophosphate using nicotinamide

phosphoribosyltransferase (NAMPT). NMN is a water-soluble ribonucleotide consisting of a

ribose backbone, a nitrogenous base (nicotinamide), and a phosphate group425. The structural

formula of NMN is depicted in Figure 6. NAMPT represents the rate-limiting enzyme in

mammalian NAD biosynthesis. NMN is then converted to NAD by nicotinamide mononucleotide

adenylyltransferase-1 (NMNAT)426.

Figure 6. Chemical structure of nicotinamide mononucleotide (NMN), which consists of a ribose

backbone, a nitrogenous base (nicotinamide), and a phosphate group. Adapted from Cell. 129(3): 453–454,

2007425.

There are two forms of NAMPT: intracellular NAMPT (iNAMPT) and extracellular

NAMPT (eNAMPT). iNAMPT is found in high levels in brown adipose tissue (BAT), liver, and

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kidneys, and in intermediate levels in WAT, skeletal muscle, lung, testes, and spleen. No

iNAMPT has been detected in the pancreas or brain423. eNAMPT, thought to be produced by

post-translational modification of iNAMPT, is secreted from adipose tissue and promotes insulin

secretion427. While iNAMPT is essential to the NAD biosynthetic pathway, eNAMPT is vital to

NAD synthesis in beta cells due to the absence of iNAMPT in the pancreas. A study found that an

eNAMPT-knockdown (haplodeficient) mouse model and chemical inhibition of NAMPT in

wildtype mice exhibited impaired NAD biosynthesis and GSIS in vivo and in vitro423.

A study published in Cell Metabolism showed that wildtype B6 mice fed a HFD

demonstrated impaired NAMPT-mediated NAD biosynthesis, resulting in decreased SIRT1

activity and exacerbated T2DM (the study did not assess which form of NAMPT was

decreased)424. The same study found that administration of the rate-limiting intermediate in NAD

biosynthesis, NMN, restored normal NAD levels and synthesis in the liver, WAT, and skeletal

muscles. Interestingly, NMN treatment also protected against T2DM induced by HFD. NMN was

administered intraperitoneally to male diabetic mice at 500 mg/kg body weight/day for 10

consecutive days. No noticeable abnormalities or significant changes in body weight were

reported.

Another study, published in Diabetologia, found that mice on a fructose-rich diet (FRD)

exhibited markedly lower eNAMPT levels in blood plasma, increased iNAMPT levels in WAT,

and decreased insulin secretion, compared with controls427. This suggests that excess energy

consumption, metabolic dyslipidemia, and chronic inflammation impair NAD biosynthesis and

insulin secretion. Furthermore, administration of NMN restored normal insulin secretion in FRD-

fed mice in vivo. The protective effect of NMN was also observed in isolated islets cultured with

pro-inflammatory cytokines427.

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The mechanism of transport of NMN into eukaryotic cells is currently unknown424;

however, the very few studies performed to date suggest that NMN transport in Salmonella

typhimurium occurs via PnuC and NadI428,429. One study found that PnuC is a membrane-

associated protein which is a major component of the NMN transport system. NadI is a protein

that depends on pyridine levels (presumably NAD) in order to serve two functions: 1) increase the

transcription of the nadA and nadB biosynthetic genes, and 2) positively regulate the activity of

NMN permease, which is part of the NMN transport system428. Another study found that pnuC of

Haemophilus influenzae, which is distally related to S. typhimurium, was able to uptake only

nicotinamide riboside and not NMN429. Thus, while few studies on NMN’s mechanism of

transport have been done, specifically in prokaryotic cells, and are unclear, it is possible that a

transporter mediates the uptake of NMN in eukaryotic cells.

There is evidence suggesting that SIRT1 may be one of the mediators of NMN’s

protective effects against T2DM427. Transcription factors expressed during HFD treatment and

during NMN administration were analyzed. The transcription factors that exhibited greatest

changes in expression particularly during the HFD and NMN treatments were all targets of SIRT1

deacetylation: NF-κB, PPARγ, p53, and c-Myc427. Moreover, assessment of the acetylation status

of NF-κB in the liver indicated increased acetylation during HFD feeding (suggesting reduced

SIRT1 deacetylation), and considerably decreased acetylation during NMN administration (likely

reflecting increased SIRT1 activity).

NAMPT-mediated NAD synthesis is involved in many physiological processes, including

metabolism, energy balance, stress responses, circadian rhythm, and cell differentiation430,431.

Based on several studies, NAMPT-mediated NAD synthesis seems to be impaired during

oxidative stress and/or inflammation. This consequently contributes to decreased SIRT1 activity

and to the progression of T2DM420–424. Nevertheless, it remains unclear how changes in the

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environment and nutritional context affect the network of NAMPT/NAD/SIRT1 pathways

(referred to as the “NAD World”431).

NAD affects several metabolic pathways which involve SIRT1. In addition, NAD levels

can affect the SIRT2-7 and PARP enzymes (Figure 7). For instance, increases in NAD levels

have been shown to increase the activities of mitochondrial sirtuins SIRT3–5. As impaired

mitochondrial sirtuin activity can lead to mitochondrial dysfunction, this seems to protect against

mitochondrial dysfunction and ROS generation432. PARPs, which are DNA repairing enzymes,

are also stimulated by NAD. With regard to beta cell function, PARP stimulation may not be

beneficial. This is because PARP-1-deficient mice were shown to be protected against cytokine-

induced islet cell death, which, however, may be due to increased NAD. However, PARP-1-

deficiency did not protect against cytokine-induced inhibition of insulin secretion433.

The importance of NAD in cell metabolism is demonstrated by the model of diabetes

which is induced by streptozotocin (STZ) administration in rodents434. STZ in blood plasma is

transported into beta cells via the GLUT2 glucose transporter. In the beta cell, STZ results in

significant DNA damage, leading to the excessive activation of the DNA-repair enzyme PARP.

As PARP consumes NAD during its DNA repair action, excessive PARP activity leads to a

depletion of NAD, which due to its necessary role in the TCA cycle, subsequently leads to a

deficiency in ATP. Consequently, the beta cells undergo necrosis434. Interestingly, the co-

administration of nicotinamide with STZ is known to produce a milder form of diabetes,

compared with STZ-only administration435,436. In this context, nicotinamide partially protects beta

cells by inhibiting PARP1 activity, consequently preventing the depletion of NAD and ATP in

STZ-exposed cells. In addition, as nicotinamide is a precursor to NAD, its administration also

results in increased intracellular NAD levels434,435.

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Figure 7. Summary of the Nampt-mediated NAD synthesis, where the rate-limiting enzyme is Nampt. The

effects of NAD on enzymes other than SIRT1 are also depicted.

1.11.7 Potential Role of SIRT3

SIRT3 is an important mitochondrial protein deacetylase that regulates enzymes involved in

beta oxidation as well as ATP production437. SIRT3 is also known to regulate ROS production

and to exert anti-inflammatory effects438. As with SIRT1, caloric restriction has been shown to

increase SIRT3 protein levels in mice439, and a chronic HFD is associated with decreased SIRT3

mRNA levels440. Intriguingly, an acute HFD was shown to increase SIRT3 mRNA levels440. Very

interestingly, SIRT3 is known to be upregulated by a substrate of SIRT1: PGC-1α. A moderate

overexpression (~2.5 fold) of SIRT1 in skeletal muscle of rats was associated with an increase in

SIRT3 and PGC-1α expression441. Thus, it seems very possible that SIRT1 may upregulate SIRT3

expression via PGC-1α.

In fact, recent studies have been investigating the role of SIRT3 in metabolic disorders. A

study published in Diabetologia in 2013 assessed changes in SIRT3 expression in mouse islets

and INS1 cells incubated in IL1β and TNFα, and in islets isolated from patients with T2DM438.

The study found significantly decreased SIRT3 expression in mouse islets and INS1 cells, and in

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the islets of type 2 diabetic patients. In addition, the study assessed the effects of SIRT3-

knockdown in INS1 cells. The authors reported a significant decrease in GSIS and in the

expression of beta cell genes, as well as a significant increase in beta cell apoptosis and

intracellular ROS and IL1β levels. Interestingly, the above study also revealed that SIRT3-

knockdown blocked the protective effects of NMN on beta cell function in INS1 cells incubated

in the proinflammatory cytokines. In addition, another study found that SIRT3-knockout mice

exhibited impaired glucose tolerance at 3 and 12 months of age when fed a HFD, and at 12

months of age when fed a standard diet439.

The above evidence suggests that an NMN-mediated increase in NAD levels may prevent

beta cell dysfunction by enhancing the activity of SIRT3 in addition to SIRT1. Nevertheless, there

are few studies regarding the role of SIRT3 in FFA-induced beta cell dysfunction per se,

compared with studies assessing SIRT1’s role. Thus, additional research is needed in order to

determine whether or not SIRT3 plays a complementary role to SIRT1 in mediating the protective

effects of NMN.

1.12 Potential Treatments for Lipid-Induced

Beta Cell Dysfunction

Dr. Adria Giacca’s laboratories and others have shown that therapies aimed at reducing

oxidative stress, ER stress, and inflammation have the potential to alleviate lipid-induced beta cell

dysfunction in animals and humans16.

As mentioned earlier, glucotoxicity in the presence of lipotoxicity accentuates beta cell

dysfunction. Nevertheless, this glucotoxicity-induced accentuation of FFA-induced beta cell

dysfunction seems to be reversible. A collaborative study by our lab showed that diabetic subjects

who undergo biliopancreatic diversion, which normalizes glycemia, exhibited similar beta cell

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dysfunction, compared with IH-infused controls181. Thus, the diabetic subjects which were

chronically exposed to hyperglycemia did not demonstrate accentuated beta cell dysfunction after

their glycemia was normalized.

It is known that nonpharmacological weight-loss improves beta cell function and insulin

sensitivity via reducing plasma FFA levels442–444. Some studies have shown that the lowering of

plasma FFA improved GSIS only in diabetic patients with very low levels of Hb A1c, suggesting

that in diabetes which is not well-controlled, the damage to beta cell function may not be

reversible445. A collaborative study by our lab showed that increasing plasma FFA levels resulted

in a greater impairment of GSIS in obese, non-diabetic patients, compared with diabetic patients

with already impaired beta cell function185. However, obese humans185 and obese prediabetic rats

were found to be more susceptible to the effect of FFA on decreasing beta cell function, compared

with non-obese, non-diabetic humans or rats446.

