The Role of the Glucagon-Like Peptide-1 Receptor in Atherosclerosis
by
Naim Panjwani
A thesis submitted in conformity with the requirements for the degree of Masters of Science
Institute of Medical Science University of Toronto
© Copyright by Naim Panjwani (2012)
ii
The Role of the Glucagon-Like Peptide-1 Receptor in
Atherosclerosis
Naim Panjwani
Masters of Science
Institute of Medical Science
University of Toronto
2012
Abstract
Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists have been shown to reduce
atherosclerosis in non-diabetic mice. We hypothesized that treatment with GLP-1R agonists
would reduce the development of atherosclerosis in diabetic Apoe-/-
mice.
Results: Exendin-4 treatment (10 nmol/kg/day) of high-fat diet-induced glucose-intolerant mice
for 22 weeks did not significantly reduce oral glucose tolerance (P=0.62) or HbA1c (P=0.85),
and did not reduce plaque size at the aortic sinus (P = 0.35). Taspoglutide treatment for 12 weeks
(0.4-mg tablet/month) of diabetic mice reduced body weight (P<0.05), food intake (P<0.05), oral
glucose tolerance (P<0.05), intrahepatic triglycerides (P<0.05) and cholesterol (P<0.001), and
plasma IL-6 levels (P<0.01); increased insulin:glucose (P<0.05); and unaltered oral lipid
tolerance (P=0.21), plasma triglycerides (P=0.45) or cholesterol (P=0.92). Nonetheless,
taspoglutide unaltered aortic atherosclerosis (P=0.18, sinus; P=0.19, descending aorta) or
macrophage infiltration (P=0.45, sinus; P=0.26, arch).
Conclusions: GLP-1R activation in either glucose-intolerant or diabetic mice does not
significantly modify the development of atherosclerosis.
iii
Acknowledgments and Contributions
First and foremost, I would like to thank my supervisor, Dr. Daniel Drucker for having given me
the opportunity to work in his lab. He offered remarkable support, guidance, and encouragement
throughout my graduate studies. It has been a true honour to work under his supervision and an
intellectual challenge that motivated me. I would also like to extend my gratitude to my
committee members, Dr. Mansoor Husain, Dr. Myron Cybulsky, and Dr. Michelle Bendeck for
their valuable guidance, support and discussions.
This thesis would not have been possible without the help from everyone in the Drucker lab. I am
especially thankful for the outstanding mentorship from Laurie Baggio, Erin Mulvihill and
Christine Longuet. I am also grateful for the help from Laurie, Xiemin and Minsuk Kim with
glucose and lipid tolerance tests; Christine also helped with lipid tolerance tests.
I am also grateful for the friendship and support from everyone at the Drucker lab, as well as
intellectual discussions with Adriano Maida, Ben Lamont, John Ussher, Bernardo Yusta and
Marc Angeli.
I would also like to acknowledge several funding agencies—Natural Sciences and Engineering
Research Council of Canada, Banting and Best Diabetes Centre, Roche Pharmaceuticals and
Institute of Medical Science.
Finally, I would like to thank my parents, Nooruddin and Gulshan Panjwani for their love and
support.
iv
Table of Contents
Abstract ..................................................................................................................................... ii
Acknowledgments ..................................................................................................................... iii
Table of Contents .......................................................................................................................iv
List of Abbreviations..................................................................................................................vi
Chapter 1. Introduction ............................................................................................................... 1
1.1 General background....................................................................................................... 1
1.2 Glucagon-like peptide-1 ................................................................................................ 1
1.2.1 Proglucagon and proglucagon-derived peptides .................................................. 1
1.2.2 GLP-1 action ...................................................................................................... 2
1.2.3 GLP-1 secretion, metabolism and clearance ....................................................... 4
1.2.3.1 Secretion .............................................................................................. 4
1.2.3.2 Metabolism .......................................................................................... 6
1.2.3.3 Clearance ............................................................................................. 7
1.2.4 The GLP-1 receptor ........................................................................................... 7
1.2.5 Structural and functional characteristics of GLP-1R agonists exendin-4 and
taspoglutide ..................................................................................................... 8
1.2.5.1 Exendin-4 ............................................................................................. 8
1.2.5.2 Taspoglutide ......................................................................................... 8
1.3 Atherosclerosis .............................................................................................................. 9
1.3.1 The development of atherosclerosis .................................................................... 9
1.3.1.1 Initiating events in atherosclerosis ........................................................ 9
1.3.1.2 Lesion propagation ............................................................................. 12
1.3.1.3 Vulnerable plaques and lesion disruption ............................................ 12
1.3.2 Atherosclerosis in diabetes ............................................................................... 13
1.3.2.1 Hyperglycemia ................................................................................... 14
1.3.2.2 Insulin resistance and hyperinsulinemia .............................................. 16
1.3.3 Lipoprotein metabolism ................................................................................... 17
1.3.4 Adipokines in athersoclerosis ........................................................................... 18
1.3.4.1 Leptin ................................................................................................. 19
1.3.4.2 Resistin .............................................................................................. 20
1.3.4.3 Interleukin-6 ....................................................................................... 20
1.3.5 The Apoe-/- mouse model of atherosclerosis ..................................................... 21
1.3.6 Current treatments for atherosclerosis .............................................................. 22
1.3.6.1 Lipid lowering .................................................................................... 22
1.3.6.2 Hypertension ...................................................................................... 23
1.3.6.3 Diabetes ............................................................................................. 24
1.4 GLP-1R actions on atherogenesis ................................................................................ 25
1.4.1 Indirect actions ................................................................................................. 25
1.4.1.1 Lipoprotein metabolism ...................................................................... 25
1.4.1.2 Blood pressure .................................................................................... 27
1.4.1.3 Adiposity and adipokines.................................................................... 27
v
1.4.1.4 Insulin resistance, hyperinsulinemia and hyperglycemia ..................... 28
1.4.2 Direct actions ................................................................................................... 29
1.4.2.1 Endothelial cells ................................................................................. 29
1.4.2.2 Vascular smooth muscle cells ............................................................. 31
1.4.2.3 Monocytes and monocyte-derived cells .............................................. 33
1.5 Project rationale and hypothesis ................................................................................... 34
Chapter 2. Materials and methods .............................................................................................. 35
2.1 Exendin-4 project ........................................................................................................ 35
2.1.1 Animals, diets and drug treatments ................................................................... 35
2.1.2 Metabolic measurements .................................................................................. 36
2.1.3 Histological analysis of atherosclerotic lesions ................................................. 37
2.2 Taspoglutide project .................................................................................................... 38
2.2.1 Animals, diets and drug treatments ................................................................... 38
2.2.2 Metabolic measurements .................................................................................. 39
2.2.3 Histological analysis of atherosclerotic lesions ................................................. 40
2.2.4 Immunohistochemical analysis of macrophage infiltration ............................... 40
2.2.5 Plasma adipokine measurements ...................................................................... 41
2.3 Statistical analyses ....................................................................................................... 41
Chapter 3. Results ..................................................................................................................... 42
3.1 Exendin-4 project ........................................................................................................ 42
3.1.1 Metabolic phenotype ........................................................................................ 42
3.1.2 Atherosclerosis................................................................................................. 42
3.2 Taspoglutide project .................................................................................................... 46
3.2.1 Metabolic phenotype ........................................................................................ 46
3.2.2 Lipid metabolism ............................................................................................. 46
3.2.3 Plasma adipokines ............................................................................................ 53
3.2.4 Atherosclerosis................................................................................................. 54
3.2.5 Blood Pressure ................................................................................................. 55
Chapter 4. Discussion ................................................................................................................ 60
4.1 Effect of GLP-1R activation on body weight, adipocyte mass, adipokines and
atherogenesis ............................................................................................................. 60
4.2 Effect of GLP-1R activation on glucose homeostasis and atherogenesis ...................... 62
4.3 Effect of GLP-1R activation on lipoprotein metabolism ............................................... 64
4.4 Effect of GLP-1R activation on atheroma development ............................................... 66
4.5 Expression of the GLP-1R in liver and atheroma ......................................................... 70
Chapter 5. Conclusions ............................................................................................................. 71
Chapter 6. Future Directions ..................................................................................................... 72
6.1 Atherosclerosis ............................................................................................................ 72
6.2 Fatty liver and lipoprotein metabolism ......................................................................... 73
References ................................................................................................................................ 75
vi
List of Abbreviations
ABCA1 ATP-binding cassette transporter A1
ABCG1 ATP-binding cassette subfamily G member 1
ACC Acetyl CoA carboxylase
ACCORD Action to Control Cardiovascular Risk in Diabetes
ACE Angiotensin-converting enzyme
ACS Acute coronary syndromes
ADVANCE Action in Diabetes and Vascular Disease Preterax and Diamicron Modified Release Controlled Evaluation
AGE Advanced glycation endproduct
Aib -Aminoisobutyric acid
ALE Advanced lipoxidation endproduct
AMP Adenosine monophosphate
AMPK Adenosine monophosphate-activated protein kinase
APO(a) Apolipoprotein(a)
APO-AI Apolipoprotein A-I
APO-AII Apolipoprotein A-II
APOB APO-B48 and/or APO-B100
APO-B100 Apolipoprotein B-48
APO-B48 Apolipoprotein B-100
APOC2 Apolipoprotein C-II
APOE Apolipoprotein E
ARB Angiotensin receptor blocker
ATP Adenosine triphosphate
AUC Area under the curve
BAEC Bovine aortic endothelial cell
BBB Blood-brain barrier
BCA Brachiocephalic artery
BH4 Tetrahydrobiopterin
BP Blood pressure
CAD Coronary artery disease
cAMP Cyclic adenosine monophosphate
CCL2 Chemokine C-C motif ligand-2
vii
CCR2 Chemokine C-C motif receptor 2
cDNA Complementary DNA
CE Cholesteryl ester
CETP Cholesteryl ester transfer protein
cGMP Cyclic guanine monophosphate
CIMT Carotid intima-media thickness
CPT-I Carnitine palmitoyltransferase-I
CREB cAMP response element binding
CRP C-reactive protein
CVD Cardiovascular disease
DAB diaminobenzidine
DAG 1,2-diacylglycerol
DC Dendritic cell
DCCT Diabetes Control and Complications Trial
DDP-4 Dipeptidyl peptidase-4
DNA Deoxyribonucleic acid
EC Endothelial cell
ECM Extracellular matrix
EIRAKO Endothelial insulin receptor and Apoe knockout
ENHANCE Ezetimibe and Simvastatin in Hypercholesterolemia Enhances Atherosclerosis Regression
eNOS Endothelial nitric oxide synthase
EPAC2 Exchange protein directly activated by cAMP 2
ER Endoplasmic reticulum
ERK1/2 Extracellular-signal-regulated kinase-1/2
ESL1 E-selectin ligand-1
ET-1 Endothelin-1
FAS Fatty acid synthase
FC Free cholesterol
FMD Flow-mediated vasodilation
FFA Free fatty acids
FFAR1 Free fatty acid receptor-1
GIP Glucose-dependent insulinotropic polypeptide
GIPR GIP receptor
viii
GLP-1 Glucagon-like peptide-1
GLP-2 Glucagon-like peptide-2
GLP1R Glucagon-like peptide-1 receptor
GP Glycoprotein
GP2B Platelet glycoprotein IIb of IIb/IIIa complex
GP3A Platelet glycoprotein IIIa of IIb/IIIa complex
GPAT Glycerophosphate acyltransferase
GPCR Guanine nucleotide-binding (G-protein)-coupled receptor
GPR119 GPCR 119
GPR120 GPCR 120
GRP Gastrin releasing peptide
GRPP Glicentin-related pancreatic polypeptide
HbA1c Hemoglobin A1c
HCAEC Human coronary artery endothelial cells
HDL High density lipoprotein
HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A reductase
HOMA-IR Homeostatic model assessement of insulin resistance
HTGL Hepatic triglyceride lipase
HUVEC Human umbilical vein endothelial cells
hVSMC Human vascular smooth muscle cells
I/R Ischemia-reperfusion injury
ICAM-1 Intracellular adhesion molecule-1
IFN Interferon
IB I-kappaB
IL-1 Interleukin-1
IL1 Interleukin-1 beta
IL1Ra Interleukin-1 receptor antagonist
IL6 Interleukin-6
IL12 Interleukin-12
i.p. Intraperitoneal
IP1 Intervening polypeptide-1
IP2 Intervening polypeptide-2
ISI Insulin sensitivity index (glucose infusion rate to plasma insulin concentration)
ix
JNK c-Jun N-terminal kinase
LAD Left anterior descending
LCAT Lecithin:cholesterol acyltransferase
LDL Low-density lipoprotein
LDLR LDL receptor
Lp(a) Lipoprotein(a)
LPS Lipopolysaccharide
LRP LDL receptor related protein
LVDP Left ventricular developed pressure
MAC-1 Macrophage-1 antigen
MAC-2 Macrophage-2 antigen
MAPK Mitogen activated protein kinase
MCD Malonyl CoA decarboxylase
MCP-1 Macrophage chemoattractant molecule-1
MHC Major histocompatibility complex
MMP Matrix metalloproteinase
MPGF Major proglucagon fragment
mRNA Messenger ribonucleic acid
MTTP Microsomal triglyceride transfer protein
NAFLD Non-alcoholic fatty liver disease
NASH Non-alcoholic steatohepatitis
NEFA Non-esterified fatty acid
NFB Nuclear factor kappa-B
NO Nitric oxide
NOD Nonobese diabetic
O-GlcNAc N-acetylglucosamine
OGTT Oral glucose tolerance test
oxLDL Oxidized LDL
PC1 Prohormone convertase-1
PC2 Prohormone convertase-2
PC3 Prohormone convertase-3
PCR Polymerase chain reaction
PDGF Platelet-derived growth factor
x
PGDP Proglucagon-derived peptides
PI3K Phosphatidylinositol-3 kinase
PKA Phosphokinase A
PKC Protein kinase C
PLC Phospholipase C
PPAR Peroxisome proliferator-activated receptor
RAAS Renin-angiotensin-aldosterone system
RAGE Receptor for AGEs
RCT Reverse cholesterol transport
RNA Ribonucleic acid
ROS Reactive oxygen species
SAA Serum amyloid A
SCD Stearoyl CoA desaturase-1
SGLT1 Sodium/glucose co-transporter-1
SMC Smooth muscle cell
SOCS-3 Suppressor of cytokine signaling-3
SPT Serine palmitoyltransferase
SRA Scavenger receptor A
SR-B1 Scavenger receptor class B1 (also known as CD36)
sTNFR Soluble tumor necrosis factor receptor
STZ Streptozotocin
SVC Stromal vascular cells
T1DM Type 1 diabetes mellitus
T2DM Type 2 diabetes mellitus
T3 Triiodothyronine
TED 500,000 IU/ml Trasylol, 1.2 mg/ml EDTA, and 0.1 mM Diprotin A
TG Triglyceride (triacylglycerol)
TIMP Tissue inhibitor of metalloproteinases
tGLP-1 GLP-1(9-36)NH2 and GLP-1(9-37)
TNF- Tumor necrosis factor alpha
Tregs Regulatory T cells
TRL Triglyceride-rice lipoprotein
TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
xi
UKPDS The United Kingdom Prospective Diabetes Study
VADT Veterans Administration Diabetes Trial
VALT Vascular-associated lymphoid tissue
VCAM-1 Vascular cell adhesion molecule-1
VDC Vascular dendritic cell
VEGF Vascular endothelial growth factor
VLDL Very low density lipoprotein
VSMC Vascular smooth muscle cell
WAT White adipose tissue
XBP1 X-box-binding protein-1
ZDF Zucker diabetic fatty
1
Chapter 1 Introduction
1.1 General background
Diabetes is at epidemic proportions with an estimated 366 million people living with the disease
in 2011, and by 2030 this figure is expected to rise to 552 million (Whiting et al. 2011).
Moreover, macrovascular disease (mainly myocardial infarction and stroke) is the principal
cause of death in patients with diabetes with an increased risk that is two to six times higher than
in the general population (Ruderman and Haudenschild 1984). The main cause for
macrovascular disease is atherosclerosis (Jarrett 1981), and the World Health Organization also
predicts that a worldwide epidemic of atherosclerosis will evolve as developing countries
acquire the dietary and sedentary lifestyle habits of the western world (Molecular Mechanisms
of Atherosclerosis 2005). The severity of atherosclerosis is worse in patients with diabetes as
cardiovascular events occur at an earlier age and at much higher incidences than patients with
no diabetes (Ruderman and Haudenschild 1984). Furthermore, the relative lower incidence of
cardiovascular events in pre-menopausal females is abolished by diabetes (Ruderman and
Haudenschild 1984).
There is currently no cure for atherosclerosis, and therapies today aim at reducing and thus
decelerating atherosclerosis in order to decrease cardiovascular events. While therapies such as
statins, fibrates and anti-hypertensive drugs are effective at reducing cardiovascular events in
patients at risk, there remains a significant event rate, especially in patients with diabetes and
insulin resistance (Rubenfire, Brook, and Rosenson 2010; Tenenbaum et al. 2006). The aim of
this thesis is to investigate whether the new class of anti-diabetic drugs that activate the
glucagon-like peptide-1 receptor (GLP-1R) play a role in the development of atherosclerosis in
the setting of diabetes.
1.2 Glucagon-like peptide-1
1.2.1 Proglucagon and proglucagon-derived peptides
Glucagon-like peptide-1 (GLP-1) is a 30-amino acid protein that belongs to the glucagon
superfamily of peptide hormones due to its considerable sequence homology with glucagon
2
(Kieffer and Habener 1999). GLP-1 is derived from the proglucagon gene, which is expressed in
the pancreas, intestine and brain (Kieffer and Habener 1999; Baggio and Drucker 2007). In the
pancreatic cells of mammals, proglucagon is cleaved posttranslationally by prohormone
convertase-2 (PC-2) into glicentin-related pancreatic polypeptide (GRPP), glucagon, intervening
peptide-1 (IP-1) and major proglucagon fragment (MPGF); whereas in enteroendocrine L cells
and brain, proglucagon is cleaved by PC1/3 to yield oxyntomodulin, glicentin, GLP-1,
intervening peptide-2 (IP-2) and glucagon-like peptide-2 (GLP-2) (Baggio and Drucker 2007;
Kieffer and Habener 1999).
The physiologic actions of MPGF, GRPP, IP-1 or IP-2 are yet to be uncovered (Baggio and
Drucker 2007). Glucagon, on the other hand, is known for its counterregulatory actions to
insulin. Glucagon is essential for the maintenance of glucose homeostasis in the fasting state by
regulating hepatic glucose production via activation of glycogenolysis and gluconeogenesis and
inhibition of glycolysis (Ramnanan et al. 2011; Baggio and Drucker 2007). Glucagon mediates
its actions through the glucagon receptor (Rodbell et al. 1971). Oxyntomodulin reduces food
intake and hunger in both humans (Cohen et al. 2003) and rodents (Dakin et al. 2004) and
inhibits gastric acid secretion (Schjoldager et al. 1988; Schjoldager et al. 1989), gastric
emptying (Schjoldager et al. 1989), and gut motility (Pellissier et al. 2004). Glicentin is also
known to reduce gastric acid secretion and gut motility (Pellissier et al. 2004). GLP-2 modulates
gastrointestinal structure and function by stimulation of crypt cell proliferation and inhibition of
epithelial cell apoptosis (Baggio and Drucker 2007; Brubaker 2006). GLP-2 also enhances
intestinal glucose transport, increases intestinal blood flow, reduces gastric emptying and
secretion, and enhances epithelial barrier function (Baggio and Drucker 2007; Brubaker 2006).
1.2.2 GLP-1 action
GLP-1 acts on the GLP-1R (Göke and Conlon 1988; Drucker et al. 1987), a G-protein coupled
receptor that is expressed by many tissues of the body (Mayo et al. 2003). Upon nutrient
ingestion, GLP-1 is secreted from the enteroendocrine L cells of the ileum and colon and acts on
the endocrine pancreas through the GLP-1R to stimulate glucose-dependent insulin secretion
and biosynthesis. Nutrient ingestion gives rise to higher insulin levels than glucose delivery by
intravenous infusion partly due to the action of GLP-1 on the GLP-1R (Gutniak et al. 1994;
3
Kreymann et al. 1987; Mojsov, Weir, and Habener 1987; Drucker et al. 1987; Unger et al.
1968). This increase in insulin secretion has been termed the “incretin effect” and is mediated by
both GLP-1 and another incretin, GIP (glucose-dependent insulinotropic polypeptide), in an
independent and additive fashion through the GLP-1R and the GIP receptor (GIPR),
respectively (Baggio and Drucker 2007; Kreymann et al. 1987; Mojsov, Weir, and Habener
1987; Gutniak et al. 1994; Drucker et al. 1987). Incretin action is potentiated for the treatment of
type 2 diabetes mellitus (T2DM) through use of agonists for the GLP-1R, such as exenatide and
liraglutide (marketed as Byetta and Victoza, respectively).
