Nicotinamide Mononucleotide in the Context of Elevated Plasma Levels of Free Fatty Acids ...

147 Pages·2016·2.63 MB·English

by  Ash Nahle

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)
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.
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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!
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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
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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

v
List of Abbreviations

8-OHdG – 8-hydroxy-2’-deoxyguanosine GIP-R – Gastric Inhibitory Polypeptide
ACS – Acyl-CoA Synthetase Receptor
ANOVA – One-way non-parametric analysis GLP-1 – Glucagon-like Peptide-1
of Variance GLP1R – Glucagon-like Peptide 1 Receptor
AROS – Active Regulator of SIRT1 GLUT1/2 – Glucose transporter 1/2
ATM – Ataxia Telangiectasia Mutated GPCR – G-protein-coupled receptor
ATP - Adenosine triphosphate GPR40/41/43/119 – G-protein-coupled
BAT – Brown Adipose Tissue receptor 40/41/43/119
BIM – Bisindolylmaleimide GPx4 – Glutathione Peroxidase-4
BMI – Body Mass Index GSIS – Glucose-Stimulated Insulin
BSA – Bovine Serum Albumin Secretion
CDA – Canadian Diabetes Association H DCF-DA - 2',7'-dichlorodihydrofluorescein
2
Cdk-1 – Cyclin-dependent kinase-1 diacetate
CEACAM1 – Carcinoembryonic Antigen- HFD – High Fat Diet
related Cell Adhesion Molecule-1 HLA – Human Leukocyte Antigens
ChIP – Chromatin Immunoprecipitation HRP – Horseradish peroxidase
CHOP – CCAAT-enhancer-binding protein IDE – Insulin Degrading Enzyme
homologous protein IH – Intralipid + Heparin
CPT1B – Carnitine palmitoyltransferase 1B IKKB – Inhibitor of nuclear factor kappa-B
CV – Coefficient of Variation kinase subunit beta
DAG – Diacylglycerol IPGTT – Intraperitoneal Glucose Tolerance
DBC-1 – Deleted in Breast Cancer-1 Test
DCF – Dichlorodihydrofluorescein IR – Insulin Receptor
DI – Disposition Index IRS – Insulin Receptor Substrate
DKA – Diabetic Ketoacidosis IκBα – Nuclear factor of kappa light
DPP-4 – Dipeptidyl Peptidase-4 polypeptide gene enhancer in B-cells
ER – Endoplasmic Reticulum inhibitor, alpha
ETC – Electron Transport Chain JNK – c-Jun-N-terminal Kinase
FFA – Free Fatty Acid K – Michaelis constant
M
FPG – Fasting Plasma Glucose LC-CoA – Long-Chain Coenzyme A
FFAR – Free Fatty Acid Receptor LDCV – Large Dense Core Vesicles
FOXO – Forkhead box-O LPL – Lipoprotein Lipase
FRD – Fructose-Rich Diet L-SACC1 - Liver-specific dominant-negative
GAPDH – Glyceraldehyde-3-phosphate phosphorylation-defective S503A CEACAM1
dehydrogenase LXR – Liver X Receptor
GDH – Glucose dehydrogenase M/I – Glucose metabolism value/[insulin]
GDM – Gestational Diabetes Mellitus (Index of insulin sensitivity)
GINF – Glucose Infusion Rate MCAD – Medium-chain acyl-CoA
GIP – Gastric Inhibitory Polypeptide dehydrogenase
MDA – Malondialdehyde
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MEHA – 3-methyl-N-ethyl-N-(B- RER – Rough Endoplasmic Reticulum
hydroxyethyl)-aniline ROS – Reactive Oxygen Species
MnSOD – Manganese superoxide dismutase RPMI media – Roswell Park Memorial
MUFA – Monounsaturated Fatty Acid Institute media
NAC – N-acetylcysteine RPTPs – Receptor-like Protein Tyrosine
NAD – Nicotinamide Adenosine Phosphatases
Dinucleotide RPTPs – Receptor-like Protein Tyrosine
NADPH oxidase – Nicotinamide Adenine Phosphatases
Dinucleotide Phosphate oxidase SAL – Saline
NAMPT – Nicotinamide SAS – Statistical Analysis System
phosphoribosyltransferase SDS-PAGE – Sodium dodecyl sulfate
NCLX – Na+/Ca2+ exchanger polyacrylamide gel electrophoresis
NCoR – Nuclear receptor co-repressor SE – Standard Error
NFκB – Nuclear factor kappa-light-chain- SF-1 – Steroidogenic Factor-1
enhancer of activated B cells SFA – Saturated Fatty Acid
NMN – Nicotinamide mononucleotide SIR genes – Silent Information Regulator
NMNAT – Nicotinamide mononucleotide genes
adenylyltransferase-1 siRNA – Small interfering ribonucleic acid
OGTT – Oral Glucose Tolerance Test SIRT – Silent mating type information
OLE – Oleate regulation 2 homolog
PARP – Poly (ADP-Ribose) Polymerase SMRT – Silencing Mediator of Retinoid and
PBA – Phenylbutyrate Thyroid hormone receptors
PC – Proprotein Convertase SPT – Serine palmitoyltransferase
PCOS – Polycystic Ovary Syndrome STZ – Streptozotocin
PDK4 – Pyruvate Dehydrogenase Kinase 4 T1DM – Type 1 Diabetes Mellitus
Pdx-1 – Pancreas duodenum homeobox-1 T2DM – Type 2 Diabetes Mellitus
PGC-1α - Peroxisome proliferator-activated TCA Cycle – Tricarboxylic Acid Cycle
receptor gamma coactivator 1-alpha TGN – Trans-Golgi Network
PI3K – Phosphoinositide 3-kinase TLR-4 – Toll-like receptor-4
PKA – Protein Kinase-C Activator TMB – 3,3',5,5'-tetramethylbenzidine
PKC – Protein Kinase C TZDs – Thiazolidinediones
PML - Promyelocytic leukemia UCP1/2 – Uncoupling protein 1/2
POD – Peroxidase UPR – Unfolded Protein Response
POMC – Proopiomelanocortin VDCC – Voltage-Dependent Ca2+ Channels
PP Cells – Pancreatic Polypeptide Cells WAT – White Adipose Tissue
PTP1B – Protein-tyrosine phosphatase 1B WHO – World Health Organization
PUFA – Polyunsaturated Fatty Acid ZDF rats – Zucker Diabetic Fatty rats
PVDF – Polyvinylidene fluoride

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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
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List of Tables

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

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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.

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