It seems that the reduction of plasma FFA levels, such as by pharmacological intervention,

is beneficial in preventing and treating FFA-induced beta cell dysfunction. However, current

antilipolytic treatments, such as Acipimox447, are not very effective mainly due to their short half-

life and the common post-treatment rebound in FFA levels in overweight individuals.

Furthermore, current antilipolytic treatments have been known to sometimes cause adverse side

effects, such as insulin resistance16. Lastly, pharmacologically decreasing plasma FFA levels by

increasing net FFA uptake by adipocytes may not be beneficial in the long-run. This would be due

to the resulting increase in adiposity, which may exacerbate the negative effects of obesity, such

as complications from inflammation and metabolic disorders.

Based on many studies, including some done by our lab, focusing on the reduction of

oxidative stress, ER stress, and inflammation seems to be a more promising approach, compared

with decreasing FFA mobilization16,141,187,212,239,241,242,265,271,282,284,294,350,448,449. Previous research

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done in our lab has shown that administration of the IKKβ inhibitor salicylate to rats i.v. infused

with oleate or 20% olive oil + heparin (50 U/ml) for 48h prevented FFA-induced beta cell

dysfunction (unpublished data). Beta cell function was assessed using hyperglycemic clamps (in

vivo). We had also observed this protective effect of salicylate ex vivo in islets isolated from rats

which had been i.v. infused with oleate or olive oil + heparin for 48h (unpublished data).

Another study found that pioglitazone (an anti-inflammatory TZD drug) and sodium

salicylate prevented an increase in apoptosis and partially protected against beta cell dysfunction

in human islets exposed to IL-1β or 33.3 mM of glucose (glucotoxicity)287. This was shown to

occur via pioglitazone and salicylate blocking the activation of NFκB by cytokines or

glucotoxicity287. Furthermore, a recent clinical trial suggests that preventing the activation of the

IL-1 receptor by using a synthetic antagonist (Anakinra) results in an improvement in beta cell

function, however, the largest improvement was observed in HbA1C levels450. Since Anakinra has

a short half-life of 4-6h and is a relatively weak antagonist, it is thought that using a stronger

antagonist with a longer half-life or inhibiting multiple targets in the inflammatory pathway may

yield a more therapeutic effect, compared with the use of Anakinra alone450. These results suggest

that oxidative stress and inflammation both play a role in FFA-induced beta cell dysfunction.

Currently, several insulin-sensitizing drugs seem to have beneficial side effects in treating

FFA-induced beta cell dysfunction. For example, besides improving insulin sensitivity, metformin

and TZDs decrease oxidative stress and islet inflammation. Metformin and TZDs also activate

AMP kinase, resulting in the depletion of triglycerides stored in islets451. The combination of the

aforementioned effects results in increased beta cell function in FFA-exposed rats452,453 and in

humans454,455. Thus, although metformin and TZDs are beneficial for treating insulin resistance

and beta cell dysfunction, their inhibitory effects are not specific to oxidative, ER, or

inflammatory stress. Elucidating the mechanisms of FFA-induced beta cell dysfunction may

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allow for the development of more specific drugs for the prevention and/or treatment of beta cell

dysfunction.

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Chapter 2

Rationale 2.1 Previous Results from the Giacca Lab

Small interfering ribonucleic acid (siRNA)-mediated and pharmacological inhibition of

SIRT1 activity were both found to decrease insulin transcription in INS-1 cells (rat beta cell

model456). This mimicked the effect of palmitate in vitro399. While SIRT1 activity may decrease

insulin transcription via the FOXO1-induced nuclear exclusion of Pdx-1, inhibition of SIRT1 may

decrease the FOXO1-mediated transcription of antioxidant enzymes, and thus increase

vulnerability to ROS-induced decrease in insulin gene transcription. This study reflected the dual

effects of FOXO1. The study also showed that SIRT1 activation by resveratrol administration

protected INS-1 cells from palmitate-induced beta cell dysfunction in vitro. Thus, decreased

SIRT1 activity may be causal to palmitate-induced beta cell dysfunction. Furthermore, another

study found that rats fed a HFD or FRD for 12 weeks exhibited a decrease in SIRT1 protein

expression in the pancreas and liver, whereas rats treated with caloric restriction showed an

increase in SIRT1 expression457.

Our laboratory aimed to investigate the potentially protective effect of SIRT1 on beta cells

in vivo in the context of high plasma levels of FFA. In hyperglycemic clamps in Wistar rats,

previous lab members found that i.v. co-infusion of resveratrol with oleate for 48h partially

protected against beta cell dysfunction induced by oleate in vivo (Figure 8). In hyperglycemic

clamps in mice, they found that BESTO mice are partially protected from oleate-induced beta cell

dysfunction in vivo (Figure 9).

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Figure 8. Resveratrol, a SIRT1 activator, partially protects against FFA-induced beta cell

dysfunction in rats in vivo. Disposition index as assessed by two-step hyperglycemic clamps following

48h treatments. Rats were i.v. infused with saline (SAL; 0.5 μmol/min), oleate (OLE; 1.3 μmol/min), OLE

+ resveratrol (RSV; 0.025 mg/kg/min), or RSV only. Data are means ± SE. ¶ p vs. SAL and RSV < 0.05; ‡

p<0.01 vs. SAL. Adapted from Desai T., M.Sc. Thesis, Dr. Giacca Lab, Department of Physiology, 2013.

Figure 9. Beta cell-specific SIRT1 overexpressing (BESTO) mice were partially protected against

FFA-induced beta cell dysfunction in vivo, compared with wildtype mice. Disposition index as

assessed by one-step hyperglycemic clamps after 48h treatment. BESTO mice (TG) and C57BL/6 controls

(WT) were i.v. infused with saline (SAL) or oleate (OLE; 0.4 μmol/min). Data are means ± SE. ‡ p vs. WT

SAL and TG SAL < 0.01. Adapted from Desai T., M.Sc. Thesis, Dr. Giacca Lab, Department of

Physiology, 2013.

As SIRT1 activation prevented oleate-induced beta cell dysfunction, our lab hypothesized

that decreased SIRT1 activity was present in islets and was responsible for the oleate-induced

beta cell dysfunction. Thus, previous lab members performed an ex vivo study in which rats were

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i.v. infused with treatments for 48h. Since SIRT1 is NAD-dependent, our lab investigated whether

or not NAD deficiency may be responsible for the oleate-induced decrease in SIRT1 activity. This

was done in collaboration with Dr. Jamie Joseph’s lab, based at the University of Waterloo. No

significant difference in NAD and NADH levels or ratios between any of the treatment groups

was observed (Figure 10A & B). As a previous study had reported that palmitate decreases

SIRT1 mRNA levels399, our lab measured SIRT1 mRNA levels using RT-PCR. This was done in

collaboration with Dr. Michael Wheeler’s lab. Preliminary experiments suggest that oleate might

not affect SIRT1 mRNA levels in rats (Figure 10C). Our lab’s current hypothesis is that SIRT1

activity or protein level in the beta cell is affected by FFA independently of NAD levels;

however, this remains to be investigated.

62

Figure 10. Islets of Wistar rats i.v. infused with oleate did not demonstrate decreased NAD

bioavailability, compared with the saline-infused control. (A & B) Unaltered NAD and NADH levels

and ratios in rat islets indicate that the oleate-induced decrease in SIRT1 activity does not seem to be

mediated by decreased NAD bioavailability. (C) Preliminary qRT-PCR-measured mRNA expression of

SIRT1 (relative to Histone H3a mRNA) in rat islets suggest that oleate does not decrease SIRT1

transcription. Data are means ± SE. Data in A and B were obtained by Dr. Jamie Joseph’s lab, University

of Waterloo. Data in C were obtained by Desai T. in our lab.

C

A

. B

.

63

2.2 My Experimental Design

It has been shown that administration of NMN, which is a key intermediate of NAD

biosynthesis, restores NAD levels and increases GSIS and glucose tolerance in HFD-fed C57BL/6

mice in vivo424 (Figure 11). NMN was administered to C57BL/6 mice via intraperitoneal

injection at 500 mg/kg/day for 10 days.

Figure 11. Glucose tolerance and plasma levels of insulin, cholesterol, triglycerides, and free fatty

acids of mice fed a high-fat-diet (HFD) and treated with nicotinamide mononucleotide (NMN). (A)

Glucose tolerance in high fat diet (HFD)-fed male mice before and after NMN treatment. Intraperitoneal

glucose tolerance tests (IPGTT) were conducted on mice before and after treatment with NMN for 10

consecutive days (500 mg/kg body weight/day). The Area Under the Curve (AUC) is presented next to the

graph. (B) Plasma insulin levels during IPGTT before and after NMN treatment. (C) Cholesterol (Chol),

triglycerides (TG), and FFA levels in aged, HFD-fed female mice before and after NMN infusion. Data are

means ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Adapted from Yoshino J et al. 2011. Cell Metab 14:

528-536.

A

.

B C

64

Mainly based on the previous results of our lab, this thesis will test the hypothesis that

NMN administration would counteract oleate-induced beta cell dysfunction in vivo in mice.

Although NAD levels were shown to have not been decreased in a previous study of ours,

increasing NAD levels should still stimulate SIRT1 activity if SIRT1’s activity is low. Therefore,

I investigated the effects of NMN infusion on oleate-induced beta cell dysfunction in C57BL/6

mice in vivo. Male, 11 – 13 wk old mice, cannulated at the jugular vein, were i.v. infused with

saline (SAL), oleate (OLE), NMN + OLE, or NMN only, for 48h. The mice then underwent a

hyperglycemic clamp assessing beta cell function in vivo.

NMN administration is an alternative method of SIRT1 activation that does not have the

same drawbacks as resveratrol infusion, which has non-specific antioxidant effects, and the

BESTO mouse model, which has a high, non-physiological level of SIRT1 expression (~50 fold).

In addition, as NMN is produced endogenously, the administration of NMN may be considered a

nutriceutical approach of treating T2DM424. A caveat of this approach is that NAD, the levels of

which are elevated by NMN administration, is involved in many physiological processes, and

therefore would have non-specific effects besides increasing SIRT1 activity.