GLP-1R agonists exert actions in the cardiovascular system that are cardioprotective (Best et al.
2011; Ban et al. 2009; Noyan-Ashraf et al. 2009). GLP-1R agonists also exert actions on lipid
metabolism (Farr and Adeli 2012; Hsieh et al. 2010; Matikainen et al. 2006; Meier et al. 2006)
that are potentially anti-atherogenic (Rizzo et al. 2009). Briefly, twice-daily intraperitoneal (i.p.)
injections of 75 g/kg liraglutide for 7 days in 12-14-week old C57BL6 male non-diabetic and
diabetic (streptozotocin-induced) mice improved survival and reduced cardiac rupture after left
anterior descending artery (LAD) ligation (to induce experimental myocardial infarction)
(Noyan-Ashraf et al. 2009); survival of mice was associated with increases in the expression of
cardioprotective genes both before and after LAD ligation; and, liraglutide pre-treatment in vivo
for 1 or 7 days improved recovery of hearts (as measured by left ventricle developed pressure)
after ischemia-reperfusion injury ex vivo. In humans, a retrospective database analysis found
exenatide therapy was associated with a reduced number of cardiovascular events in patients
with T2DM compared to other standard forms of treatment for diabetes (hazard ratio 0.81). The
reduction in cardiovascular events was observed despite a higher prevalence of ischemic heart
disease, hyperlipidemia, obesity and hypertension at baseline in the group prescribed exenatide
(Best et al. 2011). GLP-1R agonists also reduce body weight through actions in the brain
(Gutzwiller et al. 1999; Alhadeff, Rupprecht, and Hayes 2012; Turton et al. 1996), reduce blood
pressure (Hirata et al. 2009) and improve lipid profile (Farr and Adeli 2012; Hsieh et al. 2010;
Meier et al. 2006; Matikainen et al. 2006) (discussed in section 1.4); these extra-pancreatic
effects all predict a reduction in atherosclerosis (Rizzo et al. 2009). Furthermore, one study in
non-diabetic mice has suggested potential direct effects of exenatide on the initiation of
atherosclerosis by action on monocytes (Arakawa et al. 2010). This thesis aims to investigate the
in vivo role of two GLP-1R agonists, exenatide and taspoglutide, on the development of
atherosclerosis in high-fat fed glucose intolerant mice and streptozotocin-induced diabetic mice.
4
1.2.3 GLP-1 secretion, metabolism and clearance
1.2.3.1 Secretion
Secretion of GLP-1 is regulated by nutrients, endocrine and neural factors. GLP-1 is secreted
from enterendocrine L cells located in the distal ileum, colon, and duodenum (Kauth and Metz
1987; Theodorakis et al. 2006; Eissele et al. 1992). While a higher density of L cells is found in
the ileum and colon, human duodenum has about the same number of L cells as K cells (as
assessed by endoscopy of human volunteers followed by immunofluorescence with anti-GLP-1
antibodies on duodenal tissue) (Theodorakis et al. 2006). Food intake increases plasma levels of
GLP-1 within 15-30 minutes, well before nutrients reach the ileum. A second peak then follows
at 90-120 minutes after a meal, when nutrients have reached the ileum (Brubaker 2006;
Herrmann et al. 1995).
The first release of GLP-1 is likely mediated by both direct and/or indirect mechanisms. A direct
mechanism similar to the secretion of GIP may occur for GLP-1; GIP is released from K cells
(primarily found in the duodenum) by direct nutrient stimulation (Kreymann et al. 1987).
Secretion of GLP-1 from duodenal L cells may account for most of the early phase increase of
GLP-1 in plasma after nutrient ingestion (Theodorakis et al. 2006). At the same time, an indirect
stimulation of the L cells in the ileum and colon may also occur. The vagus nerve plays an
important role in a proximal-distal loop pathway for early stimulation of the L cell via a
cholinergic pathway; infusion of 3-4 ml of corn oil directly into the duodenum of male Wistar
rats with bilateral subdiaphragmatic vagotomy plus gut transection 10 cm from the infusion site
abolished the GLP-1 rise in plasma above basal, while electrical stimulation of the celiac
branches of the vagus that innervate the jejunum, ileum and colon resulted in a peak GLP-1
increase of 71 pg/ml above basal after 10 minutes (Rocca and Brubaker 1999). In humans, the
role of the vagus nerve in the early release of GLP-1 had been demonstrated with administration
of atropine, a muscarinic receptor antagonist, which attenuated the increase of GLP-1 in plasma
above basal (Balks et al. 1997). In rats, GIP also plays a role in the early release of GLP-1;
intravenous infusion of GIP to achieve post-prandial plasma GIP concentrations induced a 2-
fold increase in plasma proglucagon-derived peptide (PGDP) levels, including GLP-1 (Roberge
and Brubaker 1993). Furthermore, infusion of fat into the duodenum of rats whose duodena
were surgically ligated increased GIP in plasma followed by increased levels of proglucagon-
5
derived peptides, including GLP-1; however, resection of the ileum and colon abolished the
increase of proglucagon-derived peptides in plasma (Rocca and Brubaker 1999). In humans,
however, GIP infusion during a hyperglycemic clamp did not raise plasma GLP-1 levels above
basal in either normal or T2DM subjects (Nauck et al. 1993). Finally, infusion of gastrin-
releasing peptide (GRP) (470 ng/h) in rats also stimulated an increase in proglucagon-derived
peptides in plasma, while blocking the GRP receptor with an antagonist failed to increase the
levels of proglucagon-derived peptides in plasma (Roberge, Gronau, and Brubaker 1996). In
addition, GRP receptor knockout mice demonstrated impaired glucose tolerance and reduced
insulin response at 10 minutes after gastric glucose administration (50 or 150 mg) (Persson et al.
2000). Thus, nutrients activate the L cell to secrete GLP-1 via a cholinergic pathway that is
dependent on the GRP receptor in rodents (Brubaker 2006).
The second phase of GLP-1 secretion is stimulated by direct contact of nutrients with L cells in
the ileum and colon. Carbohydrates and amino acids can stimulate the L cell in vitro, and may
be the main stimulators for the early phase of GLP-1 secretion by acting on the L cells in the
duodenum and jejunum. In contrast, fatty acids reaching the ileum may be the main stimulus for
the second phase of GLP-1 secretion (Brubaker 2006; Iakoubov et al. 2011).
Our current knowledge of the molecular mechanism(s) for GLP-1 release is advancing in part
due to the development of transgenic mice in which GLP-1-producing cells are labeled with a
fluorescent marker, enabling the identification of murine L cells (Diakogiannaki, Gribble, and
Reimann 2011; Parker, Reimann, and Gribble 2010; Tolhurst, Reimann, and Gribble 2008). The
L cell is equipped to detect carbohydrates, protein and fat and respond to each macronutrient
accordingly. L cells are open-type cells with apical processes extending to the gut lumen, a
morphology that facilitates sensing of nutrients (Diakogiannaki, Gribble, and Reimann 2011;
Parker, Reimann, and Gribble 2010; Tolhurst, Reimann, and Gribble 2008). Glucose is a potent
secretagogue for GLP-1, and is currently believed to act through the sodium/glucose co-
transporter-1 (SGLT1). SGLT1 transports two sodium ions per glucose molecule across the
membrane, depolarizing the cell and triggering action potentials that elevate cytosolic calcium
ions, which activate voltage-gated calcium channels (Diakogiannaki, Gribble, and Reimann
2011; Parker, Reimann, and Gribble 2010; Tolhurst, Reimann, and Gribble 2008; Gorboulev et
al. 2012). Fat is a good stimulant of GLP-1 secretion as well, and the response is proportional to
the caloric content of the ingested lipid. Fatty acid receptors like the G-protein coupled receptor
6
120 (GPR120) and free fatty acid receptor-1 (FFAR1) are G-protein coupled receptors (GPCRs)
that trigger GLP-1 release by coupling to Gq, activating protein kinase C (PKC) (among which,
PKC zeta is essential) and inositol triphosphate (IP3)-induced calcium release (Diakogiannaki,
Gribble, and Reimann 2011; Iakoubov et al. 2011). The G-protein coupled receptor 119
(GPR119) is another GPCR activated by oleoylethanolamide or 2-monoacylglycerol that
couples to Gs rather than Gq, stimulating adenylyl cyclase and intracellular cyclic adenosine
monophosphate (cAMP) production (Diakogiannaki, Gribble, and Reimann 2011; Hansen et al.
2011). Finally, male Sprague-Dawley rats given a lipid stimulus (Liposyn) plus a surfactant
(Pluronic L-81) known to inhibit chylomicron formation in the intestine while not interfering
with uptake of lipolytic products and reesterification to triglyceride (TG), showed a 75%
decrease in GLP-1 secretion compared to rats given Liposyn alone during the first 30 minutes of
lipid infusion; this evidence suggests a role for chylomicron formation in the secretion of GLP-1
(Lu et al. 2012).
1.2.3.2 Metabolism
GLP-1 is processed from proglucagon and secreted as GLP-1(1-36)NH2 and GLP-1(1-37),
which are thought to be inactive (Ghiglione et al. 1984); and GLP-1(7-36)NH2 and GLP-1(7-
37), which are the bioactive equipotent peptides liberated from proglucagon by PC1/3 cleavage
in the intestine and brain (Orskov, Wettergren, and Holst 1993; Dhanvantari, Seidah, and
Brubaker 1996).
The half-life of GLP-1 is less than 2 minutes due to rapid degradation by the serine protease
dipeptidyl peptidase-4 (DPP-4), which cleaves GLP-1 in the alanine position 2 yielding GLP-
1(9-36)NH2 or GLP-1(9-37) (Deacon et al. 1995), which are also thought to play a role in the
cardiovascular system (Ban et al. 2009). DPP-4 is expressed in many tissues, including the
surface of endothelial cells that line the intestinal mucosa adjacent to the L cell; DPP-4 also
exists as a soluble form in the circulation. Thus, more than half of GLP-1 that enters the portal
circulation is in the cleaved form (Hansen et al. 1999; Mentlein 1999). Intravenous infusion of
GLP-1(7-36)NH2 in both healthy and T2DM patients results in rapid cleavage by DPP-4
(Deacon et al. 1995); infusion of [125
I]GLP-1(7-36)NH2 in male Wistar rats results in 50% of the
peptide being cleaved by DPP-4 within 2 minutes to [125
I]GLP-1(9-36)NH2 metabolite, while
7
infusion of the non-truncated tracer into DPP-4-deficient animals results in the absence of
[125
I]GLP-1(9-36)NH2 metabolite (Kieffer, McIntosh, and Pederson 1995). Inhibition of DPP-4
or its absence in DPP-4-deficient animals greatly increases the half-life of non-truncated GLP-1
(Marguet et al. 2000).
1.2.3.3 Clearance
GLP-1(9-36)NH2 and GLP-1(9-37) (truncated GLP-1 or tGLP-1) are cleared from the
circulation by the kidneys by glomerular filtration, tubular uptake and catabolism (Ruiz-Grande
et al. 1993). Due to renal clearance, the plasma half-life of tGLP-1 is about 5 minutes.
Furthermore, patients with renal failure or chronic renal insufficiency have an increased half-life
of tGLP-1 in the circulation (Meier et al. 2004; Orskov, Andreasen, and Holst 1992).
1.2.4 The GLP-1 receptor
The GLP-1R belongs to the class B family of guanine nucleotide-binding (G-protein)-coupled
receptors (GPCRs) known as the secretin receptor family of 7 transmembrane receptors,
subfamily B1, which consists of multiple receptors for peptide hormones (Mayo et al. 2003).
The rat and human receptors are structurally similar, 463 amino acids in length with 90%
sequence identity at the amino acid level (Mayo et al. 2003).
Agonists for the GLP-1R include GLP-1(7-37), GLP-1(7-36)NH2, and the Heloderma
suspectum peptides exendin-3 and exendin-4 (Mayo et al. 2003). A known antagonist for the
GLP-1R is the truncated lizard peptide exendin(9-39) (Mayo et al. 2003).
The GLP-1R is expressed in many cells and tissues such as -cells of pancreatic islets
(Tornehave et al. 2008), lung, brain, heart, kidney and stomach of rodents and humans as
assessed by RNAse protection assay (Bullock, Heller, and Habener 1996; Wei and Mojsov
1995). Although it has been shown that the GLP-1R can be desensitized and internalized by
phosphorylation of three serine doublets within the cytoplasmic tail in vitro (Mayo et al. 2003),
GLP-1R desensitization has not been observed in vivo in the endocrine pancreas of mice treated
with exendin-4 (2 g twice daily), even after long-term (26-week) administration (Hadjiyanni et
8
al. 2008). Similarly, twice-daily administration of exenatide in human patients with T2DM was
effective in significantly reducing HbA1c and glycemic deterioration after approximately 5
years of treatment (Gallwitz et al. 2012).
1.2.5 Structural and functional characteristics of GLP-1R agonists exendin-4 and taspoglutide
The short plasma half-lives of GLP-1(7-37) and GLP-1(7-36)NH2 (2 min) makes the use of
these peptides suboptimal for treatment. One major reason for the short plasma half-lives is
cleavage at the 8th residue (alanine) by DPP-4. Thus, GLP-1R agonists resistant to cleavage by
DPP-4 have been developed for pharmaceutical use. Two GLP-1R agonists have been used to
investigate GLP-1R activation in the development of atherosclerosis in diabetic mice in this
study: exendin-4 and taspoglutide.
1.2.5.1 Exendin-4
Exendin-4 is a 39-residue peptide produced in the salivary gland of the Gila monster
(Heloderma suspectum) that is not cleaved by DPP-4 (Baggio and Drucker 2007). Exendin-4
has 53% identity to GLP-1(7-36)NH2 with 16 identical amino acids in the first 30, and an
additional C-terminal extension of nine amino acid residues. Exendin-4 is equipotent to GLP-1
in binding the GLP-1R and acts as a GLP-1 mimetic (Thorens et al. 1993). The synthetic version
of exendin-4 is exenatide and has a plasma half-life of 4-6 hours after subcutaneous
administration in patients with T2DM with the kidney being the major route of elimination
(Kolterman et al. 2005). Mice and rats show similar plasma half-lives (Copley et al. 2006).
1.2.5.2 Taspoglutide
Taspoglutide is a peptide very similar to human GLP-1(7-36)NH2 with the exception of
substitutions at position 8 and 35 with -aminoisobutyric acid (Aib), which render the peptide
resistant to degradation by DPP-4 (Dong et al. 2011). This drug was formulated by Roche in the
form of a microtablet for mice that releases peptide slowly and is suitable for once-monthly
dosing. In humans, the drug was formulated as a sustained-release once-weekly formulation
9
with zinc chloride. Subcutaneous administration of taspoglutide precipitates and forms a depot
that slowly dissolves into the circulation (Dong et al. 2011). Unfortunately, taspoglutide was
withdrawn from human clinical testing after results from phase 3 clinical trials revealed a high
rate of withdrawal from treatment (11-13% versus 3.3% in placebo) and hypersensitivity
reactions in four patients, two of which withdrew (Raz et al. 2012).
1.3 Atherosclerosis
1.3.1 The development of atherosclerosis
Atherosclerosis is a disease of very complex etiology (Weber and Noels 2011) and this chapter
does not aim to review all aspects of its development and/or progression. Rather, the aim of the
chapter is to selectively review important aspects of atherogenesis and how diabetes impacts its
development.
Atherosclerosis is a disease characterized by a chronic inflammatory process in the large arteries
that is progressive and involves excess deposition of low-density lipoprotein (LDL) cholesterol
and fibrous elements in the intima of the artery (Lusis 2000; Ross 1997, 1999). Some of the
known factors that influence development and progression of atherosclerosis include
hyperlipidemia, high blood pressure, diabetes, smoking and genetic factors (Molecular
Mechanisms of Atherosclerosis 2005). In humans, the first signs of atherosclerosis may be
found in the first decade of life in the aorta and the lesions progress with age (Lusis 2000). In
this section, a brief description of the different stages of atherogenesis is discussed.
1.3.1.1 Initiating events in atherosclerosis
Atherosclerosis is thought to be initiated by a combination of endothelial dysfunction, which
upregulates adhesion molecules and chemokines by endothelial cells to attract inflammatory
cells such as lymphocytes and monocytes; a non-confluent luminal elastin layer such as those
found at branching points of arteries that facilitates entry of lipids and immune cells; and
exposure of negatively-charged proteoglycans at the luminal surface of arteries that promote
binding of APOB100-containing lipoproteins (Kwon et al. 2008). The accumulation and
oxidative modification of lipids and LDL in the intima of the artery is mediated by reactive
10
oxygen species (ROS) and/or enzymes released from inflammatory cells such as
myeloperoxidase and lipoxygenases (Weber and Noels 2011). Atherogenesis is largely mediated
by a very complex interaction between the cells of the inflammatory immune system that may
be analogous to an autoimmune reaction (Bobryshev 2000; Weber and Noels 2011; Hansson
and Hermansson 2011).
Accumulation of lipoproteins and their aggregates in the intima occurs mainly at sites of
predilection, where blood flow is turbulent rather than uniform and laminar. Turbulent flow
occurs most commonly at branching and curvature sites (eg. aortic arch) of arteries (Collins and
Cybulsky 2001). This occurs because endothelial cells take an ellipsoid shape reflecting the
flow’s uniform laminar directionality, but where flow is disturbed, the cells have polygonal
shapes with no particular orientation, and are more permeable (Collins and Cybulsky 2001).
Interestingly, these sites also have intimal thickenings of vascular smooth muscle cells
(VSMCs) that are thought to be important in the initiation and late stages of atherogenesis due to
their ability to proliferate, migrate and produce extracellular matrix (ECM) (Doran, Meller, and
McNamara 2008).
LDL accumulation in the intima itself has limited atherogenicity; it is the modification of LDL,
such as its oxidation, that initiates the inflammatory process. This involves recruitment of T
cells and monocyte-derived cells that engulf the lipoproteins at a rate higher than it can be
eliminated, thus forming lipid-laden cells called “foam cells” (Lusis 2000; Bobryshev 2000;
Cybulsky and Jongstra-Bilen 2010; Paulson et al. 2010; Weber and Noels 2011). VSMCs can
also take up lipid and appear foamy under the microscope (Doran, Meller, and McNamara
2008). Thus, hypercholesterolemia is associated with higher rates of atherosclerosis initiation
and progression, and cardiovascular events (Collins and Cybulsky 2001).
Factors that accelerate the development of atherosclerosis include both genetic and
environmental components (Lusis 2000; Weber and Noels 2011). Some examples include:
elevated levels of LDL and/or very low-density lipoprotein (VLDL), reduced levels of HDL (the
“good cholesterol”), elevated levels of lipoprotein(a) (Lp(a)), elevated blood pressure, elevated
homocysteine, diabetes, elevated levels of haemostatic factors, depression, gender (men develop
more atherosclerosis than pre-menopausal women), systemic inflammation, and insulin
11
resistance (Lusis 2000). Some environmental factors include: high-fat diet, smoking, low
antioxidant levels and lack of exercise (Lusis 2000).
Adherence and transmigration (diapedesis) of leukocytes from the blood circulation is mediated
by activated endothelial cells (ECs) (Molecular Mechanisms of Atherosclerosis 2005). EC
activation occurs under a variety of conditions, such as exposure to tumor necrosis factor alpha
(TNF-, interleukin-1 beta (IL-1, endotoxin, or substances that induce oxidative stress that is
overwhelming to the protective antioxidant mechanisms of the cell (Molecular Mechanisms of
Atherosclerosis 2005). EC activation results in the activation of nuclear factor kappa-B (NFB)
transcription factor, which mediates the upregulation of adhesion molecules for increased
leukocyte rolling, firm adherence, and diapedesis (Molecular Mechanisms of Atherosclerosis
2005; Collins and Cybulsky 2001). Families of adhesion molecules include the selectins (E-
selectin, P-selectin), integrins (heterodimeric molecules like vitronectin, macrophage antigen-1
(MAC-1), GP IIb/IIIa), and immunoglobulin superfamily members: intracellular adhesion
molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Molecular
Mechanisms of Atherosclerosis 2005). Leukocyte rolling is mediated by E-selectin and
leukocyte-expressed E-selectin ligand-1 (ESL-1); firm adhesion happens through VCAM-1 and
ICAM-1 on ECs with MAC-1, and other 4, D-integrins on leukocytes; and, transmigration is
induced by chemotactic factors such as macrophage chemoattractant molecule-1 (MCP-1),
which acts on the CCR2 receptor (4).
Defense mechanisms have also been identified that counteract the formation of atherosclerotic
lesions, mainly at the initiation stage. One such mechanism is mediated by the antioxidant
protein serum paraoxonase, an esterase carried by HDL that degrades oxidized phospholipids
(Lusis 2000). In addition, reverse cholesterol transport by HDL through the ATP-binding
cassette transporter A1 (ABCA1) and the ATP-binding cassette subfamily G member 1
(ABCG1) transporters, which mediate cholesterol efflux on macrophages is another means of
diminishing deposited LDL in the intima and thus, prevent lesion initiation and/or progression
(Yvan-Charvet, Wang, and Tall 2010; Yvan-Charvet et al. 2010). Secretion of APOE by
macrophages also promotes cholesterol efflux as evidenced by bone marrow transplantation
experiments to wildtype mice with Apoe-deficient marrow (Lusis 2000; Fazio et al. 1997; Basu
et al. 1982; Basu et al. 1981). Mice transplanted bone marrow deficient in APOE developed
atherosclerosis without inducing a change in the lipoprotein profile (Fazio et al. 1997). APOE is
12
well known to be atheroprotective due to its role in pro-atherogenic lipoprotein remnant uptake
by the liver, but it is also involved in the generation and maturation of APOE-containing HDL
particles in conjunction with ABCA1 and lecithin:cholesterol acyltransferase (LCAT), which is
an important pathway for the secretion of cholesterol by macrophage foam cells (Kypreos and
Zannis 2007). Thus, ABCA1, ABCG1, LCAT and APOE may all work in conjunction for the
efflux of cholesterol and formation of HDL particles (Yvan-Charvet, Wang, and Tall 2010;
Basu et al. 1981; Basu et al. 1982; Fazio et al. 1997; Kypreos and Zannis 2007; Yvan-Charvet et
al. 2010).
1.3.1.2 Lesion propagation
Oxidized LDL (OxLDL) induces EC oxidative stress leading to expression of genes under the
transcriptional regulation of NFB, such as the adhesion molecules ICAM-1 and VCAM-1
(Molecular Mechanisms of Atherosclerosis 2005; Collins and Cybulsky 2001). The
overexpression of adhesion molecules and chemotactic factors recruit leukocytes from the
circulation to the site of EC activation. Leukocytes recruited include neutrophils, monocytes, T
cells, B cells, dendritic cells (DCs) and mast cells (Weber and Noels 2011). The recruitment of
diverse cell subsets into the plaque lesion hints to the complexity, robustness and specificity of
the chemokine system (Koenen and Weber 2010). When the rate of endocytosis exceeds that of
cholesterol efflux, the macrophages accumulate significant amounts of cholesterol to become
foam cells and appear morphologically distinct under the microscope. Foam cells express IL-1
and TNF-, which further activate ECs, secrete proteoglycans that further induce APOB100-
containing lipoprotein binding, and induce VSMCs to produce matrix proteins (Molecular
Mechanisms of Atherosclerosis 2005; Lusis 2000; Moore and Tabas 2011).
1.3.1.3 Vulnerable plaques and lesion disruption
Cytokines and growth factors released by macrophages and T cells induce VSMC migration,
proliferation and ECM protein production such as collagen and fibronectin (Molecular
Mechanisms of Atherosclerosis 2005; Lusis 2000). As the lesion develops, a central necrotic
core of deposited lipid (mostly esterified cholesterol) and cell debris is covered by the migrating
VSMCs and secreted ECM, which form a fibrous cap that covers the growing lesion. As the
13
lesion grows, it obstructs blood flow and may produce ischaemic syndromes, especially when
blood flow demand is high (Molecular Mechanisms of Atherosclerosis 2005; Lusis 2000).
The fibrous cap prevents thrombus formation by separating the dense lipid core from luminal
blood flow. In certain cases, however, the inflammatory response results in the rupture and
erosion of the plaque, leading to acute coronary syndromes (ACS) (Virmani et al. 2006). A
recent study showed the presence of thin-cap atherosclerosis lesions preceding acute coronary
events (Virmani et al. 2006). Upon rupture, a superimposed thrombus may form and occlude
blood flow, leading to infarction of the affected tissue (Molecular Mechanisms of
Atherosclerosis 2005; Lusis 2000). It is currently unclear, largely due to the lack of animal
models, as to how these inflammatory events are initiated resulting in thinning of the fibrous cap
and its subsequent rupture (Molecular Mechanisms of Atherosclerosis 2005; Lusis 2000).
1.3.2 Atherosclerosis in diabetes
While atherosclerosis is accelerated in both type 1 and 2 diabetes, the factors involved are not
the same. In T2DM, the increased prevalence of obesity, hypertension, dyslipidemia,
hyperinsulinemia, and insulin resistance are associated factors that contribute to the increased
risk for macrovascular disease. These factors usually precede the onset of type 2 diabetes
(Molecular Mechanisms of Atherosclerosis 2005).
In contrast, in T1DM, the increased cardiovascular risk (10-fold compared to the general
population) is thought to be due in part to increased lipids and lack of perfect glycemic control,
but controlling for these factors still leaves an exceptionally increased risk that cannot be fully
explained (Nadeau and Reusch 2011). A follow-up of subjects with T1DM from the Diabetes
Control and Complications Trial (DCCT) treated intensively for 6.5 years to achieve tight
glucose control showed that this period of optimal glycemic control reduced the incidence of
cardiovascular disease by 57% even with deteriorating glycemia thereafter (Nathan et al. 2005).
However, the increased cardiovascular risk cannot be completely explained by hyperglycemia.
Whole-body insulin resistance is present in T1DM patients despite intensive glucose control (as
in the DCCT) and appears to correlate with cardiac and vascular defects (Orchard et al. 2003;
Nadeau et al. 2010). Furthermore, in most patients with T1DM, nephropathy and hypertension
also greatly accelerate atherosclerosis (Molecular Mechanisms of Atherosclerosis 2005).
14
Overall, a good understanding of the factors contributing to cardiovascular dysfunction in
T1DM is currently lacking.
1.3.2.1 Hyperglycemia
A predisposing factor in patients with diabetes that accelerates atherosclerosis was long thought
to be hyperglycemia. Hyperglycemia induces endothelial cell dysfunction and increases
nitrosative and oxidative stress (Ceriello et al. 2002). Hyperglycemia mediates accelerated
atherosclerosis by oxidative stress, which results in the formation of advanced glycation
endproducts (AGEs), synthesis of 1,2-diacylglycerol (DAG) and PKC activation, NFB-
mediated gene transcription, increase in the polyol and hexosamine pathways, and apoptosis
(Brownlee 2001). These changes due to hyperglycemia-induced oxidative stress lead to EC
activation and acceleration of atherosclerosis.
Clinical trials, however, reveal a weak association between blood glucose levels and
macrovascular outcomes. The United Kingdom Prospective Diabetes Study (UKPDS) trial
consisted of 5,102 newly-diagnosed T2DM patients with fasting plasma glucose concentrations
between 6.1 and 15.0 mM and randomized to either conventional glucose control (primarily
with diet) or intensive glucose control with sulphonylurea or insulin for an average of 10 years
(Stratton et al. 2000). The aim for the conventional control group was a fasting glucose of ≤15
mM and the intensive group <6 mM. The control group achieved a median hemoglobin A1c
(HbA1c) of 7.9% (HbA1c is a marker of average glucose control over 2-3 months measured by
the level of glycosylation of red blood cells) and the intensive group achieved a median HbA1c
of 7.0%. The intensive control group had a reduced risk of combined fatal or nonfatal
myocardial infarction and sudden death of 16% (P = 0.052), suggesting that hyperglycemia
alone is a small factor in the associated increased risk in people with diabetes as there is still a
significant event rate (Stratton et al. 2000).
Three other trials that specifically focused on glucose control and its association with
cardiovascular outcomes also failed to show a significant reduction in cardiovascular risk
(Macisaac and Jerums 2011; Control et al. 2009). The Action to Control Cardiovascular Risk in
Diabetes (ACCORD) trial was a randomized study of 10,251 patients living with type 2 diabetes
for 10 years and with established cardiovascular disease or additional cardiovascular risk factors
15
(anatomical evidence of significant atherosclerosis, albuminuria, left ventricular hypertrophy, or
two risk factors such as dyslipidemia, hypertension, status as a smoker or obesity) and a median
HbA1c of 8.1% at baseline. Patients in the ACCORD trial were assigned to either intensive
glucose control treatment to achieve a HbA1c below 6.0% or to standard treatment to achieve a
HbA1c of 7.0-7.9%. Treatments to achieve these targets included metformin, sulfonylureas,
meglitinides, thiazolidinediones, -glucosidase inhibitors, insulin, insulin analogues and
lifestyle intervention. After a 3.5-year follow-up, the ACCORD study’s intensive treatment
group had increased rates of severe hypoglycemia requiring assistance. But more importantly,
increased mortality was observed in the intensive treatment group and the trial was discontinued
(Gerstein et al. 2008).
The Action in Diabetes and Vascular Disease Preterax and Diamicron Modified Release
Controlled Evaluation (ADVANCE) trial was a randomized study of 11,140 patients with type 2
diabetes with an age ≥55 with a median follow-up of 5 years. The study also consisted of an
intensive glucose lowering group aiming for an HbA1c of ≤6.5% and a standard glucose therapy
group. Patients in the intensive therapy group were treated with gliclazide MR, a sulphonylurea,
plus any additional therapy required to achieve the HbA1c target; patients in the standard
therapy group were treated according to local guidelines with any required medication other
than gliclazide. Patients in the ADVANCE trial achieved a mean HbA1c of 6.5% in the
intensive therapy group for 2 years, but the study did not find a significant difference in the
number of macrovascular events between the groups (hazard ratio 0.94 [0.84-1.06], P = 0.32)
after a 5-year follow-up (Heller 2009).
Finally, the Veterans Affairs Diabetes Trial (VADT) trial was a study with 1791 randomized
patients with poorly-controlled type 2 diabetes with a median HbA1c of 9.4% who were
diagnosed a mean of 11.5 years earlier. Patients were allocated into an intensive treatment or
standard treatment group for glucose lowering and achieved a median HbA1c of 6.9% and
8.4%, respectively. Patients were treated with metformin plus rosiglitazone or glimepiride plus
rosiglitazone, with the intensive group started on maximal doses and the standard group on half-
maximal doses. Insulin was also added if the intensive group did not achieve a HbA1c of <6%
or the standard group patient was above 9%. As in the ACCORD and ADVANCE trials, the
VADT trial also failed to show a significant reduction in cardiovascular risk after a median
follow-up of 5.6 years in the veterans population (Duckworth et al. 2009).
16
1.3.2.2 Insulin resistance and hyperinsulinemia
The observation that cardiovascular risk increases well before the onset and diagnosis of type 2
diabetes has led to the belief that perhaps insulin resistance or hyperinsulinemia or both
contribute to the increased risk (Molecular Mechanisms of Atherosclerosis 2005; Bornfeldt and
Tabas 2011). Insulin resistance is often accompanied by hyperinsulinemia, and each contribute
independently to the risk for vascular disease; the relative contribution of each, however, has
been a matter of debate (Christian and George 2007). The term “insulin resistance” can either
mean decreased or increased insulin signaling through the insulin receptor; most studies,
however, have examined the effect of reduced insulin signaling (Bornfeldt and Tabas 2011).
Hypercholesterolemic (Apoe-/) mice with a knockout of the insulin receptor specifically in
endothelial cells (EIRAKO) showed a marked increase in atherosclerosis (as seen in the en face
flat preparation of the aorta) when fed a low-fat diet (9 kcal% with 0.221 ppm cholesterol) for
24 and 52 weeks (Rask-Madsen et al. 2010). Similar increases in atherosclerosis in mice are
obtained when the insulin receptor is eliminated in monocyte-derived cells (Seongah et al.
2006). A subsequent report showed hypercholesterolemic (Apoe-/-
) mice with one allele of the
insulin receptor knocked out in all tissues resulted in hyperinsulinemia due to reduced insulin
clearance (50% increase in plasma insulin without altering insulin resistance in other tissues),
and this hyperinsulinemic state did not change atherosclerosis burden when measured as en face
lesion area, cross-sectional plaque area in the aortic sinus or cholesterol abundance in the
brachiocephalic artery compared to control after 24 and 52 weeks of age (always fed a 9 kcal%
low-fat diet with 0.221 ppm cholesterol) (Rask-Madsen et al. 2012). Other studies show that
insulin resistant subjects often have elevated free fatty acids (FFAs) in the plasma, which impair
the action of insulin on the endothelium (Molecular Mechanisms of Atherosclerosis 2005;
Hamilton, Chew, and Watts 2007). These studies suggest that insulin signaling in ECs and
monocytes may play an important role in atherosclerosis, while hyperinsulinemia may have a
less significant role.
17
1.3.3 Lipoprotein metabolism
Lipids are of central importance in the initiation and progression of atherosclerosis. This section
briefly describes the important aspects of lipoprotein metabolism.
Lipids are hydrophobic and thus, are carried within lipoprotein complexes surrounding the
hydrophobic particles within a spherical core and exposing hydrophilic residues to solubilize
nonpolar lipids in plasma (Vance, Vance, and Bernardi 2002). In the intestine, bile salts
synthesized from cholesterol in the liver are secreted through the gallbladder following ingestion
of fat. Bile salts facilitate the emulsification of fat for lipolysis by pancreatic lipases. Once
absorbed by enterocytes, the FFAs are re-synthesized into TGs. TGs in the enterocytes are
incorporated into chylomicrons (which also have some cholesteryl ester (CE), free cholesterol
(FC) and phospholipids) on the APOB48 template and secreted into lymph, where they drain
into the circulation at the left subclavian vein. In the circulation, chylomicrons acquire APOE,
(apolipoprotein A-I) APO-AI, (apolipoprotein A-II) APO-AII, and (apolipoprotein C-II) APO-
CII from HDL (among other lipoproteins). Another key function of apolipoproteins is binding to
specific receptors. For example, APO-CII in chylomicrons activates lipoprotein lipase (LPL) on
ECs to release FFAs to peripheral cells such as muscle and adipose. As chylomicrons lose TGs,
they lose APO-AI, APO-AII and APO-CII and are termed chylomicron remnants. Chylomicron
remnants are internalized by the liver through the LDL-receptor related protein (LRP) and the
LDL receptor (LDLR) by endocytosis mediated by APOE (Vance, Vance, and Bernardi 2002).
Endogenous TGs are secreted in VLDL by the liver and contain apolipoprotein B-100 (APO-
B100), and later acquire APO-AI, APO-AII, APOC, and APOE from HDL (Vance, Vance, and
Bernardi 2002). Analogous to chylomicrons, VLDL particles also have greater TGs within their
core and deliver them to the periphery through LPL. As VLDL particles shrink, they become
VLDL remnants and are taken up by the liver through APOE binding to LDLR and/or LRP
(Vance, Vance, and Bernardi 2002).
After internalization of remnants in the liver, excess TGs in remnants undergo lipolysis by
hepatic triglyceride lipase (HTGL), remnants lose APO-CII and APOE (transferred back to
HDL), decrease in size, and are secreted as LDL into the circulation (Vance, Vance, and
Bernardi 2002). LDL also forms in the circulation from the lipolysis of TGs within VLDL. Also,
because LDL lacks APOE, its residence time is longer in the circulation than remnant particles
18
as its uptake must be mediated by APO-B100 alone, which has less affinity for the LDLR.
Furthermore, LDL is richer in cholesterol and serves as an extracellular cholesterol depot for
peripheral cells, which take up LDL through LDLR-mediated endocytosis. The most
atherogenic lipoproteins are cholesterol-rich remnant lipoproteins and LDL due to their smaller
size, which eases penetration into the subendothelium, and the presence of APOB, which bind
matrix proteoglycans through electrostatic interactions (Vance, Vance, and Bernardi 2002).
To complete the cycle, reverse cholesterol transport (RCT) is mediated by HDL and is
characterized by removal of excess cholesterol from peripheral tissues, including cholesterol-
laden macrophages, to the liver for excretion via the bile into feces (Vance, Vance, and Bernardi
2002; Brufau, Groen, and Kuipers 2011). While the traditional view of HDL function has been
in the removal of cholesterol, HDL also contributes to the extracellular cholesterol pool by
exchange of cholesterol through cholesteryl ester transfer protein (CETP) with VLDL and
remnant particles (Dominiczak and Caslake 2011). HDL is assembled on the APO-AI template,
which is secreted by both the liver and the intestine (Vance, Vance, and Bernardi 2002). Once
secreted, nascent HDL consists of phospholipid-rich discoid particles that bind to ABCA1
triggering cholesterol efflux from cells. Free cholesterol is then esterified by LCAT within the
HDL particle. As nascent HDL acquires cholesterol, it enlarges to become spherical HDL3.
HDL3 transfer cholesteryl esters to VLDL, chylomicrons and remnants in exchange for TGs.
HDL3 thus enlarges into HDL2 and offloads its cholesteryl esters into the liver by docking into
scavenger receptor B1 (SR-B1). Thus, HDL is considered anti-atherogenic as it removes
cholesterol from the body (Vance, Vance, and Bernardi 2002).
1.3.4 Adipokines in athersoclerosis
Obesity is associated with a predisposition to developing atherosclerosis, diabetes and non-
alcoholic fatty liver disease (NAFLD) (McGill et al. 2002). This association may relate to the
complex interaction of adipocytes with immune cells (stromal vascular cells or SVCs) through
secretion of adipokines. Obese individuals have more macrophages in adipocytes and they
secrete adipokines and cytokines that are pro-inflammatory (Tilg and Moschen 2006). Known
adipokines include adiponectin, leptin, resistin and visfatin. TNF-, interleukin-6 (IL-6) and
MCP-1 (also known as chemokine C-C motif ligand 2 or CCL2) are secreted by both adipocytes
19
and resident macrophages, and are considered cytokines. The role of leptin, resistin and IL-6 in
obesity are discussed.
1.3.4.1 Leptin
Leptin is a 16-kDa hormone secreted by adipocytes in proportion to adipocyte mass and is
virtually undetectable in the SVC fraction that contains the immune cells (Maury and Brichard
2010). Leptin signals energy deficiency to the brain and its circulating levels parallel adipose
tissue mass and nutritional status (leptin levels decrease during fasting). Obesity is associated
with high leptin levels and leptin resistance due to diminished transport of leptin across the
blood-brain barrier (BBB) and elevated suppressor of cytokine signaling-3 (SOCS-3) and
endoplasmic reticulum (ER) stress in the hypothalamus. Low leptin, leptin deficiency (ob/ob
mice), and/or leptin receptor deficiency (db/db mice) results in overfeeding, suppression of
energy expenditure, suppression of triiodothyronine (T3) thyroid hormone levels, decreased
testosterone and leutenizing hormone pulsatility, and a reduction in the number of CD4+ T cells
(Maury and Brichard 2010; Chan et al. 2003; Farooqi et al. 2002). Patients with lipodystrophy
have low circulating leptin levels and this results in severe insulin resistance, hyperinsulinemia,
hyperglycemia, and an enlarged fatty liver; these symptoms are ameliorated with exogenous
leptin supplementation in patients with lipodystrophy (Oral et al. 2002).
Leptin receptors have been detected in human umbilical vein endothelial cells (HUVECs) and
within human aortic atherosclerotic plaques by immunohistochemistry (Park et al. 2001; Sierra-
Honigmann et al. 1998). Increased leptin in humans is associated with impaired arterial
distensibility measured by vascular ultrasound in healthy adolescents aged 13-16 years, and this
result is independent of fat mass, blood pressure, C-reactive protein (CRP), fasting insulin, or
LDL cholesterol concentrations (Singhal et al. 2002). Leptin (10 ng/ml) induces expression of
pro-inflammatory markers such as NF-B and MCP-1, as well as the generation of ROS in
cultured HUVECs and bovine aortic endothelial cells (BAECs) possibly by increasing fatty acid
oxidation by stimulation of carnitine palmitoyltransferase-1 (CPT-1) activity and inhibition of
acetyl-CoA carboxylase (ACC) activity as shown in BAECs treated with 10 ng/ml leptin
(Bouloumie et al. 1999; Yamagishi et al. 2001). One report showed that recombinant murine
leptin administration (125 g) for 4 weeks in 16-week-old Apoe-/-
mice significantly enhanced
20
atherosclerotic lesion development despite significant reductions in body weight (Bodary et al.
2005). It is thus postulated that hyperleptinemia may be atherogenic (Ritchie et al. 2004).
1.3.4.2 Resistin
Resistin is a 114-amino-acid protein that was originally shown to induce insulin resistance in
mice (Tilg and Moschen 2006). Resistin synthesis in mice occurs only in adipose tissue;
whereas in humans, adipocytes, muscle, pancreatic cells and macrophages also synthesize the
protein. Resistin expression increases when induced by pro-inflammatory cytokines and
lipopolysaccharide (LPS). Resistin is thought to be atherogenic (Tedgui and Mallat 2006) as it
stimulates synthesis of pro-inflammatory cytokines TNF-, interleukin-1 (IL-1), IL-6 and
interleukin-12 (IL-12) through NF-B in various cell types. It also upregulates VCAM-1,
ICAM-1 and MCP-1 in human ECs. Lastly, macrophages infiltrating human atherosclerotic
aneurysms secrete resistin (Tilg and Moschen 2006).