In Summary:

My Hypothesis: NMN administration would counteract oleate-induced beta cell dysfunction

in mice in vivo.

o Aim 1: To investigate the role of SIRT1 in FFA-induced beta cell dysfunction in vivo

without the non-specific effects of resveratrol and the excessive overexpression of SIRT1.

o Aim 2: To investigate the potential effects of NMN (not necessarily specific to SIRT1) on

the metabolism of glucose and plasma FFA in vivo.

65

Chapter 3

Materials and Methods

Note: The following procedures were performed in the same manner performed by previous lab

members56.

3.1 Animal Models

Wildtype, male, C57BL/6 mice at 11 – 13 weeks of age were utilized. The mice were

housed in the Department of Comparative Medicine at the University of Toronto, under a 12h

light/dark cycle. All mice were fed a rodent diet which consisted of 25% protein (percent of

energy), 58% carbohydrate, and 17% fat (LM-485, Harland Teklad Global Diets, Madison, WI,

USA). Mice were allowed to reach 11 – 13 weeks of age before being utilized in our studies.

Littermates of experimental groups were used as controls.

3.2 Mouse Cannulation Surgery

All procedures were performed in accordance with the Canadian Council of Animal Care

Standards and were approved by the Animal Care Committee of the University of Toronto. Under

isoflurane anesthesia and sterile conditions, 11 - 13 wk old, male, C57BL/6 mice underwent

jugular vein cannulation surgery. The left jugular vein was cannulated with a catheter consisting

of two parts: a long polyethylene tubing (PE-10; Cay Adams, Boston, MA, USA) and a shorter

silastic tubing (length of 1.5 cm; Dow Corning, Midland, MI, USA). Only the silastic tubing

portion (which was soft and elastic to minimize vessel damage) was inserted inside the jugular

vein. The catheter was exteriorized by running through a subcutaneous tunnel (created using a

16G needle) and emerging at the back of the neck. This was done to protect the catheter from

being bitten by the mouse. The catheter was filled with heparinized saline (40 units/ml) in order to

prevent blood clots. The catheter was subsequently tied at its free end and wrapped with tape for

66

additional protection. Post-surgery, every cannulated mouse was housed individually and

provided with a recovery period of 3 – 5 days before i.v. infusion.

3.3 Treatment Infusion in Mice

Prior to i.v. infusion, each cannulated mouse was fitted with a polyethylene harness that

allowed the mouse’s catheter to pass through a metal, protective tether and attach to a swivel

placed above the cage. This allowed the mice freedom of movement without risking damage to

the cannula. Mice were i.v. infused via their catheters with SAL, OLE (0.4 μmol/min),

NMN+OLE (0.025 mg/kg body weight/min of NMN, as performed in previous studies424,458–460),

or NMN alone, using syringe infusion pumps for 48h. Oleate was infused while bound to BSA

(according to the Bezman-Tarcher method461) at a dose that elevates plasma FFA levels by 1.5 – 2

fold, simulating the pathophysiological levels present in obesity56,201,205,212,462. The binding of

BSA to oleate simulates physiological conditions and protects the mice from the detergent effects

of oleate. This allows the i.v. infusion of the oleate FFA into animals461. The OLE and NMN

infusates were freshly prepared and were protected from light, as had been performed in our

previous studies56,205,463,464. The pH of infusates was adjusted to 7.4 ± 0.05 prior to i.v. infusion.

Mice had ad libitum access to standard rodent chow and water. Whole blood samples were

taken at 0h and 46h of infusion via the tail vein (immediately prior to commencing the

hyperglycemic clamp, which is described below). Immediately after collection, the whole-blood

samples were centrifuged in order to obtain the plasma samples of FFA, insulin, and C-peptide.

3.4 One-Step Hyperglycemic Clamp

Mice were fasted (but had access to water) for 4 hours prior to beginning the two-hour-

long hyperglycemic clamp at 46h of i.v. infusion. Twenty minutes before initiating the

hyperglycemic clamp, a sample of blood was taken from each mouse in order to establish basal

67

plasma levels of glucose, FFA, insulin, and C-peptide. Beta cell function was assessed in

conscious mice using a one-step hyperglycemic clamp without interrupting the i.v. infusion of

treatments. This method mainly analyzes the rise in insulin and C-peptide levels in response to

elevated plasma glucose levels. Glucose was i.v. infused via a Y-shaped connector at the catheter

at 37.5% (g/ml) concentration in order to gradually elevate plasma glucose levels to ~22 mmol/L

(which is the maximum stimulatory level).

Glycemia was assessed every 5-10 minutes by measuring a drop of blood from the tail

using a HemoCue Glucose 201+ System (HemoCue, Lake Forest, CA, USA). Minimal blood

volume was collected in order to reduce the risk of anemia. By the end of the experiment, a

maximum total of 0.3 ml of blood was collected from each mouse. Once glycemia was

approximately 22 mmol/L, the plasma glucose levels were maintained, i.e. “clamped”, for ~30

minutes. This was done by varying the infusion rate of glucose according to the glycemia

readings obtained every 5-10 minutes. After maintaining glycemia at an equilibrium of ~22

mmol/L, a blood sample was obtained for plasma levels of FFA, insulin, and C-peptide.

At the end of the experiments, mice were anesthetized via an i.v. infusion of

ketamine:xylazine:acepromazine (87:1.7:0.4 mg/ml, 1 ul/g of body weight). The liver and soleus

muscles were collected, snap-frozen in liquid nitrogen, and stored at -80°C for future analyses. In

addition, the mouse pancreases were immediately fixed in formalin over 24h, and then transferred

to a 70% ethanol solution. The samples were embedded in paraffin within 7 days of collection.

Measurements

3.5 Glycemia

The concentration of glucose in plasma samples of mice were measured using the HemoCue

Glucose 201+ System. A very small volume of blood (~5 µL) per sample was required by the

machine. Upon placing a blood sample in the HemoCue microcuvette, saponin hemolyzes the

68

erythrocytes, and the mutarotase enzyme converts α-D-glucose to β-D-glucose. Glucose

dehydrogenase (GDH) then catalyzes the oxidation of β-D-glucose in the presence of NAD,

resulting in the formation of NADH. This production of β-D-glucose and NADH has a molar

relationship to the glucose concentration of the sample. Subsequently, diaphorase catalyzes the

oxidation of NADH in the presence of a specific tetrazolium salt (MTT chromogen). This results

in a redox reaction by which the tetrazolium salt is converted to a colored formazan. The

concentration of the formazan is then quantified photometrically at 660 nm and 840 nm in order

to determine the plasma glucose concentration of the sample.

3.6 Plasma Insulin Levels

Plasma insulin levels were measured using an ELISA kit (Antibody and Immunoassay

Services, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong). A 96-well

microplate was pre-coated with a monoclonal antibody against insulin. Insulin standards of

known concentrations (0, 34.5, 86, 172, 345, 603, and 1210 pM) and the plasma samples were

added to wells in duplicates. Next, an insulin-specific monoclonal antibody conjugated to

horseradish peroxidase (HRP) was added to each well, and the plate was incubated at room

temperature with shaking at 600 rpm for 90 minutes. The HRP-conjugated antibody binds to

insulin that was already bound by the antibody which was anchored to the wells. This effectively

anchors a certain amount of HRP-conjugated antibody to the wells. Subsequently, the wells were

washed with washing buffer in order to remove all substances that were unbound to the wells. A

chromogenic substrate of HRP, 3,3',5,5'-tetramethylbenzidine (TMB), was added, and the mixture

was protected from light. After 15 minutes of incubation at room temperature, a solution which

stops the reaction between TMB and HRP was added. Immediately afterwards, the concentration

of HRP-antibody was visualized at 450 nm using a microplate reader.

69

During every assay, a new standard curve was generated using the insulin standards that

were pipetted in the microplate in duplicate. As the increase in light absorbance from each well is

directly proportional to the concentration of insulin, the levels of insulin in the plasma samples

were determined using the standard curve. The sensitivity of the assay is 34.5 pM, and the

interassay coefficient of variation (CV) determined on reference plasma is less than 10%.

3.7 Plasma C-Peptide Levels

Plasma C-peptide levels were measured using an ELISA kit (ALPCO Diagnostics, Salem,

NH, USA). The kit’s protocol was identical to that of the insulin ELISA kit described above, with

exception that the antibodies used were specific to C-peptide instead of insulin. The sensitivity of

the assay is 7.6 pM, and the interassay CV is less than 10%.

3.8 Plasma FFA Levels

Plasma FFA levels were measured using a colorimetric kit (Wako and Boehringer

Chemicals, Neuss, Germany). Catalyzed by acyl-CoA synthetase (ACS), plasma FFAs acylate

coenzyme A, resulting in the formation of acyl-CoA. Acyl-CoA is then oxidized by the action of

acyl-CoA oxidase, producing H2O2. Subsequently, H2O2 is used by the enzyme peroxidase (POD)

to drive the oxidative condensation of 3-methyl-N-ethyl-N-(B-hydroxyethyl)-aniline (MEHA)

with 4-aminophenazone. This reaction results in the production of a purple coloured adduct, the

concentration of which is then measured colorimetrically at 550 nm with a variability of 1.1%.

The above reactions are summarized below:

𝐹𝐹𝐴 + 𝐴𝑇𝑃 + 𝐶𝑜𝐴𝐴𝐶𝑆→ Acyl-CoA + 𝐴𝑀𝑃 + 𝑃𝑃𝑖

Acyl-CoA + 𝑂2Acyl-CoA oxidase→ 2,3-trans-Enoly-CoA + 𝐻2𝑂2

2 𝐻2𝑂2 + 4-aminoantipyrine + MEHA 𝑃𝑂𝐷→ Purple adduct + 3 𝐻2𝑂

70

3.9 Western Blotting

Mouse liver samples were lysed in RIPA buffer containing protease and phosphatase

inhibitors (Roche). 7 µg of protein lysate from the liver samples were separated across a 7% SDS-

PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. The proteins were

transferred to a polyvinylidene fluoride (PVDF) membrane, and then immunoblotted with a

custom-made rabbit polyclonal primary antibody specific to α-CEACAM1 (1:500; Ab-2457,

Abcam). After washing the membranes, a secondary antibody specific to the primary antibody

was applied (1:5,000; LiCOR Biosciences, Lincoln, NE, USA). After washing, bands of light

produced from the secondary antibodies were visualized. In order to assess the levels of the

loading control tubulin, the membranes were stripped of antibodies, washed, and then reprobed

with a monoclonal antibody against tubulin (1:500; Santa Cruz Biotechnology). The membranes

were then washed and probed with secondary antibody as performed previously. The different

band densities were quantified using the ImageJ software and normalized to total protein content.