1.3.4.3 Interleukin-6
IL-6 is a cytokine produced by several cells (fibroblasts, ECs, monocytes, SMCs, T cells and
adipocytes) and adipose tissue contributes about 15-35% of the systemic IL-6 in humans (Maury
and Brichard 2010; Tedgui and Mallat 2006). IL-6 levels predict future coronary artery disease
(Harris et al. 1999) and have been shown to be an independent predictor of mortality among
patients with unstable coronary artery disease (Lindmark et al. 2001). IL-6 is thought to be
implicated in insulin resistance, and wildtype mice fed a high-fat diet increase IL-6 production
by adipose tissue, which induces hepatic insulin resistance. This insulin resistance is thought to
be due to the upregulation of SOCS-3, which results in the ubiquitination of insulin receptors for
proteosomal degradation (Maury and Brichard 2010). While chronic IL-6 elevation has no effect
in muscle in vivo, acute IL-6 elevation from exercise enhances insulin action in skeletal muscle
(Maury and Brichard 2010). IL-6 secreted from visceral adipose tissue also regulates hepatic
production of acute-phase reactants such as CRP; evidence for this is the 50% higher levels of
IL-6 detected in the portal vein than the radial artery of obese subjects, which correlated with
systemic CRP (Maury and Brichard 2010). In turn, CRP also predicts cardiovascular disease
(CVD) and atherosclerotic plaque instability in humans and has been shown to enhance
21
atherosclerosis in Apoe-/-
mice overexpressing human CRP (Paffen and DeMaat 2006). IL-6
protein and mRNA is detected in human atherosclerotic plaques by immunohistochemistry, and
reverse transcription and PCR amplification, respectively at 10- to 40-fold higher levels than in
non-atherosclerotic lesions (Rus, Vlaicu, and Niculescu 1996; Seino et al. 1994).
IL-6 has been shown to have both inflammatory and anti-inflammatory roles in atherogenesis.
Exogenous administration of recombinant IL-6 (5000 U) to wildtype C57BL6 mice fed a high-
fat diet (20% fat, 1.5% cholesterol), or Apoe-/-
mice on either normal chow or high-fat diets for
6 or 21 weeks (started at 3 weeks of age) increased fatty streak size at the aortic sinus 5.1-fold in
C57BL6 mice and atherosclerotic plaque area 2.4- and 1.9-fold in Apoe-/-
mice on the low-fat or
high-fat diets, respectively (Huber et al. 1999). Conversely, 1-year-old Il6-/-
Apoe-/-
mice on
normal chow have enhanced plaque formation with more calcified lesions but decreased
macrophage and leukocyte infiltration than Il6+/+
Apoe-/-
littermate control mice (Elhage et al.
2001; Schieffer et al. 2004). However, younger mice, 16-week-old Il6+/-
Apoe-/-
or Il6-/-
Apoe-/-
,
did not show differences in fatty streak lesion size (Elhage et al. 2001). Thus, the role of IL-6 in
atherosclerosis is unclear. While IL-6 can be viewed as a pro-inflammatory cytokine, it also has
anti-inflammatory actions (reviewed in (Barton 1996)). For example, continuous intravenous
administration of IL-6 in humans for 120 hours leads to an increase in IL-1 receptor antagonist
(IL-1Ra) and soluble TNF- receptors (sTNFR) (Tilg et al. 1994). IL-1Ra antagonizes the
effects of IL-1 by blocking the binding of IL-1 to cell surface receptors, thus protecting against
septic shock and inflammatory bowel disease. Soluble forms of TNF- receptors block LPS-
mediated lethality. In another experiment, i.p. administration of a sublethal dose of endotoxin (4
g/g) resulted in higher TNF- and interferon-gamma inflammatory cytokines in serum of Il6-/-
mice compared to Il6+/+
mice, and recombinant IL-6 administration to Il6-/-
mice abolished the
difference in circulating pro-inflammatory cytokines (Xing et al. 1998).
1.3.5 The Apoe-/- mouse model of atherosclerosis
Unlike humans, wildtype mice are highly resistant to developing atherosclerosis even when
given diets very high in cholesterol (Meir and Leitersdorf 2004). Also unlike humans, mice
carry most of their cholesterol in anti-atherogenic HDL, whereas humans carry 75% of
cholesterol in LDL (Jawien, Nastalek, and Korbut 2004). To circumvent these challenges, Apoe-
22
deficient mice were created (Plump et al. 1992). As discussed earlier, APOE is synthesized by
the liver for the clearance of chylomicron and VLDL remnant lipoprotein particles via the
LDLR and LRP (Meir and Leitersdorf 2004). Mice deficient in APOE and fed a low fat, low
cholesterol diet exhibit much higher plasma cholesterol than wildtype control mice (494 versus
60 mg/dl); and when fed a Western diet with 0.15% cholesterol (developed by Hayek et al),
Apoe-deficient mice have extremely high levels of plasma cholesterol (1821 versus 132 mg/dl)
(Plump et al. 1992). In addition, Apoe-deficient mice have a lipid profile that is more similar to
humans: a shift for much higher cholesterol in chylomicrons, VLDL and LDL, plus a reduction
in HDL cholesterol 45% of that compared to wildtype mice (Jawien, Nastalek, and Korbut
2004). But most important of all, these mice develop atherosclerotic lesions that are similar to
human lesions (Nakashima et al. 1994). Another advantage is that lesions in these mice develop
early and spontaneously even when fed a regular chow diet. Monocytic adhesion is observed in
5-6 weeks, fatty streaks by 3 months, intermediate lesions by 15 weeks, fibrous plaques at 20
weeks, and calcification and wall thinning after 32 weeks of age (Meir and Leitersdorf 2004).
For these reasons, the Apoe-deficient mouse model has been used extensively for the study of
atherogenesis (Meir and Leitersdorf 2004). In this study, we used the Apoe-knockout mouse
model with streptozotocin-induced diabetes to study the effects of GLP-1R activation on
atherogenesis.
1.3.6 Current treatments for atherosclerosis
1.3.6.1 Lipid lowering
Therapy with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors
(statins) has shown significant benefits for CVD (Ray, Cannon, and Braunwald 2007). HMG-
CoA reductase inhibitors inhibit the rate-limiting enzyme in cholesterol synthesis, HMG-CoA
reductase. These inhibitors thus upregulate LDLRs in hepatocytes and increase LDL uptake and
clearance from the circulation. With the fall in LDL levels, HDL levels rise slightly (Molecular
Mechanisms of Atherosclerosis 2005). They have also been discovered to exert a number of
immunomodulatory, potentially anti-inflammatory effects. HMG-CoA reductase inhibitors
inhibit interferon gamma (IFN--induced major histocompatibility complex-2 (MHC II)-
mediated T-cell activation, and monocyte adhesion molecule expression (Molecular
Mechanisms of Atherosclerosis 2005). Akt and Rho/Rho-kinase systems are also induced by
23
statins, resulting in potentiation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS)
phosphorylation, inhibition of EC tissue factor, inhibition of VSMC migration, and mobilization
of endothelial progenitor cells (Molecular Mechanisms of Atherosclerosis 2005). Thus, it is not
surprising that HMG-CoA reductase inhibitors still reduce plaque burden even in the absence of
lipid lowering ("MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in
20,536 high-risk individuals: a randomised placebo-controlled trial" 2002).
Fibric acid compounds also improve the lipid profile, but they have not shown significant
benefits in the reduction of cardiovascular risk in patients with diabetes either alone (Zoungas
and Patel 2010) or in combination with HMG-CoA reductase inhibitors (Farmer 2011).
Although dietary cholesterol is an independent risk factor for atherosclerosis, the attributable
risk is low. Increasing evidence points more at cholesterol-rich post-prandial chylomicron
remnants being most atherogenic, where dietary cholesterol contributes only about one third,
and the rest is derived endogenously from de novo synthesis (Huff 2003). To address the
intestinal pathway, the drug ezetimibe was developed, which inhibits carrier-mediated
cholesterol absorption in the intestine. Despite ezetimibe’s effect on LDL-cholesterol lowering,
its effect on atherosclerosis remains indeterminate. The ENHANCE (Ezetimibe and Simvastatin
in Hypercholesterolemia Enhances Atherosclerosis Regression) trial consisted of 720 patients
with familial hypercholesterolemia treated with 80 mg simvastatin (an HMG-CoA reductase
inhibitor) plus either placebo or 10 mg of ezetimibe given daily for 2 years. The ENHANCE
trial assessed carotid and femoral intima-media thickness by ultrasonography after 24 months
and found a trend suggesting a negative effect of ezetimibe treatment as the mean intima-media
thickness of the carotid and femoral arteries showed a trend toward greater thickness (0.0033
mm for simvastatin monotherapy versus 0.0182 mm for simvastatin plus ezetimibe therapy, P =
0.15) (Guyton 2010).
1.3.6.2 Hypertension
Therapeutic options for the treatment of hypertension include several major classes of drugs:
diuretics, β-adrenoceptor antagonists (β-blockers), angiotensin-converting enzyme (ACE)
inhibitors, angiotensin II type 1 receptor antagonists (ARBs), renin inhibitors, calcium channel
blockers, and central sympatholytics, alone or in combination (Chrysant 2010). Patients with
24
diabetes often have elevated blood pressure (BP) that adds an additional risk factor for
accelerated atherosclerosis and cardiovascular events (Taylor and Bakris 2010). ACE inhibitors
reduce the risk of coronary events by regulation of blood pressure. These drugs regulate blood
pressure by reducing the activity of components of the renin-angiotensin-aldosterone system
(RAAS). It has been suggested that these drugs not only have effects on lowering blood
pressure, but may also have other effects that might help further reduce progression of
atherosclerosis (Koh et al. 2010). For example, the ARB drug candesartan used to treat
hypertension also improves markers of oxidative stress, inflammation, and fibrinolysis
independent of BP lowering effects; losartan, another ARB, decreases myocardial collagen
content and corrects the altered endothelial structure and resistance of arteries; and valsartan,
another ARB, has BP-independent effects on left ventricular hypertrophy, reactive oxygen
species formation by monocytes, and C-reactive protein in hypertensive patients with left
ventricular hypertrophy when compared with amlodipine, a calcium channel blocker also used
to treat hypertension (Koh et al. 2010).
1.3.6.3 Diabetes
Despite current therapies to lower lipids and blood pressure, there remains a significant event
rate for cardiovascular disease especially in patients with diabetes and/or insulin resistance
(Rubenfire, Brook, and Rosenson 2010; Tenenbaum et al. 2006). Patients with diabetes often
present with more risk for atherosclerosis; obesity, dyslipidemia, hypertension, hyperglycemia
and a chronic inflammatory state may all be present in diabetes (Taylor and Bakris 2010).
Simultaneous management of diabetes and other risk factors for atherosclerosis that are usually
present in patients with diabetes requires administration of several drugs and makes control
challenging. Treatment is often associated with side effects and further progression to
developing type 2 diabetes (Taylor and Bakris 2010). For example, less than 33% of patients
with diabetes treated for hypertension attained controlled levels of BP (Taylor and Bakris 2010).
In this regard, GLP-1R agonists may hold the key to further reducing the rate of cardiovascular
events. GLP-1R agonists have been shown to reduce body weight, blood pressure and improve
lipid profile, all of which contribute to a reduction in atherosclerosis (Rizzo et al. 2009). In
addition, the GLP-1R agonist liraglutide reduced infarct size in experimental murine myocardial
infarction (Noyan-Ashraf et al. 2009), and the GLP-1R agonist exenatide was associated with a
25
reduced incidence for cardiovascular disease events in a retrospective analysis of type 2 diabetic
patients treated with exenatide twice daily (hazard ratio 0.81) (Best et al. 2011). It remains to be
shown, however, whether GLP-1R agonists can exert direct effects on atherogenesis in the
setting of diabetes.
1.4 GLP-1R actions on atherogenesis
Atherogenesis is influenced by many indirect factors such as lipoprotein concentrations, blood
pressure, visceral adiposity, insulin resistance and hyperglycemia which do not directly act on
the plaque but influence its progression and/or regression. Atherosclerosis also involves the
interplay of many cell types (neutrophils, monocytes, T cells, B cells, DCs, mast cells, ECs and
smooth muscle cells (SMCs)) (Weber and Noels 2011). In this section, the indirect and direct
actions of GLP-1R agonists on atherogenesis are discussed.
1.4.1 Indirect actions
1.4.1.1 Lipoprotein metabolism
The effects of atherogenic plasma LDL and anti-atherogenic HDL in atherogenesis are well-
known (Weber and Noels 2011). Several lines of evidence show that activation of the GLP-1R
in either rats (Qin et al. 2005), mice (Hsieh et al. 2010; Parlevliet et al. 2009), hamsters (Hsieh
et al. 2010) or humans (Koska et al. 2010; Matikainen et al. 2006; Meier et al. 2006; Tremblay
et al. 2011) results in a decrease in postprandial lipids. In adult rats, GLP-1(7-36)NH2 infusion
through the jugular vein (20 pmol/kg/min) with simultaneous direct infusion of lipids (3 ml/h)
into the duodenum of lymph duct-cannulated rats reduced triglyceride absorption as measured
by lymphatic triolein recovery and levels of lymphatic APOB lipoproteins after overnight
fasting (Qin et al. 2005). In hamsters and mice, treatment with the DPP-4 inhibitor sitagliptin
both acutely (10 g/kg) and chronically (5 g/kg for 2 to 3 weeks), resulted in marked
reductions in APO-B48-containing lipoproteins after olive oil gavage followed by Triton
WR1339 (0.5 g/kg) administration to inhibit peripheral and hepatic lipase. This reduction in
APO-B48-containing lipoproteins after olive oil gavage was abolished in GLP-1R knockout
mice (Hsieh et al. 2010). An ex vivo experiment using cultured chow-fed hamsters’ enterocytes
26
showed exendin-4 (100 pM) inhibited the secretion of newly-synthesized APO-B48 (Hsieh et al.
2010). In the absence of lipase inhibition by Triton, 8-week vildagliptin treatment (another DPP-
4 inhibitor) (1 mol/ml supplied in drinking water) also resulted in a significant reduction in
plasma triglycerides and cholesterol in high-fat-fed (45 kcal%) male C57BL6 wildtype mice
after refeeding from an overnight fast with a 45 kcal% high-fat diet (Flock et al. 2007). In
healthy (male) humans, intravenous administration of GLP-1(7-36)NH2 (1.2 pmol/kg/min) over
390 minutes (-30 to 360 min) with a mixed test meal (250 kcal) ingested at 0 min was shown to
abolish the rise in plasma triglycerides (area under the triglyceride concentration curve for
placebo, 7331±1850 mg dl-1
min and GLP-1, 1263±436 mg dl-1
min) and non-esterified fatty
acids (NEFA) (39% lower in the fasting state and 31±5% lower throughout the meal test);
however, these effects are in part due to delayed gastric emptying for GLP-1-treated individuals
versus placebo (50% of the initial content was still in the stomach compared to only 20% for
placebo) (Meier et al. 2006). In patients with recent-onset T2DM (<3 years), acute subcutaneous
administration of exenatide (10 g) also reduced plasma triglycerides (3.8 mM for placebo
versus 2.5 mM after exenatide) while improving endothelial function (measured by peripheral
arterial tonometry or PAT index; PAT index was higher in the exenatide group than placebo)
after a high-fat meal (45% fat) (Koska et al. 2010). To circumvent the effects of gastric
emptying and other hormones or FFAs on lipoprotein metabolism, a recent study in healthy
humans (15 males) administered a liquid formula (49 kcal% from fat) directly into the
duodenum through a nasoduodenal tube, and showed exenatide (10 g subcutaneous)
suppressed intestinal lipoprotein particle production (24-37% decrease in triglyceride-rich
lipoprotein (TRL)-APOB-48, and a 38% reduction in TRL-APOB-48 production rate) during
constant infusion of somatostatin during a standard pancreatic clamp (Xiao et al. 2012). Finally,
treatment with the DPP-4 inhibitors vildagliptin (50 mg twice daily for 4 weeks) or sitagliptin
(100 mg/day for 6 weeks) in T2DM patients decreased post-prandial (over 8 hours) APOB-
containing lipoproteins of both intestinal and hepatic origin (5.1 to 7.8% APOB reduction)
(Matikainen et al. 2006; Tremblay et al. 2011). In short, while a molecular mechanism of action
is currently not known, these data suggest that, in general, GLP-1R activation reduces intestinal
lipoprotein secretion independent of gastric emptying, which may lead to reductions in TRL-
APOB-48 lipoprotein particles.
27
1.4.1.2 Blood pressure
Hypertension promotes atherogenesis and is a risk factor for cardiovascular events (Zanchetti et
al. 1998). Studies in animals have shown that acute activation of the GLP-1R increases BP
(Ussher and Drucker 2012). Chronic treatment with agonists for the GLP-1R and DPP-4
inhibitors in rodents and humans, however, results in reductions in BP that can be observed
before significant weight loss is recorded (Ussher and Drucker 2012).
1.4.1.3 Adiposity and adipokines
The role of select adipokines in atherogenesis was discussed in section 1.3.4. Here, current
findings on how GLP-1R activation affects adipokine secretion and/or action is reviewed.
Treatment in both animals (Li et al. 2008) and humans (Bunck et al. 2010) with GLP-1R
agonists often results in a decrease in body weight with changes in adipokine expression. In
humans (Bunck et al. 2010), rats (Li et al. 2008) and mice (Samson et al. 2008), plasma
adiponectin has consistently been shown to be increased in vivo with exendin-4 treatment. In
vitro, 3T3-L1 adipocytes cultured with 2.5 or 5 nM exendin-4 for 8 hours also showed an
increase in adiponectin mRNA (2-fold) and protein in media (by 20%) (Kim Chung et al. 2009).
The decrease in adiponectin levels in the setting of obesity has been associated with insulin
resistance and cardiovascular risk (Kim et al. 2007; Menzaghi, Trischitta, and Doria 2007; Hotta
et al. 2000; Pischon et al. 2004); however, in vivo studies in mice have not shown a direct role of
adiponectin in atherogenesis (Nawrocki et al. 2010).
Exendin-4 treatment of 3T3-L1 adipocytes also decreased protein expression of IL-6 and MCP-
1 (Kim Chung et al. 2009). Furthermore, clinical studies have revealed a consistent decrease in
CRP after chronic treatment with exenatide in patients with T2DM, although plasma IL-6,
MCP-1 and resistin levels were unaffected (Bunck et al. 2010; Horton et al. 2009; Courrèges et
al. 2008). The observed reductions in CRP, however, could be due to the significant decrease in
body weight. A recent study, however, examined the effects of 12 weeks of treatment with
exenatide in T2DM patients. These patients did not lose weight during this period, yet showed a
marked reduction in plasma concentrations of MCP-1, matrix metalloproteinase-9 (MMP-9),
serum amyloid A (SAA), and IL-6 (Chaudhuri et al. 2012). It is thus not clear whether
28
activation of the GLP-1R has a direct role in vivo in the expression and secretion of IL-6 and
MCP-1 from adipocytes.
Long-term GLP-1R activation (15 weeks) by constitutive expression of exendin-4 through a
helper-dependent adenoviral vector expressed in liver did not change plasma leptin levels in
C57BL6 mice fed a 42 kcal% high-fat diet (Samson et al. 2008). In T2DM patients, liraglutide
treatment for 14 weeks did not change plasma leptin levels despite significant reductions in
body weight (Courrèges et al. 2008). On the other hand, reductions in resistin levels were seen
in mice treated with constitutive expression of exendin-4 through a helper-dependent adenoviral
vector in liver, but the reduction was linearly correlated to decreases in body fat (Samson et al.
2008).
Thus, current evidence suggests that the overall effect of activation of the GLP-1R on
adipokines appears to be indirect through the loss of adipocyte mass due to the anorectic effects
of the GLP-1R in the brain. Reductions in plasma CRP from GLP-1R activation also occur, but
a clear direct role of CRP in atherogenesis is currently lacking; there is only data on the
association of CRP with CVD and plaque instability (Paffen and DeMaat 2006).
1.4.1.4 Insulin resistance, hyperinsulinemia and hyperglycemia
The contributions of insulin resistance, hyperinsulinemia and hyperglycemia to atherogenesis
were discussed in section 1.3.2. Briefly, insulin resistance (decreased insulin signaling) in
endothelial cells and macrophages resulted in marked increases in atherogenesis in mice (Rask-
Madsen et al. 2010; Seongah et al. 2006); whereas hyperinsulinemia did not alter atherogenesis
(Rask-Madsen et al. 2012). Furthermore, clinical evidence pointed to moderate effects of
improved glucose control in the reduction of cardiovascular events in the UKPDS trial after 10
years of treatment (Stratton et al. 2000).
Activation of the GLP-1R in pancreatic -cells stimulates glucose-dependent insulin secretion to
promote glucose homeostasis (Baggio and Drucker 2007; Drucker et al. 1987; Lamont et al.