The western blotting procedure was performed by Ms. Hilda Ghadieh in Dr. Sonia Najjar’s lab

(University of Toledo, Toledo, OH, USA).

3.10 Insulin Sensitivity Index

The insulin sensitivity index (glucose metabolism value/[insulin]; abbreviated as M/I

where M is GINF + change in plasma glucose pool during an imperfect clamp) was first defined

by Dr. Ralph A. DeFronzo in 1979 at Yale University (New Haven, CT, USA)465. In the condition

that hyperglycemia is at a steady-state and is similar between all experimental groups (which is

our case), the sensitivity index can be calculated by dividing the average glucose infusion rate

(GINF; normalized for body weight) by the plasma insulin concentration during the

hyperglycemic clamp465. This is summarized by the following formula:

71

M/I =Steady-Stat𝒆 𝑮𝑰𝑵𝑭

[𝑰𝒏𝒔𝒖𝒍𝒊𝒏]

M/I is expressed as deciliter per kilogram per minute per microunit per milliliter. This

equation assumes that the insulin-induced change in glucose uptake is proportional to the plasma

concentration of insulin, which is not always the case466.

3.11 Disposition Index

Beta cell function was assessed during the hyperglycemic clamp by using the established

index of relative insulin secretion, the Disposition Index (DI). The DI was used instead of

absolute insulin secretion because the DI accounts for compensatory hyperinsulinemia induced by

changes in insulin sensitivity whereas absolute insulin secretion does not.

The DI was calculated as the product of the index of insulin sensitivity (M/I) and the

concentration of C-peptide in plasma. Plasma levels of C-peptide, as opposed to the insulin

secretion rate, were used since the kinetics of C-peptide (which are necessary for calculation of

insulin secretion rate) are unknown in rodents because C-peptide (species-specific) is not

available for injection and C-peptide kinetics have only been studied in humans and dogs467,468.

The DI has been used in many of our previous studies168,177,180,181,183,184,205,240,449,469,470. The

formula used in the calculation of the DI is depicted below:

𝑫𝑰 = 𝑴/𝑰 ∗ [C-peptide]

3.12 Insulin Clearance Index

The index of endogenous insulin clearance was calculated as the ratio of C-peptide to

insulin plasma levels during basal glycemia and during the hyperglycemic clamp. The ratio of C-

peptide to insulin plasma levels is an established index of the insulin clearance rate465. In cases of

increased insulin clearance rate, the ratio of plasma C-peptide to insulin would be increased as

72

insulin is removed at a higher rate, compared with that of C-peptide (which is cleared via a

different mechanism at a constant rate). By the same logic, a reduction in the insulin clearance

rate would result in a decrease in the ratio of plasma C-peptide to insulin levels. The equation

used to calculate the insulin clearance index is:

𝑰𝒏𝒔𝒖𝒍𝒊𝒏 𝑪𝒍𝒆𝒂𝒓𝒂𝒏𝒄𝒆 𝑰𝒏𝒅𝒆𝒙 =[C-peptide]

[𝑰𝒏𝒔𝒖𝒍𝒊𝒏]

3.13 Statistics

One-way non-parametric analysis of variance (ANOVA) followed by Tukey’s t-test was

used to assess statistical significance between different groups. Data are presented as means ±

standard error (SE). Calculations were performed using the Statistical Analysis System software

(SAS; Cary, NC, USA).

73

Chapter 4

Results

Glucose was i.v. infused into the mice in order to measure the DIs of the beta cells at a

similar degree of hyperglycemia. Prior to the beginning of i.v. glucose infusion, glycemia (basal)

was slightly lower in the NMN+OLE group, compared with the SAL (p < 0.04) and NMN (p <

0.001) groups (Figure 12, below). The OLE group exhibited mildly lower basal glycemia,

compared with the NMN group (p < 0.02). The blood glucose levels were elevated by the 37.5%

glucose infusion to a steady state that was similar between all groups (~22 mM). There was no

significant difference in glycemia between any two groups during the last 30 minutes of the clamp

(Figure 12). The elevation of glycemia during the entire clamp in the NMN+OLE group was

slower than those of the OLE (p < 0.04) and NMN (p < 0.01) groups even though the NMN+OLE

group was i.v. infused with glucose at a significantly higher rate compared with all other groups

(Figure 13). Thus, the NMN+OLE group’s resistance to hyperglycemia, coupled with its

relatively high GINF, compared with the other groups, reflects improved glucose tolerance. This

is consistent with our hypothesis.

74

Figure 12. Gradual elevation in glycemia of wildtype mice i.v. infused with 37.5% glucose during the

hyperglycemic clamp. Male, 11-13 week old, C57BL/6 mice were i.v. infused for 48h with SAL, OLE

(0.4 µmol/min), NMN+OLE (0.025 mg/kg body weight/min of NMN), or NMN alone. After 4h of fasting

at 42h of infusion, the hyperglycemic clamp was initiated at 46h without interrupting the administration of

treatments. Hyperglycemia was maintained during the last 30 minutes of the clamp. ¶ p SAL vs

NMN+OLE during basal < 0.04; ‡ p NMN+OLE vs NMN during basal < 0.001 and during glucose

infusion < 0.01; ¥ p OLE vs NMN during basal < 0.02; † p OLE vs NMN+OLE during glucose infusion

< 0.04. Data are means ± SE.

The glucose infusion rate (GINF) required for maintaining hyperglycemia is a reflection

of glucose tolerance. As expected, the average GINF required for maintaining a steady-state of

hyperglycemia for 30 minutes was significantly lower in the OLE group, compared with the SAL

group (Figure 13 A&B, below). Remarkably, mice co-infused with NMN+OLE had a substantial

increase in GINF, compared with the OLE (p < 0.001) or SAL group (p < 0.03). Interestingly, the

infusion of NMN in the absence of elevated FFA resulted in a significant decrease in GINF,

compared with SAL. The average GINF for the groups over the entire period of i.v. glucose

infusion (Figure 13 C) had a very similar pattern to the average GINF over the last 30 minutes of

the clamp (Figure 13 B).

75

Figure 13. Glucose infusion rate (GINF) required for obtaining and maintaining hyperglycemia in

mice. Co-infusion of NMN + OLE in C57BL/6 mice for 48h substantially improved glucose tolerance,

compared with infusion of OLE or SAL. C57BL/6 mice were i.v. infused for 48h with SAL, OLE (0.4

µmol/min), NMN+OLE (0.025 mg/kg body weight/min of NMN), or NMN alone. (A) Glucose tolerance

was reflected by the GINF required for obtaining and maintaining hyperglycemia. Comparison of the

average GINF required for maintaining hyperglycemia for (B) the last 30 minutes of the clamp and for (C)

the entire i.v. glucose infusion period. * p SAL vs OLE < 0.04; † p OLE vs NMN+OLE < 0.001 (for B

& C); ‡ p NMN+OLE vs NMN < 0.001 (for B & C); ¶ p SAL vs NMN+OLE < 0.03 (for B & C); # p

SAL vs NMN < 0.001 (for B) and < 0.01 (for C). Data are means ± SE.

A.

B. C.

76

The plasma concentration of insulin was measured in order to calculate the Insulin

Sensitivity Index (M/I = Steady-State GINF/[Insulin]), the DI (which is M/I * [C-peptide]), and

the Insulin Clearance Index (which is [C-peptide]/[Insulin]). Plasma insulin levels were similar

between the SAL and OLE groups prior to i.v. glucose infusion (basal glycemia, which was after

4 hours of fasting) and during the hyperglycemic clamp (Figure 14). While insulin levels were

similar between the SAL and OLE groups during hyperglycemia, the GINF required by the OLE

group was lowered due to oleate-induced insulin resistance, as will be shown in Figure 17.

During basal glycemia, the plasma concentration of insulin was significantly higher in the

NMN+OLE group, compared with the SAL group. During hyperglycemia, plasma insulin levels

in the NMN+OLE group were more than twice as high as those in the other groups. The insulin

concentration of the NMN group was not significantly different from that of the SAL or OLE

groups during both basal glycemia and hyperglycemia.

Figure 14. Plasma insulin levels prior to i.v. glucose infusion (basal glycemia) and after the

maintenance of hyperglycemia (clamp) for ~30 minutes in C57BL/6 mice. Mice were i.v. infused for

48h with SAL, OLE (0.4 µmol/min), NMN+OLE (0.025 mg/kg body weight/min of NMN), or NMN

alone. Administration of treatment was maintained during the hyperglycemic clamp experiments. † p OLE

vs NMN+OLE < 0.01; ‡ p NMN+OLE vs NMN <0.001; ¶ p SAL vs NMN+OLE < 0.04 during basal

and < 0.001 during clamp. Data are means ± SE.

77

The plasma concentration of C-peptide was measured in order to determine absolute insulin

secretion (not controlling for insulin resistance), the DI (which is M/I * [C-peptide]), and the

Insulin Clearance Index (which is [C-peptide]/[Insulin]). During basal glycemia, plasma C-

peptide levels tended to be lower in the OLE and NMN+OLE groups, compared with the SAL

group, although they were not statistically significant (Figure 15). During the hyperglycemic

clamp, the NMN+OLE group had a significantly greater C-peptide level, compared with the OLE

and NMN groups. However, this increase in C-peptide level was not proportional to the increase

in insulin levels in the NMN+OLE group, compared with the other groups.

Figure 15. Plasma C-peptide levels prior to i.v. glucose infusion (basal glycemia) and after the



alone. Administration of treatment was maintained during the hyperglycemic clamp experiments. † p OLE

vs NMN+OLE < 0.01; ‡ p NMN+OLE vs NMN < 0.04. Data are means ± SE.

78

During the hyperglycemic clamp, OLE infusion resulted in a significant decrease in the

Insulin Clearance Index (which is [C-peptide]/[Insulin]), compared with SAL infusion (Figure

16). However, during basal glycemia, OLE infusion did not significantly decrease insulin

clearance, compared with SAL infusion. Interestingly, the co-infusion of NMN+OLE resulted in a

significant decrease in insulin clearance during basal glycemia and hyperglycemia, compared with

SAL infusion. Thus, NMN accentuated the oleate-induced decrease in insulin clearance in the

context of elevated FFA levels, although both the OLE and NMN+OLE groups significantly

decreased insulin clearance during hyperglycemia. As NMN infusion on its own did not

significantly alter insulin clearance, the effect of NMN on insulin clearance seems to be specific

to the context of elevated plasma levels of FFA.

Figure 16. Insulin Clearance Indices prior to i.v. glucose infusion (basal glycemia) and after the



alone. Administration of treatment was maintained during the hyperglycemic clamp experiments. * p SAL

vs OLE < 0.05; ‡ p NMN+OLE vs NMN < 0.05 during basal and < 0.001 during clamp; ¶ p SAL vs

NMN+OLE < 0.04 during basal and < 0.001 during clamp. Data are means ± SE.

79

As expected, oleate infusion resulted in a significant decrease in the Insulin Sensitivity

Index (M/I = Steady-State GINF/[Insulin]), compared with SAL infusion (Figure 17). NMN

infusion with or without OLE did not significantly alter insulin sensitivity, compared with the

SAL and OLE groups. Thus, NMN in the context of elevated plasma FFA levels seems to have

overcome insulin resistance through hyperinsulinemia, evidently resulting in a substantial

improvement in glucose tolerance (Figure 13).

Figure 17. Insulin Sensitivity Indices (M/I) obtained after the maintenance of hyperglycemia (clamp)

in C57BL/6 mice for ~30 minutes. Mice were i.v. infused for 48h with SAL, OLE (0.4 µmol/min),

NMN+OLE (0.025 mg/kg body weight/min of NMN), or NMN alone. Administration of treatment was

maintained during the hyperglycemic clamp experiments. * p SAL vs OLE < 0.04; ¶ p SAL vs

NMN+OLE < 0.01. Data are means ± SE.

80

To assess beta cell function in vivo, the DI is typically evaluated as it controls for potential

changes in insulin sensitivity and thus more accurately represents beta cell function, compared

with absolute insulin secretion471. Consistent with our expectations, OLE infusion decreased beta

cell function in mice, compared with SAL infusion (Figure 18, below). NMN+OLE co-infusion

resulted in a DI that was not significantly different, compared with both SAL and OLE groups.

Hence, NMN+OLE co-infusion resulted in an intermediate DI, suggesting partial protection from

FFA-induced beta cell dysfunction.

It is worth noting that, had insulin clearance not changed, the improvement in DI of the

NMN+OLE group relative to that of the OLE group could have been higher because of the

suppressive effects of hyperinsulinemia on insulin secretion in the NMN+OLE group, compared

with the OLE group472. The hypothesis that beta cell function was improved by NMN in the

context of elevated plasma levels of FFA is supported by the significantly higher C-peptide levels

(indicator of insulin secretion) in the NMN+OLE group, compared with the OLE group. Indeed,

the improvement in DI was less than that in C-peptide because of the slightly lower insulin

sensitivity in the NMN+OLE group, compared with the OLE group. Mice infused with NMN in

the absence of elevated FFA levels exhibited a significantly lower DI, compared with the SAL

group.

81

Figure 18. Disposition Indices obtained after the maintenance of hyperglycemia (clamp) in C57BL/6

mice for ~30 minutes. Mice were i.v. infused for 48h with SAL, OLE (0.4 µmol/min), NMN+OLE (0.025

mg/kg body weight/min of NMN), or NMN alone. Administration of treatment was maintained during the

hyperglycemic clamp experiments. * p SAL vs OLE < 0.01; # p SAL vs NMN < 0.04. Data are means ±

SE.

82

The plasma concentration of FFA was measured in order to verify the intended plasma

FFA levels in each group as well as to assess the potential effects of NMN on plasma FFA levels.

48h i.v. infusion of OLE resulted in a 1.5 – 2 fold elevation of FFA levels, compared with SAL

infusion (Figure 19). Interestingly, co-infusion of NMN resulted in a significant decrease in

plasma FFA levels, compared with the OLE group, and NMN alone had a similar trend compared

with the SAL group. These results are consistent with previous findings published in Cell

Metabolism424. Thus, it seems that NMN has a significant FFA-lowering effect in the context of

elevated FFA levels.

At the end of the hyperglycemic clamps, plasma FFA levels in all groups had moderately

decreased, compared with their levels prior to the commencement of the clamp (basal glycemia).

This decrease in FFA levels is expected and is due to hyperinsulinemia increasing FFA uptake and

decreasing lipolysis by adipocytes473. As the NMN+OLE group exhibited higher plasma levels of

insulin, compared with the other groups, its relatively greater decrease in FFA levels is logical.

Figure 19. Plasma FFA levels of mice during basal glycemia (after 4 h of fasting and prior to glucose

infusion) and during hyperglycemia (after ~30 minutes at steady-state). Wildtype mice were i.v.

infused for 48h with SAL, OLE (0.4 µmol/min), NMN+OLE (0.025 mg/kg body weight/min of NMN), or

NMN alone. * p SAL vs OLE < 0.001; † p OLE vs NMN+OLE < 0.04; ‡ p NMN+OLE vs NMN <

0.001; ¶ p SAL vs NMN+OLE < 0.01; ¥ p OLE vs NMN < 0.02. Data are means ± SE.

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Since we unexpectedly found a decrease in insulin clearance in the NMN + OLE group

versus the OLE group alone, we collaborated with a renowned expert in CEACAM biology, Dr.

Sonia Najjar (University of Toledo), in order to measure CEACAM1 protein levels in livers of

mice belonging to our treatment groups. As shown in Figure 20 (below), CEACAM1 protein

expression was significantly decreased in the NMN+OLE group, compared with controls.

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Figure 20. Western blots of CEACAM1 (Cc1) performed on livers of mice infused with SAL, OLE,

NMN+OLE, or NMN for 48h. 7 µg of protein lysates from each liver were separated using 7% SDS-

PAGE gel. Proteins were immunoblotted (Ib) with polyclonal antibodies against α-CEACAM1 (1:500, Ab-

2457) and reimmunoblotted (reIb) with monoclonal antibodies against Tubulin (loading control; Santa

Cruz Biotech). Proteins were visualized using LiCOR secondary antibodies according to manufacturer’s

instructions (LiCOR Biosciences, Lincoln, BE). Protein band density was quantified using Image J.

Analysis was performed using one-way ANOVA. Mouse treatment and liver collection were performed by

A.N.; western blotting for CEACAM1 was carried out by Ms. Hilda Ghadieh, at Dr. Sonia Najjar’s lab,

University of Toledo. * p SAL vs NMN+OLE < 0.05; ¶ p NMN+OLE vs NMN < 0.05. Data are means ±

SE.

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Chapter 5

Discussion

In C57BL/6 mice, we have obtained sufficient evidence supporting the notion that NMN

administration in the context of elevated plasma FFA levels substantially improves glucose

tolerance. Interestingly, NMN administration in the context of normal plasma levels of FFA had

an opposite effect on glucose tolerance. As NMN is metabolized into NAD, which is involved in

numerous physiological processes, the mechanisms behind the dual effects of NMN

administration are not clear. Nevertheless, there are a several possibilities that may explain these

results. These possibilities are described in the next few pages.

Our hyperglycemic clamps have demonstrated that NMN administration in the context of

elevated plasma FFA levels accentuated the FFA-induced decrease in insulin clearance. This was

evident by the co-infusion of NMN with OLE resulting in a significant difference in insulin

clearance, compared with the SAL group. This was in contrast to the OLE group not being

significantly different in insulin clearance, compared with the SAL group during basal glycemia.

In addition to substantially improving glucose tolerance, decreased insulin clearance may relieve

beta cell overload by helping overcome insulin resistance, thus slowing the deterioration of beta

cell function.

The substantial decrease in insulin clearance in the NMN+OLE group, compared with the

OLE group, may have been due to decreased CEACAM1 expression in the NMN+OLE group.

Indeed, Western blots of CEACAM1 which were produced in collaboration with Dr. Najjar’s lab

support our hypothesis that NMN administration decreased CEACAM1 expression in the liver in

the context of elevated plasma FFA levels (Figure 20); however, NMN resulted in no significant

difference in the context of normal FFA levels, compared with controls. NMN administration

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results in increased NAD availability, which consequently increases SIRT1 activity. Thus, the

NMN+OLE-induced decrease in CEACAM1 expression may be due to increased SIRT1 activity

and/or other pathways affected by NAD.

Since OLE infusion decreased beta cell function, and since NMN+OLE co-infusion did

not result in a significant decrease in DI, compared with SAL infusion, NMN+OLE co-infusion

partially rescued beta cell function. As mentioned earlier, the DI of the NMN+OLE group could

have been higher than reported had insulin secretion not been suppressed by hyperinsulinemia

(due to decreased insulin clearance) or had insulin sensitivity (M/I) not been slightly (although

not significantly) lower, compared with the OLE group (since DI = M/I * [C-peptide]).

Intriguingly, NMN infusion in the presence of normal plasma FFA levels significantly decreased

DI, compared with the SAL group. Consistent with our results, a 2008 study has reported a

decrease in glucose tolerance and insulin secretion (assessed using an IPGTT) after NMN

administration in 20-month-old male mice fed a normal diet, compared with saline

administration458.

After 4h of fasting and before the hyperglycemic clamp, the insulin concentration in the

NMN+OLE group tended to be higher than those of the other groups due to decreased insulin

clearance. The aforementioned three parameters: decreased insulin clearance, increased beta cell

function, and elevated basal insulin levels seem to have collectively resulted in high levels of

plasma insulin during the hyperglycemic clamps. Although oleate-induced insulin resistance was

not ameliorated, the over-two-fold elevation of plasma insulin concentration in the NMN+OLE

group resulted in a considerable improvement in glucose tolerance.

Interestingly, NMN infusion in the context of normal FFA levels decreased beta cell

function, compared with the SAL group. Consistently, the NMN-only group tended to have lower

plasma levels of insulin, compared with the SAL and OLE groups. Although NMN did not

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significantly alter insulin sensitivity, the decrease in beta cell function seems to have significantly

decreased glucose tolerance, compared with the SAL group.