2012). The improved blood glucose control from GLP-1R activation may have modest effects
on reducing atherogenesis as proved by the UKPDS (Stratton et al. 2000), whereas the increased
29
insulin secretion may not play an important role as proved in hyperinsulinemic mice prone to
atherosclerosis (Rask-Madsen et al. 2012).
While the GLP-1R is not expressed in muscle, fat or liver, studies suggest prolonged GLP-1R
activation may improve insulin sensitivity. Non-diabetic Zucker fa/fa rats treated with exenatide
(3 g/kg twice daily) for 6 weeks had a 61% increase in insulin sensitivity index (ISI—glucose
infusion rate to plasma insulin concentration) as assessed in a hyperinsulinemic euglycemic
clamp compared to pair-fed controls (Gedulin et al. 2005). Similarly, infusion of GLP-1(7-
36)NH2 (4.8 pmol/kg/min) for 6 weeks in T2DM patients improved the insulinogenic index
(area under the curve, AUC insulin to AUC glucose) during a hyperinsulinemic euglycemic
clamp (Zander et al. 2002). Nonetheless, increased insulin sensitivity by GLP-1R activation is
controversial; GLP-1 (1.2 pmol/kg/min) or exendin-4 (0.12 pmol/kg/min) in eight healthy
subjects failed to show changes in glucose infusion rate, glucose disappearance or endogenous
glucose production during a hyperinsulinemic euglycemic clamp with infusion of somatostatin
(120 ng/kg/min) to inhibit endogenous insulin, while maintaining growth hormone (3
ng/kg/min) and glucagon (0.65 ng/kg/min) basal levels (Vella et al. 2002). It is likely that
studies showing a positive effect on insulin sensitivity are due to a period of improved glucose
control due to glucose-stimulated insulin secretion from GLP-1R activation. In turn, improved
glucose control independently improves insulin sensitivity of peripheral tissues (Yki-Jarvinen
1990, 1997). Thus, while GLP-1R activation does not directly modulate insulin sensitivity, it
may do so indirectly in diabetic patients through improvements in glucose homeostasis. Thus,
improved glucose control through GLP-1R activation may indirectly ameliorate insulin
resistance and thus, reduce the accelerated development of atherosclerosis in diabetic patients.
1.4.2 Direct actions
1.4.2.1 Endothelial cells
In vitro studies: Activation of the GLP-1R by liraglutide (0.01 to 100 g/ml) in HUVECs
induced NO formation dose-dependently through eNOS phosphorylation and suppressed high
glucose- (27.5 mM) or TNF--mediated (10 ng/ml) activation of NF-B and I-kappaB (IB)
degradation. HUVECs treated with liraglutide (1 g/ml) for 24 hours also reduced TNF--
induced upregulation of MCP-1, VCAM-1, ICAM-1 and E-selectin mRNA (Hattori et al. 2010).
30
In human coronary artery endothelial cells (HCAECs) incubated at euglycemic conditions (5
mM glucose), 1-10 nM exendin-4 treatment for 48 hours also resulted in a greater than 2-fold
phosphorylation of eNOS at Ser-1177 (activating eNOS) by a PI3K-Akt-dependent pathway,
and an increase in eNOS protein expression (Erdogdu et al. 2010). The study also showed that
incubation with 1 nM exendin-4 for 48 hours increased the rate of [3H]thymidine incorporation
to 190% and 10 nM further augmented the rate to 264%. The proliferation of HCAECs was
through Akt and MAPK phosphorylation, and the proliferative effects were abolished with
PI3K, Akt, PKA or eNOS inhibitors (Erdogdu et al. 2010). Another study, however, found no
Akt phosphorylation in HUVECs treated with 10 nM GLP-1, although cAMP response element
binding (CREB) phosphorylation was increased by 52% in a PKA-dependent manner (effect
was abolished by PKA inhibition) (Oeseburg et al. 2010). The same group treated 10-week-old
ZDF rats with the DPP-4 inhibitor vildagliptin (3 mg/kg/day) for 15 weeks which resulted in a
6-fold increase in plasma GLP-1 levels and reduced senescence of abdominal aortic endothelial
cells as assessed by -galactosidase staining (Oeseburg et al. 2010).
Incubation of HUVECs with 0.03-0.3 nM GLP-1(7-36)NH2 for 4 hours resulted in inhibition of
AGE-induced ROS generation and the inhibition of AGE-induced VCAM1 mRNA upregulation;
incubation of HUVECs with 0.3 nM GLP-1 for 4 hours also downregulated mRNA levels of the
receptor for AGEs (RAGE) (Ishibashi et al. 2010). In the spontaneously-transformed C11-STH
human umbilical vein endothelial cell line, incubation with 100 nM liraglutide for 1 hour
inhibited TNF--induced (10 ng/ml) or hyperglycemia-induced (10 M) upregulation of PAI-1,
ICAM-1 and VCAM-1 mRNA and protein (Liu et al. 2009). Furthermore, incubation of C11-
STH endothelial cells with 100 nM liraglutide incubated with 10 ng/ml TNFfor 16 hours
inhibited TNF-mediated induction of ICAM-1 and VCAM-1 mRNA and protein and NFkB
mRNA expression, which was independent of PKA signaling as use of the PKA inhibitor H89
(10 M) did not attenuate this effect (Gaspari et al. 2011).
In vivo studies: Apoe-/-
mice fed a 22% fat and 0.15% cholesterol diet for 12 weeks and
subsequently treated twice daily with 80 nmol/kg liraglutide for four weeks show significant
improvements in endothelial function as assessed by endothelial-dependent vasodilation of
abdominal aortic rings (using acetylcholine) pre-constricted ex vivo with a thromboxane A2
analogue (Rmax = 78.45% versus 55.36% in controls) (Gaspari et al. 2011). In addition, eNOS
and ICAM-1 were shown to be upregulated by liraglutide by immunohistochemical analysis in
31
intermediate aortic cross-sections compared to control mice (Gaspari et al. 2011). Activating the
GLP-1R in pulmonary artery rings isolated from male Sprague-Dawley rats also showed a dose-
dependent relaxation with GLP-1(7-36)NH2 (10-8
to 10-5
M) when arteries were pre-constricted
using 10-7
M norepinephrine (16.2% of maximal relaxation by acetylcholine) (Richter et al.
1993; Golpon et al. 2001).
In healthy non-diabetic humans, GLP-1 infusion improved acetylcholine-induced vasodilation
and forearm blood flow measured by venous occlusion plethysmography (9.1 versus 6.5
ml/100ml/min) (Basu et al. 2007). Similarly, in T2DM patients with established coronary artery
disease (CAD), a hyperinsulinemic euglycemic clamp with GLP-1(7-36)NH2 infusion (2
pmol/kg/min) increased brachial artery flow-mediated vasodilation (FMD) (6.6% versus 3.1%)
(Nyström et al. 2004). A third study with both healthy and T2DM patients infused with GLP-
1(7-36)NH2 at 0.4 pmol/kg/min (to reach physiological postprandial GLP-1 levels) also showed
improvements in flow-mediated vasodilation in both groups during a hyperglycemic clamp
(healthy subjects, 9.6% versus 7.0%; T2DM subjects, 3.8% versus 0.5%) (Ceriello et al. 2011).
While these experiments were conducted using the native GLP-1 peptide, DPP-4-resistant
exenatide (0.03 pM to 0.3 M) did not induce vasodilation or cyclic guanine monophosphate
(cGMP) release from mouse mesenteric arteries pre-constricted with 3 M phenylephrine ex
vivo. However, GLP-1(7-36)NH2 and the truncated peptide GLP-1(9-36) did induce vasodilation
and cGMP release (Ban et al. 2008). A similar result in male Sprague-Dawley rats’ femoral
arteries demonstrated that exenatide did not exert an effect after infusion with intralipid, but
GLP-1 and GLP-1(9-36) produced concentration-dependent vasorelaxation after pre-
constriction with phenylephrine (10-5
M) (Nathanson et al. 2009). Thus, it is not clear whether
vasodilatory effects can be mediated by degradation-resistant GLP-1R agonists as current data
suggests the effects are predominantly mediated by the truncated GLP-1(9-36) metabolite.
1.4.2.2 Vascular smooth muscle cells
VSMCs are important in both early and late stages of atherosclerosis (Doran, Meller, and
McNamara 2008). GLP-1R binding sites are known to be expressed in rat pulmonary VSMCs as
detected by incubation of pulmonary arteries with 125
I-GLP-1(7-36)NH2 and slide-mount
autoradiography (Richter et al. 1993). The receptor was also shown to be expressed by Western
32
blotting using the GLP-1R antibody from Abcam using extracts from murine aortic, rat and
human primary coronary artery SMCs (Goto et al. 2011). Isolated and cultured murine VSMCs
also express the GLP-1R as detected using the same antibody from Abcam (Arakawa et al.
2010). Little is known about the role of the GLP-1R in VSMCs. It has been reported that
exendin-4 treatment for 4 weeks (24 nmol/kg/day infused through an osmotic pump) reduces
neointimal formation (by about 50% and intima/media ratio by about 3-fold) in 9-week-old
C57BL6 mice subjected to endothelial denudation at the femoral artery (Goto et al. 2011). Ten-
week-old female ZDF rats subjected to balloon catheter injury in the carotid artery and
descending aorta showed a marked reduction in intimal hyperplasia when treated with exendin-4
(5.0 g/kg for one week prior to injury, then resuming exendin-4 treatment for another 22 days
after injury; intima/media ratio = 1.0 in controls versus 0.2 in exendin-treated rats). Examination
by Western blotting of the aortic tissue revealed no differences in the level of total eNOS
(endothelial nitric oxide synthase), but did show a trend for decreased protein levels of NFB
(0.11 versus 0.045 density units) (Murthy et al. 2010). In vitro, platelet-derived growth factor
(PDGF)-induced proliferation (measured by BrdU incorporation) of cultured VSMCs of the
mouse aorta was inhibited with 12-hour pre-incubation with 10 nM exendin-4. The inhibitory
effect by exendin-4 was not due to changes in intracellular cAMP, phosphorylation of mitogen-
activated protein kinase (MAPK), Akt, cAMP response element-binding (CREB) or
P70S6kinase (a serine/threonine kinase that phosphorylates S6 ribosome to induce protein
synthesis) as Western blotting did not reveal significant changes from control (Goto et al. 2011).
1.4.2.3 Monocytes and monocyte-derived cells
The GLP-1R has been shown to be expressed by Western blotting in human circulating
monocytes, in the THP-1 monocyte cell line, and in murine peritoneal macrophages using the
antibody from Abcam (Kodera et al. 2011; Arakawa et al. 2010). The GLP-1R was also shown
to be present in atherosclerotic lesions of Apoe-/-
mice using fluorescent staining with the GLP-
1R antibody from MBL International (LS-A1205) (Arakawa et al. 2010). Detection of the GLP-
1R using today’s commercially-available antibodies or the use of RT-PCR, however, is
controversial (please refer to section 4.5 for an expanded explanation). In vitro treatment of
human monocytes with 0.3-3 nM exendin-4 reduced CD11b surface receptors (which bind
ICAM-1) as assessed by flow cytometry (Arakawa et al. 2010). In cultured THP-1 monocyte
33
cells, exendin-4 was shown to reduce the mRNA expression (by RT-PCR) and protein secretion
(by ELISA on supernatant) of TNF- and IL-1 (Kodera et al. 2011). In thioglycolate-induced
peritoneal macrophages from wildtype C57BL6 mice, 0.03-3 nM exendin-4 pre-treatment for 1
hour reduced 10 g/ml LPS-induced expression of MCP-1 and TNF- through adenylate
cyclase/cAMP/PKA pathway as explored by use of adenylate cyclase and PKA inhibitors or
activators (Arakawa et al. 2010). Cholesteryl ester accumulation in peritoneal macrophages
from Apoe-/-
mice incubated with GLP-1(7-36) was reduced compared to control in association
with reduced protein levels of CD36 (a scavenger receptor) and ACAT-1 (a cholesteryl ester
transferase) (Nagashima et al. 2011). THP-1 monocyte-derived macrophages also express the
GLP-1R as shown by RT-PCR and Western blotting using the antibody from Santa Cruz
Biotechnology (Matsubara et al. 2012). Treatment with GLP-1 (10 pM) in THP-1 cells reduced
LPS-induced (10 ng/ml) IL-6 production through inhibition of LPS-induced extracellular-signal-
regulated kinase-1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), and increased cAMP as
assessed with MDL-12330A adenylyl cyclase inhibitor (5 M) and H89 PKA inhibitor (5 M)
(Matsubara et al. 2012).
1.4.2.4 Plaque formation
The effect of GLP-1R activation on plaque development has been evaluated in non-diabetic
mice. The first report identified a moderate reduction in atherogenesis in association with
reduced monocyte adhesion to the endothelium as assessed by en face immunohistochemical
staining using MAC-2 antibody on the thoracic aorta after 28-day continuous infusion of
exendin-4 with an osmotic pump (24 nmol/kg/day) in both wildtype C57BL6 and Apoe-/-
mice
(wildtype, 7 versus 23 cells/mm2; Apoe
-/-, 23 versus 46 cells/mm
2) (Arakawa et al. 2010). Plaque
size at the aortic sinus (stained with oil red O) of Apoe-/-
mice treated with exendin-4 was
significantly reduced, as was Icam1 and Vcam1 mRNA expression in the thoracic aorta
(Arakawa et al. 2010). Continuous infusion through implanted mini-osmotic pumps of active
GLP-1(7-36)NH2 for 4 weeks at 2.2 nmol/kg/day in Apoe-/-
mice aged 17 weeks fed an
atherogenic diet (30% fat, unknown amount of cholesterol) for 4 weeks resulted in markedly
reduced plaque size and macrophage infiltration in cross-sections of the aortic root (ORO and
MOMA-2 antibody staining, respectively), and reduced lesion size in the entire aorta (from root
to abdominal area) compared to control and independent of cholesterol levels (Nagashima et al.
34
2011). Furthermore, co-infusion of GLP-1(7-36)NH2 with exendin(9-39) antagonist (22
nmol/kg/day) did not reduce plaque size in the entire aorta (including the aortic root) nor
reduced macrophage infiltration beyond that of control (Nagashima et al. 2011).
1.5 Project rationale and hypothesis
While the effects of activating the GLP-1R on plaque formation have been examined in non-
diabetic mice, it is currently not known whether the effects of activating the GLP-1R on
reduction of plaque development and monocyte recruitment will remain in the presence of
diabetes. The focus of this thesis is to examine the effects of activation of the GLP-1R by two
agonists, exendin-4 and taspoglutide, on atherogenesis in glucose intolerant and diabetic Apoe-/-
mice.
The main reason for choosing the Apoe-/-
mouse model over the Ldlr-/-
mouse model (another
well-known mouse model for atherosclerosis) is to avoid the need to utilize a high-cholesterol
diet for inducing atherosclerosis. Ldlr-/-
mice have physiological levels of cholesterol unless
cholesterol is supplemented exogenously in the diet. As mentioned in section 1.3.5, mice are
highly resistant to developing atherosclerosis unless hypercholesterolemia is induced (Meir and
Leitersdorf 2004). Apoe-/-
mice, on the other hand, are hypercholesterolemic from birth and
develop atherosclerosis spontaneously even when fed a regular chow diet (Meir and Leitersdorf
2004). Because GLP-1R activation was shown to inhibit intestinal lipoprotein secretion (Qin et
al. 2005; Hsieh et al. 2010), we wanted to avoid the effect of cholesterol lowering on
atherosclerosis lesion size in order to be able to examine the more direct actions of GLP-1R
activation in atherosclerosis.
Hypothesis: We predict that activation of the GLP-1R in glucose intolerant and diabetic Apoe-/-
mice will slow the progression of atherosclerosis, similar to that shown in non-diabetic mice
(Arakawa et al. 2010; Nagashima et al. 2011).
35
Chapter 2 Materials and methods
All experimental procedures on animals conformed to the guidelines and policies approved by
the Mount Sinai Hospital Animal Care Committee. Mice were maintained on a 12-hour light
(7:00 A.M.)/dark (19:00) cycle in a pathogen-free environment and had ad libitum access to food
and water, except where noted. Two sets of experiments were carried out and will be
categorized as the “exendin-4 project” and the “taspoglutide project”, where exendin-4 and
taspoglutide are the GLP-1R agonists being tested in each project.
2.1 Exendin-4 project
2.1.1 Animals, diets and drug treatments
Six-week-old male Apoe-/-
mice were purchased from Jackson Laboratory (Stock Number
002052, Bar Harbor, ME) and housed at the animal facility of the Toronto Centre for
Phenogenomics at Mount Sinai Hospital. Upon arrival, mice were acclimatized for one week
and fed a standard chow diet with 18% of calories from fat (2018S, Harlan Teklad Laboratories,
Mississauga, Ontario, Canada) before taking basal measurements. At 8 weeks of age, all mice
were started on a 60 kcal% high-fat diet (HFD) with 0.003% by weight cholesterol (D12492,
Research Diets, New Brunswick, NJ). The reason for choosing this diet low in cholesterol and
not the Western diet with 0.15% cholesterol was because exendin-4 is known to inhibit
intestinal lipoprotein secretion (Hsieh et al. 2010); development of atherosclerosis in Apoe-/-
mice is dependent on hypercholesterolemia (Whitman 2004) and the possibility for a reduction
in excess dietary cholesterol by exendin-4 treatment would be the main factor driving lesion
size. We were more interested in the effects of exendin-4 treatment in atherosclerosis
independent of changes in lipid parameters. The other reason for choosing a high-fat diet was in
order to render the mice glucose intolerant and hyperinsulinemic, two important aspects that are
observed in insulin-resistant T2DM patients. It had been reported that male Apoe-/-
mice fed this
diet for 17 weeks exhibit obesity, glucose intolerance, inflammation and increased aortic sinus
plaque size with increased levels of serum amyloid A without inducing changes in lipoprotein
profile or adipokines (King et al. 2010).
36
Mice were divided into 3 groups and treatments started at 8 weeks of age (N = 8 per group, but
staggered in time into two groups with N = 4). Group 1 was administered PBS (i.p.) twice a day
(9:00 and 17:00); group 2 was treated with metformin supplied in food (sterilized and premixed
by Research Diets) (225 mg/kg/day; food intake was measured weekly per cage and the
metformin diet was appropriately diluted with the 60 kcal% control diet using a blender to
match the dose) and also injected with PBS twice daily; and, group 3 was administered 10
nmol/kg/day exendin-4 (i.p.) dissolved in PBS and injected twice daily (5 nmol/kg per
injection). The reason for having the metformin group in the study was to have a control for the
glucose-lowering effect of exendin-4. Mice were treated for 22 weeks and euthanized by carbon
dioxide asphyxiation.
2.1.2 Metabolic measurements
Body composition. Total body fat and lean mass were measured using a mouse whole-body
magnetic resonance analyzer (Echo Medical Systems, Houston, TX) after 5 and 10 weeks of
treatment.
Oral glucose tolerance tests and measurement of plasma insulin levels. Oral glucose tolerance
tests (OGTTs) were carried out 5, 9, and 22 weeks after the start of treatments following a 6-
hour fast (7:00-13:00). No drugs were administered in the morning of the day of the test.
Glucose (1.5 mg/g of body weight) was administered orally through a gavage tube. Blood was
drawn from the tail vein at 0, 10, 20, 30, 60, 90, and 120 min after glucose administration, and
blood glucose levels were measured by the glucose oxidase method using the Contour
glucometer (Bayer Healthcare, Toronto, Ontario, Canada). For plasma insulin determinations,
blood samples (50 μl) were drawn from the tail vein at the 0- and 10-minute time points
following glucose administration in a heparinized tube. Blood was mixed with 10% (v/v) TED
(500,000 IU/ml Trasylol, 1.2 mg/ml EDTA, and 0.1 mM Diprotin A) and plasma separated by
centrifugation at 4 °C and stored at –80°C until assayed. Plasma was assayed for insulin using a
mouse insulin ELISA kit (Alpco).
Systolic blood pressure. Systolic blood pressure was measured in awake mice using an
automated tail cuff system (BP-2000, Visitech Systems, Apex, NC) after 11 weeks of treatment.
Measurements were taken twice in order to train the mice on the apparatus the first day, and
37
measurements from the second day were recorded for analysis. During measurements, mice
were restrained in a dark chamber, in which the floor was heated to 38°C. Results from the first
10 inflation cycles were discarded, and the average obtained from the next 10 cycles was
recorded. Mice were in the chamber for about 15 min. On each study day, systolic blood
pressure and heart rate were measured between 10:30–15:30. While the apparatus measured
heart rate and diastolic blood pressure values, it failed to do so accurately and consistently (a
high variability in heart rate and erroneous values were observed; for example, diastolic blood
pressure was very frequently not measured and reported by the apparatus, and heart rates varied
from 70 to 900 beats per minute in repeated measurements).