5.1 Potential Mechanisms Behind NMN’s Effects on

Insulin Clearance

With regards to insulin clearance, the mechanisms are relatively challenging to explore as

insulin uptake and degradation are complex, are not well-understood, and involve multiple

tissues. As described earlier, insulin clearance occurs mainly in hepatocytes via CEACAM1-

mediated insulin-insulin-receptor complex endocytosis followed by IDE-mediated endosomal

degradation. Currently, there is very little research available connecting NMN metabolism with

CEACAM1 regulation.

As described earlier, insulin clearance is known to be decreased by elevated plasma FFA

levels and liver fat content462,474. Nevertheless, in our study, the accentuation of the decrease in

insulin clearance in the NMN+OLE group occurred despite a significant decrease in FFA levels,

compared with the OLE group. This finding indicates that the accentuation of the FFA-induced

decrease in insulin clearance in the NMN+OLE group was not caused by the change in FFA

levels, but by the co-infusion of NMN.

5.1.1 Synergistic Effects of SIRT1 and FFA on PPARα-Mediated Decrease in

CEACAM1 Expression

Long-chain FFA, such as oleate, are activating ligands for PPARα475–479, which is highly

expressed in the liver and directly decreases CEACAM1 transcription, as will be explained below.

A study found that treatment of cultured hepatoma cells with a PPARα agonist (Wy14,643)

resulted in decreased promoter activity of rat and mouse Ceacam1, compared with controls480. In

the same study, treatment of hepatoma cells with Wy14,643 also resulted in a rapid decrease in

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CEACAM1 mRNA and protein levels. In Pparα+/+ mice, but not Pparα-/- mice, supplementing a

chow diet with Wy14,643 resulted in a decrease in hepatic CEACAM1 mRNA and protein levels,

and revealed that ligated PPARα was bound to the Ceacam1 promoter in liver lysates480.

As described earlier in my thesis, one of the main targets of deacetylation by SIRT1 is

PGC-1α333,337,481,482. PGC-1α is activated via direct deacetylation by SIRT1 at specific lysine

residues333,337. Moreover, as FOXO1 serves as a transcription factor for both PGC-1α and SIRT1

in hepatocytes, and SIRT1 activates FOXO1, FOXO1 could amplify the effects of SIRT1 on

PGC-1α via a feedforward mechanism483,484. PGC-1α, in addition to being a co-activator for

PPARγ, interacts with and co-activates PPARα482,485,486. Hence, FFA and NMN may play a

synergistic role in the activation of PPARα and consequent decrease in CEACAM1 expression.

This would explain the accentuated decrease in insulin clearance observed in our NMN+OLE

group, compared with the OLE group.

Further research is required to identify the co-activators and co-repressors involved in the

binding of PPARα to the CEACAM1 promoter in murine hepatocytes; however, chromatin

immunoprecipitation (ChIP) analysis suggests that liganded PPARα directly regulates

CEACAM1 expression480. Consistently, mice treated with potent PPARα-selective agonists

exhibited decreased CEACAM1 mRNA in the liver in vivo487. Thus, current evidence supports the

hypothesis that an NMN-mediated increase in hepatic NAD levels resulted in increased SIRT1

activity, which led to the deacetylation of PGC-1α and the subsequent activation of PPARα,

which directly silenced the Ceacam1 promoter. This ultimately resulted in decreased hepatic

insulin clearance in vivo.

In addition to inhibiting CEACAM1 transcription, PPARα increases the transcription of

many genes (such as carnitine palmitoyltransferase 1, Acadl, and Acadv1) which shift hepatic fuel

metabolism towards mitochondrial fatty acid oxidation and away from glucose oxidation488–497.

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This results in the conservation of glucose for the central nervous system488–497. This increased

beta oxidation could also account for the decrease in plasma FFA levels in groups infused with

NMN, compared with their controls.

5.1.2 NMN Accentuates the FFA–Akt–FOXO1–PGC-1α–PPARα–Mediated Decrease

in CEACAM Expression

PGC-1α can act as a co-activator for FOXO1 by directly interacting with FOXO1, resulting

in the formation of a PGC-1α-FOXO1 complex483. Specifically, the carboxy-terminal of PGC-1α

binds with the amino-terminal of FOXO1483. Using a constitutively active form of Akt, it has been

shown that the direct phosphorylation of FOXO1 by Akt strongly impedes the physical interaction

between FOXO1 and PGC-1α483. Furthermore, phosphorylation of FOXO1 by Akt is known to

result in the nuclear exclusion of FOXO1498,499. Hence, the insulin signaling pathway regulates the

co-activation of FOXO1 by PGC-1α as well as the subcellular localization of FOXO1499,500.

Insulin action has been shown to increase CEACAM1 transcription and insulin clearance501.

Since FOXO1 functions as a transcription factor for PGC-1α in hepatocytes483,484, the Akt-

mediated nuclear exclusion of FOXO1 results in decreased PGC-1α expression and activity, and

consequently decreased co-activation of PPARα by PGC-1α482,485,486. As activated PPARα

decreases CEACAM1 transcription by directly binding to its promoter480, decreased PPARα

activity results in increased CEACAM1 expression and insulin clearance487. Thus, the Akt-

mediated decrease in FOXO1 transcriptional activity results in reduced PGC-1α expression,

which leads to decreased co-activation of PPARα. By this indirect pathway and/or perhaps by

direct action of FOXO1 on the CEACAM1 promoter, CEACAM1 expression is upregulated502.

Moreover, since FOXO1 is also a transcription factor for SIRT1381, the Akt-mediated

nuclear exclusion of FOXO1 results in decreased SIRT1 expression and activity. As SIRT1

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directly activates both PGC-1α and FOXO1 by deacetylation333,337, this leads to the decreased

activation of FOXO1 by SIRT1 and thus a further reduction in CEACAM1 expression.

In our study, the elevation of plasma FFA levels resulted in the development of insulin

resistance in the OLE and NMN+OLE groups. Elevated plasma FFA levels can induce insulin

resistance as follows: as insulin resistance involves the serine-phosphorylation of IRS286,287,300,

this leads to decreased Akt activation by IRS, which consequently leads to decreased FOXO1

inhibition and nuclear exclusion by Akt. As a result, FOXO1’s upregulation of PGC-1α

transcription is less impaired by Akt, leading to increased co-activation of PPARα by PGC-

1α482,485,486. Activated PPARα decreases CEACAM1 transcription by directly binding to its

promoter480, resulting in decreased insulin clearance487. Thus, decreased insulin action also results

in decreased insulin clearance. Conversely, insulin increases its own clearance by upregulating

CEACAM1 expression118.

In our study, administration of NMN accentuated the reduction in insulin clearance caused

by elevated plasma FFA levels in vivo. In addition to a synergistic effect on PPARα due to FFA

activation of PPARα and PGC-1α activation by NMN-induced SIRT1 activity, as described

previously, a synergistic effect between FFA and NMN may have occurred at the level of

FOXO1. FOXO1 may have been activated by FFA-induced insulin resistance as well as by the

increase in NAD levels and consequently SIRT1 activity following NMN administration. Hence,

NMN and FFAs may have synergistically activated FOXO1 in the group treated with both NMN

and OLE, resulting in further stimulation of the PGC-1α – PPARα pathway, which decreases

CEACAM1 expression and insulin clearance. Moreover, since the administration of NMN on its

own did not decrease CEACAM1 expression in our study, the activation of FOXO1 and/or PGC-

1α by the administration of NMN alone may not be sufficient. It would be insightful to conduct a

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study in which the activities of FOXO1 and PPARα are assessed in the mouse liver samples

collected in our study.

5.1.3 Potentially Opposing Effects of FFA and SIRT1 on PKCδ/ε-Mediated Decrease

in Insulin Clearance

As described earlier in my thesis, our lab had demonstrated that oleate induces the

translocation of PKCδ from the cytosol to the membrane fractions of hepatocytes316. PKCδ

increases the internalization of membrane receptors, including the insulin receptor, via a non-

specific and ligand-independent process of coated-pits internalization317. This may be mediated

by the phosphorylation of internalization motifs on the cytoplasmic juxtamembrane domain of the

membrane receptors317,318. By decreasing the number of insulin receptors available on

hepatocytes, PKCδ mediates the FFA-induced decrease in insulin binding316. Since insulin

clearance requires the binding of insulin to its receptor, and is predominantly performed by

hepatocytes, PKCδ may mediate the FFA-induced decrease in insulin clearance.

In insulin-targeted cells, FFAs are known to also activate PKCε503–505, and both PKCδ and

ε lead to decreased insulin sensitivity506,507. Since the insulin signaling pathway is in part required

for insulin clearance in addition to insulin action, this would be expected to also lead to reduced

insulin clearance.

However, surprisingly, PKCε-null mice were shown to exhibit reduced hepatic insulin

clearance, compared with wildtype controls314. Furthermore, PKCε-null mice exhibited higher

circulating insulin levels during an intraperitoneal glucose tolerance test (IPGTT) despite having

similar rates of C-peptide secretion, compared with controls314. This finding would be consistent

with a study in human endometrial carcinoma cells, which has shown that pharmacological

activation of several PKC isoforms, including PKCδ and ε, results in increased CEACAM1

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expression508. However, treatment with PKC inhibitor (calphostin C) did not alter CEACAM1

phosphorylation in colon carcinoma cells319. Thus, the effect of PKC isoforms on insulin

clearance and CEACAM1 expression remains unclear.

In contrast to the effects of FFA on activating PKCδ and ε, a recent study has shown that

adenoviral-mediated SIRT1 overexpression in an immortalized mouse hepatocyte cell line509,510

inhibits PKC activity (isoforms were not specified)511. The mechanism by which SIRT1 may have

decreased PKC activity is not known511. No other studies suggesting that SIRT1 activity inhibits

PKC have been found. If increased SIRT1 activity does in fact inhibit PKCδ and ε activities, then

SIRT1 would have an effect on insulin clearance and sensitivity opposite to that of FFA.

However, in our study, the combination of NMN and elevated FFA levels resulted in a synergistic

effect on insulin clearance and no significant effect on insulin sensitivity, compared with elevated

plasma levels of FFA alone.

5.2 Potential Mechanisms Behind NMN’s Effects on

Beta Cell Function

A number of mechanisms could explain our results regarding the effects of NMN on beta

cell function in the context of normal and elevated plasma FFA levels. These mechanisms are

explained below.

5.2.1 SIRT1-FOXO1/3-Mediated Reduction of Oxidative Stress Ameliorated FFA-

Induced Beta Cell Dysfunction

Although we have shown, in collaboration with Dr. Jamie Joseph’s lab, that OLE does not

decrease NAD or NADH levels or ratios in islets (Figure 10), the increase in NAD levels by

NMN administration seems to have increased SIRT1 activity enough to partially counter-act

FFA-induced beta cell dysfunction in vivo.

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As described earlier in my thesis, FOXO1/3, which are activated by SIRT1, are known to

decrease oxidative stress by upregulating antioxidant genes, such as MnSOD, glutathione, and

catalase348,378. Thus, it is possible that the NMN-induced increase in SIRT1 activity reduced the

FFA-induced oxidative stress by increasing the levels of antioxidants via FOXO1/3. This would

have culminated in ameliorated FFA-induced beta cell dysfunction.

It is also possible that increased SIRT1 activity resulted in the repression of UCP2

transcription by directly binding to the UCP2 promoter336. Since UCP2 is involved in dissipating

the H+ electrochemical gradient in the mitochondrial membranes, decreased UCP2 expression

results in improved efficiency of oxidative phosphorylation and thus increased ATP levels257,347.

As insulin secretion depends on the availability of ATP, it is also increased346. However, using a

novel beta cell-specific UCP2-knockout model, a recent study has demonstrated that UCP2 does

not play a large role as a metabolic uncoupler in beta cells, but rather is important in the

regulation of ROS levels276. Furthermore, preliminary measurements of UCP2 mRNA levels in

our lab have shown no significant differences between any of the SAL, OLE, OLE + resveratrol

(RSV), and RSV groups (unpublished data). Thus, NMN’s partial protection against FFA-induced

beta cell dysfunction is likely due to a decrease in oxidative stress caused by increased antioxidant

expression, and not caused by decreased UCP2 expression.

Our results regarding the effects of NMN on beta cell dysfunction in the context of

elevated plasma FFA levels complement our previous studies which demonstrated SIRT1’s

protective effects on FFA-induced beta cell dysfunction using the SIRT1 activator RSV and the

BESTO (beta cell-specific SIRT1-overexpression) genetic model (Figure 8 &Figure 9).

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5.2.2 Decreased Plasma FFA Levels Resulted in the Amelioration of FFA-Induced

Beta Cell Dysfunction

With regards to plasma FFA levels, NMN+OLE co-infusion resulted in an intermediate

elevation of FFA levels, compared with the SAL and OLE groups (p < 0.01 vs SAL and p < 0.04

vs OLE). Thus, NMN administration in the context of oleate infusion seems to decrease plasma

FFA levels. It is possible that the NMN-induced decrease in FFA levels in the NMN+OLE group,

compared with the OLE group, had reduced the amplitude of beta cell dysfunction induced by

FFAs. The decrease in plasma FFA levels could be due to increased beta oxidation mediated by

SIRT1 and PPARα in the skeletal muscles512.

In many cells, NMN is converted to NAD by NMNAT513. In skeletal muscles, increased

NAD levels increase SIRT1 activity. SIRT1 then directly activates the transcriptional co-activator

PGC-1α, which, through PPARα, increases mitochondrial biogenesis and the expression of

mitochondrial fatty acid oxidation genes514. These genes include pyruvate dehydrogenase kinase 4

(PDK4), carnitine palmitoyltransferase 1B (CPT1B), and medium-chain acyl-CoA dehydrogenase

(MCAD). Increased beta oxidation would reduce the levels of FFA in plasma515.

5.2.3 Excess Antioxidants During Normal Plasma FFA and Intracellular ROS Levels

Resulted in Sub-Optimal ROS Levels and Decreased GSIS

Unlike the positive effect on beta cell function caused by NMN administration in the

context of elevated plasma FFA levels, NMN in the presence of normal plasma FFA levels

resulted in a significant decrease in beta cell function. There is a mechanism that can explain the

dual effects of NMN on beta cell function. This mechanism depends on the intracellular levels of

ROS (i.e. presence or absence of FFA-induced oxidative stress). As the dose of NMN

administered was relatively high (0.025 mg/kg body weight/min), it may have resulted in excess

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expression of antioxidant enzymes, which in the presence of normal levels of plasma FFA, led to

sub-optimal levels of ROS. Sub-optimal levels of ROS are known to result in impaired insulin

secretion257,266,279,280.

As described earlier in my thesis, it has been shown that exposure of beta cells to high

levels of antioxidants in the absence of oxidative stress negatively affects GSIS266. Some studies

have found that ROS can be important signaling molecules in insulin secretion257,266. For instance,

a transient increase in ROS levels has been shown to promote insulin signaling via the oxidation

and inhibition of RPTPs (receptor-like protein tyrosine phosphatases), which normally impair the

tyrosine-phosphorylation-mediated activation of the insulin receptor279,280. This mechanism can

be tested by measuring ROS as well as intracellular markers of oxidative stress, such as MDA and

8-OHdG, as described earlier268.

5.2.4 SIRT1-FOXO1-Mediated Nuclear Exclusion of Pdx-1, Resulting in Decreased

Insulin Secretion

There is another mechanism that can potentially explain the NMN-induced decrease in

beta cell function in the context of normal plasma levels of FFA. It is possible that the NAD-

SIRT1-FOXO1 pathway resulted in the nuclear exclusion of the key insulin transcription factor

Pdx-1348. This would have resulted in a decrease in beta cell function during normal plasma FFA

levels and absence of oxidative stress, whereas in the presence of FFA-induced oxidative stress,

the increased transcription of antioxidant genes induced by FOXO1/3 would have resulted in

overall beneficial effects on beta cell function.

5.2.5 NAD-Mediated Increase in PARP Activity Decreases ATP for Insulin Secretion

There is a third potential mechanism by which NMN in the context of normal plasma

levels of FFA results in decreased beta cell function. As described earlier, since PARP consumes

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NAD during its DNA repair action401, an increase in NAD levels can increase PARP activity. In a

normal physiological context, excessive PARP activity can lead to NAD depletion and a

consequent impairment of the TCA cycle and ATP production. However, in our experiments,

PARP is very unlikely to have depleted NAD levels due to the supplementation of NMN.

Nevertheless, PARP may have been involved in decreased beta cell function. In cells of

the proximal tubule of the kidney subjected to ischemic renal injury, PARP1 has been reported to

directly inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity via poly(ADP-

ribosyl)ation in vivo and in vitro516. As GAPDH catalyzes the sixth step in glycolysis, this results

in decreased glycolysis and thus decreased ATP production516,517. The effects of GAPDH on ATP

production were confirmed by the inhibition of GAPDH activity using a GAPDH inhibitor

(iodoacetamide) in a pig kidney epithelial cell line (LLCPK1)516. Iodoacetamide inhibits GAPDH

activity by directly modifying cysteine residues in the GAPDH active site518,519. Iodoacetamide

treatment was found to dose-dependently inhibit GAPDH activity and thus decrease glycolysis

and ATP production. Since ATP is required for insulin secretion, this results in decreased beta

cell function. This mechanism can be assessed by measuring the levels of ATP and D-glycerate

1,3-bisphosphate (product of GAPDH) in islets or beta cell lines. In addition, it would be

informative to measure PARP activity in islets or beta cell lines520.

Similar to the case of nuclear exclusion of Pdx-1, it is possible that the beneficial effect of

increased expression of antioxidant enzymes in the presence of significant FFA-induced oxidative

stress outweighed the negative effect of decreased ATP production in beta cells. This would

explain the partially protective effect of NMN on beta cell function observed in the NMN+OLE

group, compared with the OLE group.

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5.3 Limitations

The utilization of the ratio of C-peptide to insulin plasma levels obtained during

hyperglycemic clamps has its limitation as an index of endogenous whole-body insulin clearance

as it requires a relatively steady-state of glycemia and insulin secretion. In addition, the

hyperglycemic clamp is not the gold-standard method of assessing insulin sensitivity due to the

potential differences in plasma levels of insulin across different groups and due to the non-

linearity of insulin action with respect to plasma levels of insulin. The hyperglycemic clamp was

used in this study since, based on our previous studies involving the activation of SIRT1 by

resveratrol and genetic overexpression, we expected to observe changes mainly in beta cell

function (for which the hyperglycemic clamp is the gold standard), and not in insulin clearance. It

would have not been feasible to perform both hyperglycemic and hyperinsulinemic-euglycemic

clamps on the same mice due to the limited blood volume in mice and the excessive stress which

would have been caused by their experience of both clamps.

Hyperinsulinemia caused by decreased insulin clearance has been reported to result in

secondary insulin resistance by the downregulation of insulin receptors on hepatocytes111. Global

CEACAM1-null mice and liver-specific dominant-negative phosphorylation-defective S503A

CEACAM1 (L-SACC1) mutants have been known to develop secondary insulin resistance due to

chronic hyperinsulinemia111,120,521. Hyperinsulinemia can also upregulate hepatic lipogenesis via

the insulin-mTORC2-AKT signaling pathway, which can contribute towards hepatic steatosis and

a further decrease in insulin clearance522.

As we did not find secondary insulin resistance in our studies, it appears that the

chronicity and degree of inhibition of CEACAM1 activity determine whether or not secondary

insulin resistance will develop from hyperinsulinemia. In the context of decreased CEACAM1

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expression via PPARα activation, other studies have shown improved insulin sensitivity and

reduced dyslipidemia480,523–525. Thus, it seems that the effect of PPARα activation to increase

insulin sensitivity prevails over the effect of decreased CEACAM1 expression to decrease it.

In our study, insulin sensitivity was not improved by NMN, and in fact, it was slightly but

not significantly decreased (Figure 17). It is also possible that the two effects of NMN to

decrease CEACAM1 expression and activate PPARα resulted in a balance between the opposite

effects of CEACAM1 and PPARα on insulin sensitivity. Hence, although CEACAM1 activity

tended to induce a decrease in insulin sensitivity, the activation of PPARα prevented that from

happening480,523–525.