2.1.3 Histological analysis of atherosclerotic lesions
The aortic root was isolated for quantification of atherosclerosis lesion size as described
(Daugherty and Whitman 2003) with the following modifications: the aorta was flushed with
cold PBS (about 8-10 ml) by perfusion with a needle syringe inserted into the left ventricle of
the heart. Prior to perfusion, the right atrium was nicked. The aorta was dissected while attached
to the heart and a scalpel was used to cut out two thirds of the bottom ventricles. The aortic arch
region was cleared of periadventitial adipose tissue and other surrounding vessels under a
dissecting microscope and the aorta was cut about 1 mm above the atria. This upper part of the
heart with the aortic root was embedded in Tissue-Tek OCT Compound (Sakura Finetek,
Torrence, CA), wrapped in clear plastic wrap and frozen in liquid nitrogen. Frozen samples
were processed by Lily Morikawa from the Centre for Modeling Human Disease (CMHD), a
division of the Toronto Centre for Phenogenomics (TCP). Frozen samples were sectioned with a
cryostat with 5-m thickness and mounted on slides. The first emergence of the three valve
leaflets was taken as reference point and the 1st, 14
th and 21
st sections were taken for analysis of
plaque size (each section being 5 m apart). Slides were stained with oil red O and
counterstained with hematoxylin and eosin for determination of plaque size and lipid within the
plaque. ImageScope software version 11.0.2.716 (Aperio) was used to manually draw a
perimeter around the plaques and quantify plaque area and lipids by thresholding function
available in Aperio on the three sections. The plaque areas from all three sections were then
added. Plaques that formed directly on the leaflets of the aortic sinus were not quantified; only
plaques that developed on the aortic wall were quantified.
38
2.2 Taspoglutide project
2.2.1 Animals, diets and drug treatments
Four-week-old male Apoe-/-
mice were purchased from Jackson Laboratory (Bar Harbor, ME)
and housed at the animal facility of the Toronto Centre for Phenogenomics at Mount Sinai
Hospital. Mice were acclimatized for one week before taking basal measurements. Upon arrival,
mice were immediately started on a 45 kcal% high-fat diet (HFD) (D12451, Research Diets,
New Brunswick, NJ). The reason for choosing this diet is the same as for the exendin-4 project;
this diet is low in cholesterol (unlike Western diets with 0.15% cholesterol) to avoid the
possibility for a reduction in ingested cholesterol due to taspoglutide treatment, as cholesterol is
a key mediator of atherogenesis (see section 1.5). In addition, a change from 60 to 45 kcal% was
made because the protocol to induce mild diabetes with streptozotocin (STZ, a beta-cell toxin)
(as described next) was optimized with the 45 kcal% diet by other members of our lab.
To render the mice diabetic (plasma glucose between 15-25 mM), we did not want to induce
total pancreatic beta-cell destruction, hence we employed a specific combination of diet and
STZ. After 4 weeks on the HFD, mice were given an intraperitoneal injection of 75 mg/kg STZ
in 0.05 M citrate buffer, pH 4.5, or citrate buffer alone as control. After 10 days, a second dose
with an additional 125 mg/kg STZ was administered. Plasma glucose was measured 10 days
after the second dose of STZ between 9-10 A.M. and mice in the desired range (15-25 mM) were
used in the study. This procedure rendered, as expected, 40-50% of mice in the desired glucose
range and only these mice were selected for the study. Insulin supplementation was not required
for these mice.
Mice were divided into 3 groups and treatments started at 13 weeks of age (n = 15 per group).
All mice were rendered diabetic with STZ. Group 1 mice were administered a placebo
microtablet subcutaneously (the test drug, taspoglutide, was formulated in the form of a
microtablet); group 2 was treated with metformin supplied in autoclaved water (water intake
was monitored weekly and concentration adjusted to correspond to a dose of 400 mg/kg/day;
this dose was chosen based on a previous study from our lab (Sauve et al. 2010)) and also given
the placebo microtablet; and, group 3 was made diabetic and administered with 0.4-mg dose of
taspoglutide microtablet subcutaneously (provided by Roche Pharmaceuticals). The dose of
39
metformin was changed in the taspoglutide study as the previous dose of 225 mg/kg/day in the
exendin-4 study did not improve oral glucose tolerance. The dose of taspoglutide was chosen
after results from a 4-week pilot study in 6-week-old C57BL6 wildtype mice where the 0.4-mg
and 1.0-mg doses were administered and assessed for the least change in body weight while still
lowering blood glucose compared to chow-fed controls during an oral glucose tolerance test.
The reason for the metformin group in this study was also to have a control for the glucose-
lowering effect of taspoglutide. Mice were treated for 12 weeks (30-week old) with placebo or
taspoglutide microtablets administered every 4 weeks and euthanized by carbon dioxide
asphyxiation.
Administration of microtablets: placebo or taspoglutide microtablets were administered
subcutaneously. Due to the thickness of needles loaded with the microtablets, mice were
anaesthetized with isofluorane and shaved for administration of the microtablet. Antibiotic
(Polysporin) was then applied in the area to avoid infection.
2.2.2 Metabolic measurements
Assessment of food intake and stool collection. Food intake was measured three days prior to
euthanasia, and stools were collected. Mice were placed in single cages without bedding and
pre-weighed food. On the last day, food and stool weights were recorded.
Body composition. Total body fat and lean mass were measured using a mouse whole-body
magnetic resonance analyzer (Echo Medical Systems, Houston, TX) after 5 and 10 weeks of
treatment.
Oral glucose tolerance test and measurement of plasma insulin levels. An oral glucose tolerance
test was carried out after 5 weeks from start of treatments as described in section 2.1.2 except
for the differences described here. A lower glucose concentration (0.75 mg/g of body weight)
was administered orally through a gavage tube. A lower glucose concentration than that
employed in the exendin-4 study was used to avoid inducing a glucose change above 33.3 mM,
which is the upper limit value measured by the glucometers.
Systolic blood pressure. The same procedure as described in section 2.1.2 was followed.
40
Lipid tolerance test. After 6-week treatment, mice were fasted overnight (19:00-9:00) before a
lipid tolerance test. Olive oil (5 l/g) was administered orally through a gavage tube and blood
(50 l) was drawn from the tail vein at 0, 60, 120 and 180 min after olive oil administration.
Triglycerides were measured using a enzymatic colorimetric assay kit (Roche Cat. No.
11877771216) based on the glycerol oxidase method.
Plasma cholesterol. Blood (50 l) was withdrawn from overnight-fasted mice (19:00-9:00).
Blood was mixed with 10% (v/v) TED, plasma separated by centrifugation at 4 °C and stored at
-80°C until assayed. Plasma was assayed for cholesterol using an enzymatic colorimetric assay
kit (Wako, Cat. No. 439-17501).
2.2.3 Histological analysis of atherosclerotic lesions
Lesion size in the aortic root was quantified as described above for the exendin-4 project. In
addition, percent lesion occupied in the descending and abdominal aorta (from the left
subclavian artery to the iliac branch) was quantified as described (Tangirala, Rubin, and Palinski
1995; Whitman 2004) with the following modifications: periadventitial adipose tissue was
excised using a dissecting microscope, stained with Nile red fluorescent stain for 1 hour and
destained in PBS for 5 min. The aorta was then cut open longitudinally to expose the intima,
pinned on waxed plates on a black background and imaged using a fluorescent microscope.
Percent lesion area analysis was conducted with ImageJ software by a thresholding method.
2.2.4 Immunohistochemical analysis of macrophage infiltration
In addition to determining lesion size by oil red O at the aortic sinus, the 15th
and 20th
aortic
sections (refer to atherosclerotic lesion size method above under exendin-4 project) were stained
with MAC-2 primary antibody and stained by diaminobenzidine (DAB). The sections were
quantified for percent occupancy by macrophages within lesions using ImageScope software
(Aperio).
The aortic arch cut from sinus to left subclavian artery, with about 1-mm sections of the three
branching arteries (the brachiocephalic trunk, left common carotid artery, and left subclavian
41
artery), was cleaned, fixed in 10% formaldehyde overnight and embedded in paraffin.
Longitudinal sections were cut and sections that contained all three arteries stained with MAC-
2/DAB. ImageScope (Aperio) software was used to quantify the stained area in the arch.
2.2.5 Plasma adipokine measurements
Blood (100 l) was withdrawn from 6-hour fasted mice (7:00-13:00). Blood was mixed with
10% (v/v) TED, plasma separated by centrifugation at 4 °C and stored at -80°C until assayed.
The MilliplexTM
MAP Mouse Serum Adipokine Kit (Millipore, MADPK-71K) was used to
measure plasma levels of leptin, interleukin-6, resistin, TNF- and MCP-1.
2.3 Statistical analyses
Results are presented as mean ± standard error. Statistical analyses were done using Repeated
Measures Analysis of Variance (ANOVA) followed by Bonferroni post hoc test. A P value of
<0.05 was considered to be statistically significant. To determine whether future experiments
were worth conducting to increase sample size and achieve statistical significance, a sample size
calculation was approximated using GraphPad StatMate 2.00 software. The largest standard
deviation and a power of 80% were chosen to estimate the required sample size, N, per
treatment group. The effect size was estimated from the mean difference observed between
untreated controls and either exendin-4 or taspoglutide-treated mice.
42
Chapter 3 Results
3.1 Exendin-4 project
Figure 1 shows a simplified timeline for the exendin-4 project and the specific time points at
which experiments were conducted. The actual exendin-4 project was split in two groups
staggered in time. Each staggered group consisted of all treatment groups with N of 4 mice per
treatment group such that after pooling the two staggered groups, each treatment group had a
total N of 8 mice.
3.1.1 Metabolic phenotype
Increased body weight has been shown to accelerate atherogenesis in Apoe-/-
mice fed a high-fat
diet with very low levels of cholesterol (0.003%) (King et al. 2010). In the current study, mice
were given the 60 kcal% high-fat diet with no cholesterol (0.003% by weight) for 22 weeks and
showed no significant change in a) body weight b) fat to body weight ratios as assessed by MRI
and c) epididymal white adipose tissue (WAT) weights normalized to body weight when treated
with either metformin (225 mg/kg/day supplied in food) or exendin-4 (10 nmol/kg/day, ip)
(Figure 2).
Apoe-/-
mice on the 60 kcal% diet exhibited glucose intolerance (Figure 3A). Surprisingly,
metformin treatment deteriorated oral glucose intolerance towards the end of the study, although
this difference was not statistically significant (P = 0.21). Insulin-to-glucose ratios were not
different among groups (Figure 3B). Hemoglobin A1c levels were not different among groups,
although the group treated with metformin showed lower levels (P = 0.16) (Figure 3C).
3.1.2 Atherosclerosis
No statistically significant differences in aortic sinus plaque areas were detected across the three
groups (Figure 4A and 4C). Interestingly, a non-significant (P = 0.15) increase in plaque lipid
content was observed in the aorta of mice treated with exendin-4 (Figure 4B and 4C).
43
Tim
elin
e f
or
the
Exe
nd
in-4
Pro
ject
Figu
re 1
. Sim
plif
ied
tim
elin
e f
or
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exe
nd
in-4
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. Mal
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ice
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Jac
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44
Figure 2. Body weight and body fat. Apoe-/- mice were fed a 60 kcal% high-fat diet (HFD) and treated with either 225 mg/kg/day metformin (Met) supplied in food or 10 nmol/kg/day exendin-4 (EX-4) (intraperitonealinjection given twice a day) for 22 weeks. (A) Body weights of mice measured at the end of the study. (B) Fat mass was assessed by whole-body magnetic resonance imaging (MRI) after 11 weeks of treatments and (C) by weight of epididymal white adipose tissue (WAT) normalized to body weight (BW) after conclusion of the study in male Apoe-/- mice on a 60 kcal% high-fat diet (HFD). Numbers in parentheses show sample size, N.HFD, high-fat diet; Met, metformin; EX-4, exendin-4.
HFD(8)
HFD+
Met(8)
HFD+
EX-4(8)
Body WeightsA
30
20
40
50
60
10
0
Bo
dy
We
igh
t (g
)
Epididymal WATWeight per BW
HFD(8)
HFD+
Met(8)
HFD+
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C0.07
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0.04
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Epid
. W
AT
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0
5
10
15
20
25
HFD(8)
HFD+
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HFD+
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Fat MassB
Fat
(%)
Figure 3. Glucose tolerance, insulin levels and HbA1c. Effect of treatment with metformin (225 mg/kg/day in food) or exendin-4 (10 nmol/kg/day, i.p.) on (A) oral glucose (1.5 mg/g glucose) tolerance test (OGTT), (B) insulin:glucose ratios at basal and 10 min after oral gavage of glucose, and (C) hemoglobin A1c levels compared to the HFD control group after 22 weeks. Statistical analyses were carried out by one-way ANOVA for each time point in the OGTT; AUC glucose (inset in A) and HbA1c were analyzed by one-way ANOVA; insulin:glucose ratios (B) were analyzed by two-way ANOVA. HFD, high-fat diet; Met, metformin; EX-4, exendin-4; OGTT, oral glucose tolerance test.
Oral Glucose Tolerance Test
0 20 40 60 80 100 120
HFD (8)
HFD + Met (8)
HFD + EX-4 (8)
A
AU
C (
mM
·min
)
0
5
10
15
20
25
30
35
Blo
od
Glu
cose
(m
M)
0 10 0 10 0 10
Time (min)
B
**
0
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Insu
lin:G
luco
seR
atio
(m
g/m
ol) HFD
HFD + Met
HFD + EX-4
Insulin:Glucose Ratios DuringOral Glucose Tolerance Test
Hemoglobin A1cC
HFD(8)
HFD+
Met(8)
HFD+
EX-4(7)
0
1
2
6
5
4
3
Hb
A1
c (%
)
45
Figure 4. Atherosclerotic plaque development after treatment with metformin or exendin-4 for 22 weeks on male Apoe-/-. (A) Plaque size was quantified in the aortic root and (B) intraplaque lipid was assessed by ORO staining for mice treated with either 225 mg/kg/day metformin or 10 nmol/kg/day exendin-4 for 22 weeks. (C) Representative cross-sections of aortic roots cut at the same level (reference point taken was the first sign of aortic valves protruding from the aortic wall while cutting from the top towards the aortic valves) and stained with ORO (magnification 5X). To achieve statistical significance for Figure 4A, an approximate sample size of 80 mice per group would be needed for a power of 80% as assessed from the effect size and variability of the data using GraphPad StatMate 2.00 software.
HFD Control HFD+EX-4HFD+Met
C
Aortic Root Plaque Size
HFD HFD+Met HFD+EX-4
A
0.00
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
Pla
qu
e S
ize
(m
m2 )
Aortic Root Lipid Content
HFD HFD+Met HFD+EX-4
B85
75
65
55
45
35
25
Intr
apla
qu
elip
id (
%)
46
3.2 Taspoglutide project
Figure 5 shows a simplified timeline for the taspoglutide project and the specific time points at
which experiments were conducted. The actual taspoglutide project was split in three groups
staggered in time. Each staggered group consisted of all treatment groups with N of 5 mice per
treatment group such that after pooling the three staggered groups, each treatment group had a
total N of 15 mice.
3.2.1 Metabolic phenotype
Treatment with taspoglutide significantly decreased body weight by 2.42 g or 8.44% when
compared to its diabetic control (P <0.05). The decrease in body weight for taspoglutide-treated
mice may be due to reduced food intake compared to diabetic control (Figure 6B) (P <0.05).
There were no significant differences in percent fat mass between groups as assessed by whole-
body MRI after 10 weeks of treatment (Figure 6C) or by gravimetric measurement of
epididymal WAT at the end of the study (Figure 6D).
Taspoglutide-treated mice had improved blood glucose control: treatment of diabetic Apoe-/-
mice with taspoglutide significantly improved oral glucose tolerance (P <0.05) (Figure 7A),
increased basal levels of plasma insulin (P <0.05) (Figure 7B) and showed reduced hemoglobin
A1c levels (P = 0.19) (Figure 7C).
3.2.2 Lipid metabolism
Lipid metabolism plays a major role in the development and progression of atherosclerosis
(Weber and Noels 2011). To assess changes in post-prandial triglyceride concentrations in
plasma after a meal, a lipid tolerance test was carried out in overnight-fasted mice administered
olive oil. After six weeks of treatment, no differences were seen among diabetic groups for
triglyceride levels in plasma during the lipid tolerance test (Figure 8). While this result may be
surprising at first it is important to note that, in contrast to other studies, no triton WR1339 was
administered to inhibit lipase activity; the decrease in plasma triglycerides observed by Hsieh et
47
al (Hsieh et al. 2010) after acute administration of exendin-4 in wildtype C57BL6 mice was
after triton administration to inhibit the action of lipase. In addition, Xiao et al (Xiao et al. 2012)
demonstrated that infusion of a liquid meal formula directly into the duodenum (through a
nasoduodenal tube) of human volunteers given exenatide intravenously did not change
triglyceride levels from those of control subjects during a pancreatic clamp. While not shown
here, there were no differences in gastric emptying among groups of mice in our study as
assessed by measuring blood acetaminophen levels administered during an oral glucose
tolerance test and measuring acetaminophen levels at 0 and 10 minutes after oral gavage.
Furthermore, no differences were observed after 11 weeks of treatment in plasma triglyceride
(Figure 9A) or cholesterol (Figure 9B) levels after a six-hour fast.
48
4 W
eeks
45
% H
FD3
Wee
ks f
or
STZ
PB
O, M
et o
r TA
SPO
STA
RTE
D @
13
-Wee
k-O
ld f
or
12
Wee
ks
5-W
ee
k Tr
eat
me
nt
OG
TT
10
-We
ek
Tre
atm
en
tP
lasm
a fo
r Li
pid
sM
RI
11
-We
ek
Tre
atm
en
tP
lasm
a fo
r A
dip
oki
ne
sB
P
6-W
ee
k Tr
eat
me
nt
Lip
id T
ole
ran
ce T
est
Mic
e f
rom
Jac
kso
nA
rriv
e @
4 W
ee
ks O
ld
Tim
elin
e f
or
the
Tas
po
glu
tid
eP
roje
ct
Figu
re 5
. Sim
plif
ied
tim
elin
e f
or
the
tas
po
glu
tid
est
ud
y p
roje
ct. M
ale
Ap
oe-/
-m
ice
we
re o
rder
ed f
rom
Jac
kso
n L
abo
rato
ries
an
d a
ll m
ice
we
re f
ed a
45
kc
al%
hig
h-f
at d
iet
(HFD
) im
med
iate
ly u
po
n a
rriv
al. T
her
e w
ere
th
ree
trea
tmen
t gr
ou
ps
wit
h N
= 1
5 p
er t
reat
men
t gr
ou
p:
1)
45
% h
igh
-fat
die
t (H
FD),
dia
bet
ic (
D),
inje
cte
d w
ith
th
e p
lace
bo
(P
BO
) m
icro
tab
let
ever
y 4
we
eks
2)
45
% h
igh
-fat
die
t (H
FD),
dia
bet
ic (
D),
tre
ated
wit
h 4
00
mg/
kg/d
ay m
etfo
rmin
an
d in
ject
ed
wit
h t
he
pla
ceb
o (
PB
O)
mic
rota
ble
tev
ery
4 w
eek
s (t
his
m
etfo
rmin
gro
up
is a
co
ntr
ol f
or
glu
cose
low
eri
ng)
3)
45
% h
igh
-fat
die
t (H
FD),
dia
bet
ic (
D),
inje
cted
wit
h t
he
0.4
-mg
tasp
ogl
uti
de
(TA
SPO
) m
icro
tab
let
ever
y 4
wee
ksO
GTT
, ora
l glu
cose
to
lera
nce
te
st; B
P, b
loo
d p
ress
ure
; PB
O, p
lace
bo
; Met
, met
form
in; T
ASP
O, t
asp
ogl
uti
de
.
8-W
ee
k O
ld1
3-W
ee
k O
ld
Bas
als
49
Figure 6. Body weight, food intake and fat mass in male diabetic Apoe-/- mice in the taspoglutide study project. Apoe-/- mice with streptozocin-induced diabetes were fed a 45 kcal% high-fat diet (HFD) and treated with either 400 mg/kg/day metformin (Met) supplied in water or taspoglutide microtablets(subcutaneous injection given every 4 weeks) for 12 weeks. (A) Body weights were measured at the end of the study. (B) Food intake was assessed after 12 weeks of treatment. (C) Body fat content was measured after 10 weeks of treatment by whole-body magnetic resonance imaging (MRI) and (D) epididymal white adipose tissue (WAT) weight normalized to body weight at the end of the study. *P<0.05, ***P<0.001. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide. Comparison of groups was made by one-way ANOVA using Graphpad Prism. Not all mice were used for food intake or fat mass assessment by MRI.