5.4 Future Directions

As mentioned above, the hyperglycemic clamp method is the gold-standard for the

assessment of beta cell function, but not of insulin clearance and sensitivity, in vivo. As the

hyperinsulinemic-euglycemic clamp is a more accurate and direct assessment of insulin clearance

and sensitivity (because the plasma insulin levels in all groups would be equalized), compared

with the hyperglycemic clamp method, future experiments should include performing

hyperinsulinemic-euglycemic clamps. In addition, insulin labeled with radioactive iodine (I125)

could be administered during the hyperinsulinemic-euglycemic clamps in order to more

accurately measure insulin clearance (independent of the plasma levels of insulin) and to follow

up on the insulin uptake by hepatocytes in tissue studies. Insulin uptake by hepatocytes may also

be examined in vitro. In order to study the effects of NMN on beta cell function independent of

the effects of changes in insulin clearance, it would be informative to assess beta cell function of

islets ex vivo (after i.v. administration of NMN to animals) or in vitro (after direct administration

of NMN to isolated islets).

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Regarding the SIRT1-PGC-1α-PPARα-mediated mechanism by which NMN may have

decreased CEACAM1 expression, the activity of PPARα in liver samples could be assessed by

measuring mRNA levels of Cyp4A10 and Cyp4A14, which are targets genes of PPARα and

extremely sensitive markers of its activity in mouse hepatocytes497. In addition, the activity and

expression of SIRT1 and PGC-1α in our mouse livers may be worth measuring in order to assess

their potential roles in the effects of NMN on hepatocytes. In response to elevated plasma FFA

levels, we expect the activity and expression of SIRT1 and PPARα to be decreased and increased,

respectively.

Since FFA and NMN both lead to increased PPARα activity, they may have had

synergistic effects on PPARα activation. Hence, we would expect PPARα activity to be greatest

in the NMN+OLE group, compared with the other groups. If this turns out to be the case, it would

indicate that both NMN and FFA are required to sufficiently activate PPARα in order to decrease

CEACAM1 expression, compared with the NMN group and OLE group. On the other hand, since

SIRT1 activity is decreased by FFA but increased by NMN administration, we would expect to

see an intermediate degree of SIRT1 activity in the NMN+OLE group, compared with the OLE

and NMN groups. Since FOXO1 may amplify the effects of SIRT1 on the activation of PGC-1α

and consequently PPARα, as described earlier, it would be informative to examine the activity

and expression of FOXO1, particularly in the nucleus. The potentially opposing effects of FFA

and SIRT1 on PKCδ and ε and consequently on insulin clearance can be assessed by measuring

the activities of PKCδ and ε in our liver samples.

In order to discern the effects of NMN on hepatic insulin clearance without the potentially

confounding effects of unequal plasma levels of insulin (which can affect the rate of insulin

clearance) between groups in vivo, our collaborator Dr. Sonia Najjar will be performing a similar

study using rat hepatoma cells and human HepG2 cells in vitro. Hepatocytes will be exposed to

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NMN in culture media with or without elevated FFA levels, and the insulin uptake of hepatocytes

will be measured using radioactive insulin. The effects of an increase in SIRT1 activity on

CEACAM expression in hepatocytes will also be assessed.

Preliminary results suggest that NMN and SIRT1 affect CEACAM1 expression differently

in rat, compared with human, hepatocytes in vitro. Specifically, an adenoviral-mediated increase

in SIRT1 activity resulted in decreased CEACAM1 expression in rat hepatoma cells, but

increased CEACAM1 expression in human HepG2 cells, where NMN also increased CEACAM1

expression. Since these data are still preliminary, additional experiments are currently being

conducted by the lab of Dr. Najjar. If the results of the additional experiments are consistent with

the preliminary results, an important question would be raised about the potential differences

between the effects of NMN and SIRT1 on hepatic insulin clearance in rats and in humans, and

whether or not their effects in the HepG2 cells were specific to the cell line and not representative

of human hepatocytes. Thus, it may be important to examine the effects of NMN and SIRT1 on

insulin clearance in humans526. It is worth noting that a very recently launched clinical trial

(March 3rd, 2016) at the Keio University School of Medicine in Japan will be assessing the safety

of oral consumption of NMN in healthy adult men527. New clinical trials regarding NMN will be

described in the “Conclusions” section of my thesis.

It may be worth performing a similar study ex vivo in which animal models would be

treated with NMN in vivo and then the rate of insulin uptake by hepatocytes would be examined

in vitro. This would simulate the physiological conversion of NMN to NAD by eNAMPT in

blood plasma in vivo yet avoid the potential interference of different plasma levels of insulin

between groups by assessing insulin uptake by hepatocytes in vitro. Radioactive insulin could be

used in order to measure the uptake of insulin by hepatocytes.

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Lastly, in both in vitro and ex vivo studies, the expected increase in SIRT1 activity in

hepatocytes following NMN administration can be confirmed since the in vitro system is better

suited to measuring the enzymatic activity of SIRT1. As described earlier, it has been confirmed

in other studies that administration of NMN results in elevated NAD levels and SIRT1 activity in

hepatocytes and beta cells in vivo and in vitro424,427,528,529.

As described earlier, studies regarding the effects of FFA on IDE gave contrasting results.

Nevertheless, it may be useful to gain insight into the levels and activity of IDE in the livers of

our mice. In a study published in Diabetologia in 2012, FFA were shown to decrease hepatic

insulin clearance as well as protein and mRNA levels of high-activity and low-activity IDE in

mouse livers530. We are collaborating with renowned IDE expert Dr. Malcolm A. Leissring, based

at the University of California, Irvine. His lab will be assaying the liver samples of all four of our

mouse treatment groups for IDE expression and activity.

To investigate whether or not sub-optimal levels of ROS were responsible for NMN’s

negative effect on beta cell function in the context of normal plasma FFA levels, and to confirm

the increase in ROS after OLE treatment, the levels of intracellular ROS in our mouse pancreatic

islet cells can be measured. This can be done using the method of dichlorodihydrofluorescein

(DCF) fluorescence531,532, the principle of which is as follows: pancreatic islet cells are incubated

in buffer containing the non-fluorescent chemical 2',7'-dichlorodihydrofluorescein diacetate

(H2DCF-DA). After entering the cells, H2DCF-DA is hydrolyzed by intracellular esterases and

oxidized by ROS to form the highly fluorescent molecule DCF. Fluorescence intensity is then

measured at the excitation and emission wavelengths of 485 and 535 nm, respectively. We would

expect the levels of ROS to be highest in the OLE group and lowest in the NMN group, compared

with the other groups.

102

Since the dose of NMN administered in our study (as well as in several other

studies423,424,427,533) was relatively high, it is possible that excess levels of NMN were responsible

for the adverse effects observed in the context of normal plasma FFA levels. It would be

insightful to conduct a study with the aim of determining the effects of different doses of NMN on

beta cell function. It is possible that a dose of NMN that is lower than the dose utilized in our

study would not result in a decrease in beta cell function in the presence of normal FFA levels,

compared with controls.

In beta cells, the proposed mechanism involving Pdx-1 exclusion via the SIRT1-FOXO1

pathway can be assessed in our mouse pancreas samples. Using immunohistochemistry, Pdx-1

expression and its nucleocytoplasmic localization will be determined in the mouse pancreases

which were fixed in formalin and then embedded in paraffin. The procedure would involve

deparaffinizing the pancreas sections, performing Pdx-1-antigen retrieval, and staining with anti-

Pdx-1 antibodies534.

As described earlier, the NMN-mediated activation of PARP may have been involved in

the decreased beta cell function observed in the absence of oxidative stress by directly inhibiting

GAPDH, consequently impairing glycolysis and ATP production516. This mechanism can be

tested by measuring the activities and expression of PARP and GAPDH in islets or beta cells.

Using immunohistochemistry, the expression of PARP535–537 and GAPDH538–540 in our fixed

pancreas samples can be assessed.

103

5.5 Conclusions

Overall, our study provides evidence that NMN administration in the presence of elevated

plasma FFA levels (which simulates obesity) results in substantially improved glucose tolerance

by significantly decreasing insulin clearance and partially protecting against FFA-induced beta

cell dysfunction in vivo. However, in the presence of normal FFA levels, NMN administration

does not affect insulin clearance and significantly reduces beta cell function. NMN resulted in

decreased plasma FFA levels in the context of either elevated or normal plasma FFA levels.

Although the decrease in plasma FFA levels is not responsible for many of the effects induced by

NMN (as explained earlier), the NMN-induced decrease in plasma FFA levels would be

beneficial in increasing the chance of reversing the progression of T2DM60,62,541 and in reducing

the risk of developing fat-related diseases other than T2DM, such as metabolic syndrome and

cardiovascular disease34,36,65,115,198,443,524,542,543.

A very recent clinical trial (initiated on March 3rd, 2016) at the Keio University School of

Medicine in Japan is investigating the safety of oral consumption of NMN in healthy adult

men527. This trial will also evaluate parameters related to metabolic syndrome in order to assess

the potential benefits of NMN as an orally administered drug527. The few studies which have

utilized the administration of NMN in human cells are encouraging regarding the safety of NMN

administration in humans544–546. In addition, based on an email exchange between me and Dr.

David Sinclair, Professor at Harvard University, Dr. Sinclair may begin a clinical trial within a

year in order to investigate the potential ability of NMN to slow the aging process in humans, as it

had done in mice547. Thus, depending on the results of the clinical trials, it may be safe to begin

clinical trials assessing the effects of NMN on glucose tolerance, insulin clearance, and FFA-

induced beta cell dysfunction.

104

Due to Dr. Najjar’s preliminary results showing that adenoviral- and NMN-mediated

activation of SIRT1 induced an increase in CEACAM1 expression in human HepG2 cells, it is

not clear whether or not the administration of NMN in humans will have effects similar to those

observed in our mouse study. If the effects are similar, NMN administration in humans with

elevated plasma levels of FFA (such as patients with T2DM) would significantly improve glucose

tolerance. This would occur via decreased insulin clearance and partial protection against FFA-

induced beta cell dysfunction. However, NMN administration in humans with normal plasma

levels of FFA may have negative effects on beta cell function.

Thus, if clinical trials regarding the effects of NMN on glucose tolerance, insulin

clearance, and FFA-induced beta cell dysfunction end up being consistent with our results, NMN

would be a beneficial treatment for individuals with elevated plasma FFA levels and impaired

glucose tolerance. If individuals no longer have elevated plasma levels of FFA (such as through

weight loss), their treatment with NMN might need to be ceased in order to avoid the net negative

effect of NMN on glucose tolerance in the absence of oxidative stress.

105

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