Body WeightsA
*
DHFDPBO(14)
DHFDMetPBO(15)
DHFD
TASPO(15)
35
15
10
5
0
30
25
20
Bo
dy
We
igh
t (g
)
B Food Intake
DHFDPBO(9)
DHFDMetPBO(10)
DHFD
TASPO(9)
*
0.25
0.20
0.15
0.10
0.05
0.00
Foo
d in
take
(g/
ho
ur)
D Epididymal WATWeight per BW
DHFDPBO(14)
DHFDMetPBO(15)
DHFD
TASPO(15)
0.020
0.015
0.010
0.005
0.000
Epid
. W
AT
Wt/
BW
% Fat Massper Body Weight
DHFDPBO(5)
DHFDMetPBO(5)
DHFD
TASPO(5)
C
15.0
12.5
10.0
7.5
5.0
2.5
0.0
% F
at M
ass
pe
r B
D
50
Figure 7. Oral glucose tolerance test, insulin levels and HbA1c levels in the taspoglutide study. (A) Oral glucose (0.75 mg/g glucose) tolerance test after 5 weeks of treatment. The concentration of glucose was halved compared to the exendin-4 study to avoid readings above 33.3 mM, the upper limit for the glucometers used. (B) Plasma insulin to glucose ratios at basal and 10 min after oral glucose gavage after 5 weeks of treatment (C) Hemoglobin A1c levels at the conclusion of the study, after 12-week treatment. *P<0.05, **P<0.01, ***P<0.001. Plasma insulin:glucose ratios were analyzed by two-way ANOVA. All other graphs were analyzed by one-way ANOVA. To detect a statistically significant difference in Figure 7C, we would need approximately 60 mice per group for 80% statistical power. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide.
C Hemoglobin A1c
DHFDPBO(14)
DHFDMetPBO(15)
DHFD
TASPO(15)
Hb
A1
c (%
)
0.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Plasma Insulin:Glucose RatiosDuring OGTT
B
*
D HFD PBO (14)
D HFD+Met PBO (15)
D HFD TASPO (15)
0.000
0.035
0.030
0.020
0.015
0.010
0.005
0.025
Insu
lin:
glu
cose
rat
io (
mg/
mo
l)
0 10 0 10 0 10Time (min)
Oral Glucose Tolerance TestA
D PBO (14)
D Met PBO (15)
D TASPO (15)
*
*** ***
0
30
25
20
15
10
5
*
0 20 40 60 80 100 120
Blo
od
Glu
cose
(m
M) AU
C
Time (min)
Figure 8. Plasma triglyceride levels during oral lipid tolerance test in diabetic male Apoe-/-
mice in the taspoglutide study. Oral olive oil was administered after overnight fasting (14-16 hours) in the absence of triton WR1339 (no lipase inhibition) and levels of triglycerides in plasma measured using colorimetric assay. Analysis of lipid tolerance graph was made by two-way ANOVA and AUC by one-way ANOVA. To detect a statistically significant difference, the approximate sample size per group would have to be 25 mice.
3
Oral Lipid Tolerance Test
D HFD PBO (5)
D HFD+Met PBO (5)
D HFD TASPO (5)
150
75
50
25
0
125
100
0 1 2
Time (hours)
Trig
lyce
rid
e (
mg/
dl)
AU
C
175
51
Livers from the diabetic control group (P = 0.20) and the group treated with metformin (P
<0.05) were heavier compared to mice treated with taspoglutide (Figure 10A). In addition, livers
from both control groups appeared nodular, yellow and unhealthy by simple visual inspection
(Figure 10D). Surprisingly, livers from mice treated with taspoglutide had a healthy and normal
appearance with no gross enlargement (Figure 10D). Triglycerides were decreased in the livers
of diabetic animals treated with taspoglutide (Figure 10B, P <0.05). In addition treatment with
either metformin or taspoglutide significantly reduced hepatic cholesterol levels (Figure 10C).
Triglyceride and cholesterol levels in stools were not significantly different among the three
diabetic groups (Figure 11A, B) but were slightly increased in mice treated with taspoglutide
(Figure 11A, B) (P = 0.20).
Figure 9. Plasma triglyceride and cholesterol levels in diabetic male Apoe-/- mice in the taspoglutidestudy. Plasma levels of (A) triglycerides and (B) cholesterol were measured by colorimetric assay after a 6-hour fast (7:00-13:00) after 11 weeks of treatment. *P<0.05, **P<0.01, ***P<0.001. Statistical comparisons made by one-way ANOVA. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide.
B Plasma Cholesterol
DHFDPBO(8)
DHFDMetPBO(8)
DHFD
TASPO(8)
500
400
300
200
0
100
Ch
ole
ste
rol (
mg/
dl)
Plasma TriglycerideA
DHFDPBO(8)
DHFDMetPBO(8)
DHFD
TASPO(8)
55
40
35
30
25
20
15
10
5
0
50
45
Trig
lyce
rid
e (
mg/
dl)
52
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
Liv
er
Tri
gly
ce
rid
e (
mg
/mg
)
DHFDPBO(5)
DHFDMetPBO(5)
DHFD
TASPO(5)
Intrahepatic TriglyceridesB
*
Figure 10. Liver weights and intrahepatic lipids in male diabetic Apoe-/- mice in the taspoglutidestudy. (A) Wet liver weights normalized to body weight after 12 weeks of treatment. (B) Lipids in liver tissue were extracted by methanol:chloroform (Bligh & Dyer method) and triglycerides and (C) cholesterol levels quantified using colorimetric assays. To detect a significant difference in Figure 10A between diabetic control and taspoglutide mice, 35 mice per treatment group would be needed. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide. Statistical comparisons were made by one-way ANOVA.
0.000
0.001
0.002
0.003
0.004
0.005
Liv
er
Ch
ole
ste
rol
(mg
/mg
)
DHFDPBO(5)
DHFDMetPBO(5)
DHFD
TASPO(5)
Intrahepatic CholesterolC
*** ***
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Liv
er
We
igh
t p
er
BW
Liver Weight per Body WeightA
DHFDPBO(14)
DHFDMetPBO(15)
DHFD
TASPO(15)
* **
D HFD D HFD + Met D HFD TASPO
D
53
3.2.3 Plasma adipokines
Plasma levels of IL-6 were significantly higher in the diabetic control group than the group
treated with taspoglutide (Figure 12A). Plasma leptin levels were not significantly different
among groups (Figure 12B). Plasma resistin levels were increased in taspoglutide-treated mice
(Figure 12C, P = 0.17) and were significantly higher than in the metformin-treated group (P
<0.05). Plasma levels of TNF- and MCP-1 were below the detectable levels by the assay
(sensitivity of TNF-, 4.39 pg/mL; MCP-1, 14.4 pg/mL).
Figure 11. Triglyceride and cholesterol concentrations in stools. (A) Triglyceride or (B) cholesterol excretion in stools were measured by colorimetric assays after methanol:chloroform extraction of lipids from stools after 12 weeks of treatment. To detect a statistically significant difference in Figure 11A and Figure 11B, approximately 18 and 20 mice per group, respectively, would be needed for 80% statistical power. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide. Statistical comparisons were made by one-way ANOVA
Triglyceride Levels in StoolsA
DHFDPBO(5)
DHFDMetPBO(4)
DHFD
TASPO(7)
P = 0.17
Sto
ol T
rigl
yce
rid
e (
mg/
mg)
10
7.5
5.0
2.5
0.0
Cholesterol Levels in StoolsB
DHFDPBO(5)
DHFDMetPBO(4)
DHFD
TASPO(6)St
oo
l Ch
ole
ste
rol (
mg/
mg)
1.31.21.11.00.90.80.70.60.50.40.30.20.10.0
54
Figure 12. Plasma adipokines of male Apoe-/- mice. (A) Plasma IL-6 (B) leptin and (C) resistin levels were measured after 11 weeks of treatment after a 6-hour fast (7:00-13:00) using the Millipore MilliplexTM MAP Mouse Serum Adipokine Kit. TNF- and MCP-1 levels were also measured, but levels in all mice were below the sensitivity of the assay (sensitivity TNF- 4.39 pg/mL, MCP-1 14.4 pg/mL). *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA. To detect a statistically significant difference in Figure 12C, 35 mice per group would be needed for 80% statistical power. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide. Statistical comparisons were made by one-way ANOVA.
***
Plasma IL-6A
DHFDPBO(5)
DHFDMetPBO(4)
DHFD
TASPO(4)
Pla
sma
IL-6
(p
g/m
l)
90
80
70
60
50
40
30
20
10
0
Plasma LeptinB
DHFDPBO(8)
DHFDMetPBO(8)
DHFD
TASPO(8)
Pla
sma
Lep
tin
(pg/
ml)
1300120011001000900800700600500400300200100
0
Plasma ResistinC
DHFDPBO(8)
DHFDMetPBO(8)
DHFD
TASPO(8)
*
Pla
sma
Re
sist
in(p
g/m
l)
4500
4000
3500
3000
2500
2000
1500
1000
500
0
3.2.4 Atherosclerosis
Atherosclerotic plaque area was measured in the aortic root using ORO staining. Interestingly,
increased plaque area was detected in mice treated with taspoglutide at the aortic root (P = 0.18)
and this difference was statistically significant compared to metformin-treated controls (P <0.05,
Figure 13A and 13B). There were no differences in lipid content within the plaques of
taspoglutide-treated mice compared to the diabetic control group; however, the metformin-
55
treated group exhibited significantly lower lipid levels within the plaques compared to diabetic
control mice (Figure 13C) (P <0.05).
Macrophage content of plaques at the aortic sinus was quantified using Mac-2 antibody. The
percent macrophage area within the plaques of the aortic sinus was not different among all
diabetic groups regardless of treatment (Figure 13D). Similarly, macrophage infiltration
assessed at the aortic arch was also not different among the study groups (Figure 14).
Atherosclerosis area in the descending aorta was quantified using Nile red staining. While
plaque area was slightly reduced in taspoglutide-treated mice, the decrease did not reach
statistical significance (P = 0.19) (Figure 15).
It was interesting to note that plaques in the aortic sinus were complex and in later stages
whereas plaques in the aortic arch and abdominal aorta were of very early stages, predominantly
fatty streaks. This may be because a diet very low in cholesterol was used and the aortic sinus is
the site that is most prone for lesion formation; other sites require even greater increases in
plasma cholesterol to accelerate lesion formation (Daugherty et al. 2009; Whitman 2004).
3.2.5 Blood Pressure
Systolic blood pressure was not different among groups when measured using a tail-cuff system
for the taspoglutide-treated groups or the exendin-4 treated groups (Figure 16).
56
Figure 13. Characterization of plaques in the aortic root in male diabetic Apoe-/-mice in the taspoglutidestudy. (A) Plaque size was quantified in the aortic root. (B) Representative oil-red O histology images of aortic sinus cross-sections cut at the same level (reference point taken was the first sign of aortic valves protruding from the aortic wall while cutting from the top towards the aortic valves). Magnification 5X. (C)Intraplaque lipid was assessed with oil-red O staining for lipids. (D) Mac-2 antibody was used for staining for macrophages in plaques at the aortic sinus. *P<0.05, **P<0.01. . To detect a statistically significant difference in Figure 13A, approximately 55 mice per treatment group would be needed for a statistical power of 80%. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide. Statistical comparisons were made by one-way ANOVA.
D HFD PBO D HFD+Met PBO D HFD TASPO
B
Aortic Sinus Plaque Area
DHFDPBO(13)
DHFDMetPBO(14)
DHFD
TASPO(14)
A
*
Pla
qu
e A
rea
(mm
2 )
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
DHFDPBO(13)
DHFDMetPBO(15)
DHFD
TASPO(14)
Intraplaque LipidAt the Aortic Sinus
C
*
Intr
a-p
laq
ue
lip
id (
%)
60
50
40
30
20
10
0
D Mac-2 Staining in PlaquesAt the Aortic Sinus
DHFDPBO(5)
DHFDMetPBO(5)
DHFD
TASPO(4)
Mac
-2-s
tain
ed
are
a (%
)
35
30
25
20
15
10
5
0
57
B
D HFD PBO D HFD+Met PBO D HFD TASPO
Figure 14. Macrophage area stained at the aortic arch. (A) Macrophages were quantified in the aortic arch using mac-2 antibody and positive pixel analysis to determine stained area. (B) Representative longitudinal sections of the aortic arch stained with mac-2 antibody. To detect a statistically significant difference, an approximate sample size of 100 mice per group would be needed for a power of 80%. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide. Statistical comparisons were made by one-way ANOVA.
DHFDPBO(13)
DHFDMetPBO(15)
DHFD
TASPO(13)
Aortic Arch Macrophage AreaA
Mac
-2 a
rea
(mm
2 )
0.130.120.110.100.090.080.070.060.050.040.030.020.010.00
58
D HFD PBO D HFD+Met PBO D HFD TASPO
B
Figure 15. Plaque area occupied in the descending aorta of diabetic male Apoe-/- mice in the taspoglutidestudy. (A) The area of early atherosclerotic plaques in the descending aorta was determined by the en facemethod of aortas stained with Nile red. The area was quantified by thresholding and expressed as the percent total area occupied within total intimal area. To detect a statistically significant difference, 60 mice per treatment group would be needed for a power of 80%. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide. Statistical comparisons were made by one-way ANOVA.
% Atherosclerotic Surface AreaDescending Aorta
A
DHFDPBO(13)
DHFDMetPBO(12)
DHFD
TASPO(13)
Pla
qu
e a
rea
(%)
5
4
3
1
0
2
59
0
25
50
75
100
125
Syst
olic
BP
(m
mH
g)
Systolic Blood PressureTaspoglutide Study
DHFDPBO(14)
DHFDMetPBO(15)
DHFD
TASPO(15)
Figure 16. Systolic blood pressure of male Apoe-/- mice in both studies. Systolic blood pressure was measured by tail-cuff measurements after mice were trained. (A) Systolic blood pressure of glucose-intolerant male Apoe-/- mice after 11 weeks of treatment in the exendin-4 study. (B) Systolic blood pressure of male diabetic Apoe-/- mice after 10 weeks of treatment in the taspoglutide study. D, diabetic; HFD, high-fat diet; PBO, placebo; Met, metformin; TASPO, taspoglutide. Statistical comparisons were made by one-way ANOVA.
0
25
50
75
100
150
Syst
olic
BP
(m
mH
g)
125
Systolic Blood PressureExendin-4 Study
HFD(8)
HFD+
Met(8)
HFD+
EX-4(8)
60
Chapter 4 Discussion
In order to identify potential roles of GLP-1R activation on atherogenesis, it was important to
elucidate the effect of GLP-1R activation on adipocyte mass, insulin resistance,
hyperinsulinemia, plasma glucose levels, triglyceride and cholesterol levels in the Apoe
knockout mouse model as these impact the development of atherosclerosis (Weber and Noels
2011; Molecular Mechanisms of Atherosclerosis 2005; Rask-Madsen et al. 2012; Rask-Madsen
et al. 2010). Because previous studies implicate the GLP-1R in having a role in the reduction of
body weight, increase in insulin secretion, regulation of blood glucose homeostasis, and
reduction of triglyceride and cholesterol levels (section 1.4), it was hypothesized that GLP-1R
activation would lead to decreased atherosclerotic lesion area. Our results, however, revealed an
overall neutral effect of either exendin-4 or taspoglutide treatment on atherogenesis when lesion
size and macrophage infiltration was assessed at the aortic sinus, aortic arch and the abdominal
aorta.
4.1 Effect of GLP-1R activation on body weight, adipocyte mass, adipokines and atherogenesis
Reductions in body weight in T2DM patients treated with exenatide or liraglutide is well-
known; a recent analysis of 39 randomized controlled trials that assessed different treatments for
T2DM found GLP-1 analogues significantly decreased body weight with a mean change of -
1.66 kg (Liu et al. 2012). GLP-1 agonists are known to induce satiety through their actions in
the brain; intracerebroventricular administration of GLP-1 dose-dependently inhibits food intake
in fasted rats (Alhadeff, Rupprecht, and Hayes 2012; Gutzwiller et al. 1999; Turton et al. 1996).
We showed that long-term taspoglutide treatment for 12 weeks in Apoe-/-
mice fed a high-fat
diet significantly reduced body weight. However, long-term exendin-4 treatment for 22 weeks
did not significantly alter body weight; a dose of exendin-4 was chosen (10 nmol/kg/day) that
would not induce significant changes in body weight and this dose was assessed in a separate
pilot study of 6-week-old C57BL6 wildtype mice given exendin-4 for 4 weeks at various doses
to optimize the effect of blood glucose lowering while maintaining a neutral effect on body
weight. The reduction in body weight in taspoglutide-treated mice did not correlate with a
61
reduction in fat mass normalized to body weight as assessed by either whole-body magnetic
resonance imaging (MRI) or epididymal white adipose tissue weights normalized to body
weight. Taspoglutide-treated mice’s caloric intake was significantly reduced compared to
control and may explain the reduction in body weight observed. Thus, while caloric intake was
reduced with GLP-1R activation, body fat ratio did not change. This result was probably due to
a balancing combination of increased insulin levels in taspoglutide-treated mice compared to
control (which increased the rate of adipogenesis (Guller et al. 1988)), but decreased food intake
in taspoglutide-treated mice, which limited the extent of overall body weight gain and
adipogenesis.
As introduced in section 1.3.4, obesity is associated with a predisposition to developing
atherosclerosis and this is thought to be due to an establishment of a pro-inflammatory milieu
involving macrophage infiltration into adipocytes and the secretion of adipocytokines and
cytokines (Tilg and Moschen 2006; Wellen and Hotamisligil 2005).
In the taspoglutide study, diabetic control animals had significantly elevated plasma IL-6 levels
compared to animals treated with either metformin or taspoglutide. Chronic elevations of
plasma IL-6 are thought to be pro-inflammatory due to induction of hepatic insulin resistance
and its association with systemic CRP (Maury and Brichard 2010), which has been shown to
enhance atherosclerosis in Apoe-/-
mice overexpressing human CRP (Paffen and DeMaat 2006).
Thus, the elevations in IL-6 seen in diabetic control animals may induce increased
atherogenesis. On the other hand, IL-6 has also been shown to have anti-inflammatory effects
(see section 1.3.4.3), and thus, the exact significance of elevated IL-6 levels and its net effect on
atherogenesis is unclear.
The precise role of leptin in atherosclerosis is not known. However, it is postulated that
hyperleptinemia is atherogenic (see section 1.3.4.1) (Ritchie et al. 2004). Plasma leptin levels
were unchanged by either metformin or taspoglutide treatments. Given that leptin is secreted by
adipocytes in proportion to their mass (Maury and Brichard 2010), this result is not surprising.
Interestingly, resistin levels in taspoglutide-treated mice were slightly elevated compared to
control, but this difference was not statistically significant (P = 0.17). Resistin is thought to be
pro-atherogenic (see section 1.3.4.2), but the small increase seen in taspoglutide-treated mice is
unlikely to have a significant role (Tedgui and Mallat 2006; Tilg and Moschen 2006).
62
Thus, the net effect of taspoglutide on the reduction of body weight and IL-6 levels compared to
diabetic controls predicts a reduction in atherogenesis. However, the reduction in body weight
was only an 8.44% change from controls and did not result in a significant change in fat mass to
body weight ratio. Furthermore, the pro-inflammatory effects of chronically elevated IL-6 is a
matter of controversy (see section 1.3.4.3). Therefore, the reduction in body weight and IL-6
levels may have contributed to the neutral effect we observe on atherosclerosis development
compared to control mice.
4.2 Effect of GLP-1R activation on glucose homeostasis and atherogenesis
Activating the GLP-1R aids in the maintenance of glucose homeostasis, which is mediated not
only by stimulation of increased insulin release and inhibition of glucagon release, but also
through indirect mechanisms that stimulate glucose uptake by peripheral tissues (reviewed in
(Baggio and Drucker 2007)). Insulin resistance is often accompanied by hyperinsulinemia and
T2DM is characterized by hyperglycemia (Molecular Mechanisms of Atherosclerosis 2005).
The relative contributions of insulin resistance, hyperinsulinemia and hyperglycemia to
atherosclerosis development were discussed in section 1.4.1.4.
In the exendin-4 study, 10 nmol/kg/day of extendin-4 administered as two intraperitoneal
injections per day did not improve oral glucose tolerance beyond that of control. Interestingly,
metformin showed a trend (P = 0.21) for worsening glucose tolerance. Insulin-to-glucose ratios
and HbA1c were similarly unaffected in the exendin-4 study. A likely reason for this is that the
dose of exendin-4 administered was not sufficient to improve glucose homeostasis. The chosen
dose of exendin-4 was arrived at with 6-week-old mice in the absence of glucose intolerance
induced by a high-fat diet as in the exendin-4 study; it is likely that in the setting of diet-induced
glucose intolerance, the dose needed had to be higher. However, we opted to keep the dose
constant in order to avoid the confounding effects of weight loss in atherosclerosis. Mice in the
exendin-4 study were glucose intolerant and hyperinsulinemic, which are two important
characteristics of insulin resistance. Because the exendin-4 dose used did not improve glucose
homeostasis, this allowed us to eliminate the contribution of reduction in hyperglycemia that
would impact atherogenesis. However, independent of changes in glucose, the dose of exendin-
63
4 used (10 nmol/kg/day) did not change atherosclerosis lesion size or intraplaque lipid content in
the aortic sinus. The reason for this observation may also have been due to higher dose
requirements as these mice age; Arakawa et al. (Arakawa et al. 2010) used an infusion rate of 24
nmol/kg/day of exendin-4 for their high-dose group in order to observe a significant difference
in plaque size at the aortic sinus of relatively younger (12-week-old) Apoe-/-
mice. Another
difference in our experimental setup from that of Arakawa et al. is that we intermittently
injected the mice with two i.p. injections per day, whereas Arakawa et al. continuously infused
exendin-4 through an osmotic pump, thus activating the GLP-1R continuously rather than
intermittently. Assuming no desensitization of the GLP-1R, it is possible that continuously
activating the receptor may be more effective in attenuating plaque development. For this
reason, we opted to use taspoglutide, a GLP-1R agonist formulated as microtablets with a much
longer half-life that is administered every 4 weeks in mice.
Taspoglutide improved oral glucose tolerance, increased insulin secretion and improved HbA1c
(P = 0.19). Lower insulin levels in control diabetic mice were associated with more
hyperglycemia. Despite improved glucose homeostasis, taspoglutide did not change
atherosclerosis lesion size in the aortic sinus or the descending aorta, or macrophage infiltration
in plaques at the aortic sinus or in the aortic arch. Improvements in glucose levels in
taspoglutide-treated mice was expected to decrease macrophage infiltration in early lesions, as
shown in the study by Renard et al, where they employed Ldlr-/-
mice expressing a viral
glycoprotein (GP) under the control of the rat insulin promoter such that, when infected with the
lymphocytic choriomeningitis virus, they develop type 1 diabetes due to selective destruction of
cells by T cells. These Ldlr-/-
:GP mice had greater macrophage infiltration in early lesions of
the brachiocephalic artery (BCA) compared to non-diabetic controls independent of changes in
the lipid profile (mice were induced with diabetes at 7-10 weeks of age and given a cholesterol-
free, normal chow diet for 12 weeks) (Renard et al. 2004). While there was a slight decrease in
infiltration of macrophages in the early lesions of the aortic arch of metformin and taspoglutide-
treated mice compared to control mice, the effect was not statistically significant. It is possible
that the lack of effect in macrophage infiltration may be due to changes in the dynamic of
infiltrating cells as the atherosclerotic lesions examined in the aortic arch may have progressed
past the initiating stages of atherogenesis, and it is possible that activation of the GLP-1R past
the early fatty streaks may have a different role as more advanced lesions of the aortic sinus
reveal a relative increase in lesion size compared to diabetic and metformin-treated controls.
64
Furthermore, while insulin sensitivity was not assessed, the decrease in glycemia from
taspoglutide treatment would be expected to improve insulin sensitivity according to the glucose
toxicity theory, where peripheral cells adapt to chronic increases in glucose by increasing insulin
resistance to decrease the rate of glucose entry (Yki-Jarvinen 1990, 1997). The improvement in
insulin sensitivity in taspoglutide-treated mice, in turn, was expected to halt the accelerated
progression of atherosclerosis due to diabetes, as studies in EIRAKO mice showed increased
lesion area with decreased insulin signaling (discussed in section 1.3.2.2) (Rask-Madsen et al.
2010). Rather, only a small (not statistically significant) decrease was recorded in the early
lesions of the descending aorta of taspoglutide-treated mice. This result may be due to a
relatively small effect in the increase in insulin secretion in the taspoglutide group compared to
controls (while basal insulin:glucose ratios were statistically significant, post-prandial levels did
not reach statistical significance).
4.3 Effect of GLP-1R activation on lipoprotein metabolism
One major characteristic of diabetes (type 2) is increased hepatic VLDL secretion that results in
increased plasma VLDL triglyceride and APOB secretion (Adiels et al. 2005). Mechanisms
proposed include increased free fatty acid flux into the liver due to peripheral insulin resistance
that increase availability of lipid for VLDL synthesis and secretion (reviewed in (Lewis 1997)).
Furthermore, insulin has been shown to suppress VLDL APOB production in healthy human
volunteers while insulin-resistant subjects are resistant to the suppressive effect of insulin on
VLDL APOB production (Lewis and Steiner 1996). Thus, it may seem that decreased insulin
signaling in the periphery and liver could both act to increase VLDL secretion.
Activation of the GLP-1R has been shown to decrease postprandial lipid levels (section 1.4.1.1).
In the taspoglutide study, however, treatment with taspoglutide for six weeks did not change
postprandial lipid levels during an oral olive oil gavage on overnight-fasted mice. Experiments
to date have shown an inhibitory effect of GLP-1R activation on intestinal APOB lipoprotein
production (Hsieh et al. 2010; Qin et al. 2005). Thus, while taspoglutide treatment might have
decreased the rate of intestinal lipoprotein production, it is not known whether hepatic and/or
peripheral tissues had a decreased rate of uptake of lipoproteins. The decreased levels of
65
triglycerides and cholesterol in the liver suggest this may in fact be the case. Alternatively,
taspoglutide may have reduced hepatic lipid synthesis or increased lipid oxidation.
Taspoglutide treatment also did not change triglyceride or total cholesterol levels after 11 weeks
of treatment after a six-hour fast. This result is in agreement with similar reports where Apoe-/-
mice were treated with exendin-4 (Arakawa et al. 2010), GLP-1(7-36)NH2 (Nagashima et al.
2011), or sitagliptin (Vittone et al. 2012).
Interestingly, while plasma triglyceride and cholesterol levels remained unchanged, intrahepatic
levels differed greatly in the taspoglutide group compared to diabetic controls; intrahepatic
triglycerides showed a mean decrease of about 40%, and intrahepatic cholesterol decreased
about 39%. In addition, liver weight normalized to body weight tended to be lower in
taspoglutide-treated mice (about 12% smaller). Measurement of triglycerides and cholesterol in
stools revealed that some of the lipids were being excreted; stools of taspoglutide-treated mice
had a mean triglyceride concentration about 67% higher, and a mean cholesterol concentration
about 45% higher, although these differences did not reach statistical significance (P = 0.17 for
triglyceride and P = 0.20 for cholesterol). Thus, intrahepatic lipid concentrations may be partly
explained by enhanced excretion in the stools.
To compare our current observations with those in the literature, one report (Burmeister et al.
2012) showed that in 18-week-old high-fat-fed wildtype mice (60 kcal/g for 12 weeks), an acute
intracerebroventricular infusion of GLP-1(7-36)NH2 (0.01 g/min) during a hyperinsulinemic
euglycemic clamp led to a marked decrease in intrahepatic triglycerides. In addition, chronic
activation of the GLP-1R also reduces hepatic fat. One study showed 4-5-week old wildtype
C57BL6 mice fed a 45 kcal% high-fat diet rich in hydrogenated vegetable oil for 8 weeks then
treated with liraglutide (200 g/kg/day, i.p.) for 4 weeks exhibited reduced liver weight, serum
triglycerides and cholesterol, and decreased hepatic steatosis (as examined by ORO staining).
These mice showed reduced levels of mRNA transcripts for genes associated with lipid
synthesis (fatty acid synthase, acetyl CoA carboxylase alpha and PPAR-) and increased levels
of mRNA transcripts of peroxisomal fatty acid -oxidation (acyl-CoA oxidase-2 (Acox2))
(Mells et al. 2012).
The increase in triglyceride concentration in stools in taspoglutide-treated mice could be due to
decreased absorption of triglycerides; infusion of GLP-1(7-36)NH2 (1 pmol/kg/min
66
intravenously) inhibited human gastric lipase secretion in healthy human volunteers, which may
prevent the hydrolysis of triglycerides for absorption by enterocytes (Wojdemann et al. 1998).
Intrahepatic cholesterol may also have been reduced from higher excretion rates in bile and/or
decreased reabsorption from the gut as concentrations observed in stool tended to be higher than
in diabetic control animals.
The increase in liver weights and unhealthy appearance of livers in diabetic control mice was a
notable finding. It is important to note that compared to wildtype C57BL6 mice, Apoe-/-
mice
accumulate triglycerides in liver to a greater extent; one report showed male C57BL6 wildtype
mice fed a regular chow diet (6.2% fat and 0.01% cholesterol) until 3-4 months of age
accumulated 20.6 nmol/mg of triglycerides in liver, while Apoe-/-
mice accumulated 64.1
nmol/mg (P < 0.05) (Mensenkamp et al. 1999). Thus, the combination of increased intrahepatic
triglycerides and increased glucose flux due to hyperglycemia in diabetic mice in the
taspoglutide study may have led to the increase in liver weight, triglycerides and cholesterol,
which are important features for the development of NAFLD. Since taspoglutide-treated mice
had decreased intrahepatic triglycerides and cholesterol, there may have been a smaller chance
for development of NAFLD. Thus, the decrease in intrahepatic lipids and improvement in
glucose homeostasis from taspoglutide treatment may be the reason for the observed differences
in the livers’ physical texture, colour appearance and size between groups.
4.4 Effect of GLP-1R activation on atheroma development
As discussed in section 1.4.2.4, previous studies (Arakawa et al. 2010; Nagashima et al. 2011)
demonstrated that activation of the GLP-1R resulted in decreased lesion size in non-diabetic
mice. The potential mechanisms leading to the smaller lesions in these reports was a decrease in
the mRNA expression of adhesion molecules expressed on the endothelium such as ICAM-1
and VCAM-1; reduced CD11b receptor on monocytes for binding to ICAM-1; reduction in
LPS-induced MCP-1 and TNF-; and reduced cholesteryl ester accumulation due to reduced
CD36 and ACAT-1 in macrophages (Arakawa et al. 2010; Nagashima et al. 2011).
However, in the presence of diet-induced glucose intolerance as in our exendin-4 study,
exendin-4 treatment did not influence the size of lesions in the aortic root compared to control.
In addition, in the presence of streptozotocin-induced diabetes as in our taspoglutide study, there
67
was no significant change in the size of the lesions in the aortic root or the percent area occupied
by lesions in the abdominal aorta. Plaques in the aortic sinus were complex and in later stages
whereas plaques in the aortic arch and abdominal aorta were of very early stages, predominantly
fatty streaks.
Examination of macrophage content within advanced plaques of the aortic sinus or infiltrated
macrophage area within the intima and/or fatty streaks of the aortic arch also did not reveal
significant differences among groups.
Our studies thus reveal that in the setting of diet-induced glucose intolerance or streptozotocin-
induced diabetes, chronic activation of the GLP-1R does not have an effect on atherogenesis. In
fact, there was an increase in plaque area in the aortic sinus and a slight decrease in the
abdominal aorta in taspoglutide-treated mice. Normally, an increase in plaque size in the aortic
sinus corresponds to an increase in plaque size in the abdominal aorta (Tangirala, Rubin, and
Palinski 1995). However, in our taspoglutide study, there is a slight reduction in lesion size
observed for the early stages of atherosclerosis in the abdominal aorta (P = 0.19) and a slight
enlargement in size in more advanced lesions in the aortic sinus (P = 0.18). Furthermore, a slight
trend for a decrease in macrophage area in early lesions of the aortic arch was observed while no
change in the percentage occupied by macrophages is observed at the aortic sinus. The small
decrease in macrophage area in early lesions of the aortic arch in mice treated with either
metformin or taspoglutide may be due to improved glycemia, as discussed above in section 4.2.
Previous studies (Arakawa et al. 2010; Nagashima et al. 2011) demonstrated that activation of
the GLP-1R resulted in decreased lesion size in the aortic root and abdominal aorta in non-
diabetic mice. These studies activated the GLP-1R with either exendin-4 for 4 weeks in young
(8-week-old) mice (Arakawa et al. 2010) or with GLP-1(7-36)NH2 in slightly older (17-week-
old) mice (Nagashima et al. 2011). In the report by Nagashima et al. (Nagashima et al. 2011),
mice were given a high-fat, high-cholesterol diet for 4 weeks starting at 17 weeks of age. Mice
infused with GLP-1(7-36)NH2 for 4 weeks concurrently with the high-fat, high-cholesterol diet
(until 21 weeks old) had no increase in plaque area compared to mice 17 weeks of age (before
the introduction of the high-fat, high-cholesterol diet) when quantified en face from the aortic
arch to the iliac bifurcation; in fact, lesion area was slightly lower in GLP-1-treated mice
compared to 17-week-old controls. In contrast, lesions at the aortic root still increased with 4-
68
week GLP-1 infusion compared to 17-week-old controls, although not as much as untreated 21-
week-old controls given the high-fat, high-cholesterol diet. They also saw only small (but
statistically significant) reductions in macrophage infiltration after GLP-1 treatment in the aortic
root. Similarly, Arakawa et al. (Arakawa et al. 2010) observed marked reductions in monocyte
adhesion at branching points of the thoracic aorta at the endothelium surface in the very early
stages of atherosclerosis after 4 weeks of exendin-4 treatment, while only small reductions in
plaque area could be observed in the aortic sinus after 4 weeks of exendin-4 treatment. The
decreasing trends in macrophage infiltration in the aortic arch, as well as early stage lesion area
in the abdominal aorta in our studies agree with the reports by Arakawa et al. and Nagashima et
al. However, in our studies, activating the GLP-1R for 12 weeks, rather than just 4 weeks,
resulted in increased lesion size in the aortic sinus, while the other two papers (Arakawa et al.
2010; Nagashima et al. 2011) reported a statistically significant reduction in lesion size after the
shorter 4-week treatment. Thus, it is likely that activating the GLP-1R in diabetic mice, rather
than non-diabetic mice, has different consequences for early lesions, such as in the abdominal
aorta, and different consequences in more advanced lesions, such as in the aortic sinus. Indeed,
as discussed above, the study by Renard et al found diabetic mice had accelerated formation of
early lesions by inducing greater macrophage infiltration in the BCA independent of changes in
lipoprotein profile (Renard et al. 2004).
The development of atherosclerosis involves many cell types and their involvement is very
specific according to the stage and complexity of the lesion (reviewed in (Hansson and
Hermansson 2011; Weber and Noels 2011)). One possible explanation for the reduction of
atherosclerosis development in the early stages would be the activation of the GLP-1R in
regulatory T cells (Tregs). Our lab has shown exendin-4 treatment (2 g twice daily for 26
weeks) in nonobese diabetic (NOD) mice, a model of type 1 diabetes that spontaneously
develop diabetes by autoimmune destruction of pancreatic beta cells, delays the onset of type 1
diabetes (Hadjiyanni et al. 2008). Furthermore, purified CD4+CD25+ ex vivo lymphocytes (at
least 75% of which are Tregs) from male C57BL6 mice express the Glp1r receptor mRNA
(Hadjiyanni et al. 2008; Hadjiyanni et al. 2010). It has also been shown that Glp1r-/-
lymph-
node-derived lymphocytes are hyperproliferative in response to mitogenic stimulation such as
concanavalin A or phorbol myristate acetate plus ionomycin. And, a significant reduction of
CD4+CD25+FoxP3+ Treg cells is observed in lymph nodes from Glp1r-/-
mice (Hadjiyanni et
al. 2010). Thus, GLP-1R signaling may regulate the proliferation and maintenance of regulatory
69
T cells. Experiments in Apoe-/-
mice have also revealed an atheroprotective role of Tregs. The
first demonstration consisted of transfer experiments where in vitro-expanded Tregs from
BALB/c mice were injected intraperitoneally (106 cells per mouse) to female Apoe
-/- mice; mice
that received the Treg clones had markedly reduced atherosclerosis and macrophage infiltration
independent of plasma cholesterol (Mallat et al. 2003). Other experiments have also shown an
atheroprotective role for Tregs. For example, an experiment that disrupted the CXCR3
chemokine receptor in Apoe-/-
mice (CXCR3-/-
Apoe-/-
) (CXCR3 is a chemokine receptor of the
CXC family expressed in T lymphocytes) increased the number of Tregs and delayed early
atherosclerosis (lesion size at the aortic sinus was unaffected) by attenuating recruitment of T
helper 1 (TH1) cells (Veillard et al. 2005). One limitation of our studies is that we did not
examine T-cell populations within plaques, however it is possible that treatment with exendin-4
and/or taspoglutide may have attenuated early atherosclerosis lesion formation by activation of
the GLP-1R on Tregs. This may explain our trend for decreased atherosclerosis in the
abdominal aorta of taspoglutide-treated mice, but as the lesion progressed into later stages in the
aortic sinus, the atheroprotective role of Tregs was no longer apparent (their role in later stages
is currently not clear).
For later stages of atherosclerosis, such as in the aortic sinus, it is possible that GLP-1R
activation by exendin-4 and/or taspoglutide may have led, directly and/or indirectly, to the
secretion of extracellular matrix material by macrophages and/or smooth muscle cells that led to
the slight increase in size. Activation of the GLP-1R may have also stimulated the proliferation
and/or migration of smooth muscle cells to stabilize the plaque, which may be an important
finding despite the observed trend for an increase in lesion size because stabilization of plaques
by smooth muscle cells may prevent thrombosis and/or plaque rupture, which is clinically
important to reduce the incidence of cardiovascular events. One report clearly illustrates this
point: Apoe-/-
mice fed a high-fat (40 kcal%), high-cholesterol (1.25%) diet and treated with
sitagliptin (576 mg/kg) for 12 weeks did not change lesion size in the aortic root or the rest of
the aorta, but significant reductions in macrophage infiltration, metalloproteinase-9 and a two-
fold increase in collagen were observed, which indicate that, while plaque size was unaffected,
the plaques themselves have a more stable composition (Vittone et al. 2012). While we did not
observe a reduction in macrophage infiltration, it is possible that the increase in plaque size in
the aortic sinus may be from an increase in secreted collagen to stabilize the lesions.
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4.5 Expression of the GLP-1R in liver and atheroma
A possible explanation to the conflicting findings in atherogenesis and macrophage area from
previous reports (Arakawa et al. 2010; Nagashima et al. 2011) is that the GLP-1R may not be
expressed in monocytes and/or macrophages, as reported in non-diabetic Apoe-/-
mice.
The expression of the GLP-1R in different tissues has been difficult to reconcile due to
difficulties in generating antibodies specific for the receptor. Antibodies for GPCRs are usually
raised against a fragment(s) of synthetic peptide antigen that corresponds to the membrane
protein. The epitope recognized by the antibody may not be specific and may in fact, yield
several bands during Western blot analysis. The concern of antibody specificity for GPCRs is
not new, and concerns for faulty specificity for membrane receptors including adrenergic,
muscarinic and dopaminergic receptors, have been expressed before (Kirkpatrick 2009). One
method to identify and/or verify expression of the receptor is by examining mRNA expression
using reverse-transcribed cDNA followed by PCR using primers for the full length Glp1r.
Identification of the full length expression is important as primers directed at amplifying partial
fragments may yield faulty results (there may be leaky transcription in some tissues, but the
partial mRNA may never be translated).
Thus, it is possible that the anti-GLP-1R antibody from Abcam used by both studies (Arakawa
et al. 2010; Nagashima et al. 2011) may not be specific for the receptor and the observed
decrease in monocyte adhesion and/or plaque may be rather an indirect result of GLP-1R
activation in tissues other than monocytes and/or macrophages. Furthermore, the reduction in
cholesteryl esters recorded by Nagashima et al. (Nagashima et al. 2011) in peritoneal
macrophages from non-treated Apoe-/-
mice may have been an effect of the truncated peptide,
GLP-1(9-36)NH2 through a GLP-1R-independent mechanism possibly by acting through
another receptor that has yet to be identified (Ban et al. 2008).
71
Chapter 5 Conclusions
The present study demonstrated that exendin-4 treatment twice daily (10 nmol/kg/day, i.p.) in
Apoe-/-
mice fed a 60 kcal% high-fat diet for 22 weeks did not reduce body weight, adiposity,
glucose tolerance nor insulin to glucose ratios, and did not reduce plaque size in the aortic sinus.
In the taspoglutide study, taspoglutide:
- Significantly reduced body weight (P <0.05)
- Significantly reduced food intake (P <0.05)
- Had no effect on adiposity
- Improved glucose tolerance beyond that of diabetic control animals (P <0.05)
- Significantly increased basal insulin to glucose ratio (P <0.05)
- Tended to improve hemoglobin A1c (P = 0.19)
- Did not change plasma triglyceride or cholesterol levels compared to diabetic controls
- Tended to decrease liver weight per body weight (P = 0.17)
- Significantly reduced intrahepatic TG (P <0.05) and cholesterol (P < 0.001)
- Tended to increase triglyceride (P = 0.17) and cholesterol levels in stools (P = 0.20)
- Significantly reduced plasma IL-6 levels
Unexpectedly, taspoglutide did not change overall plaque size in STZ-induced diabetic mice
compared to diabetic controls in the aortic sinus (P = 0.18) or the descending aorta (P = 0.19).
Furthermore, there was no change in macrophage-infiltrated area within plaques of the aortic
sinus or early lesions in the aortic arch.
72
Chapter 6 Future Directions
6.1 Atherosclerosis
One of the important questions that arise from our current studies is whether GLP-1R activation
changes in any way the composition and/or secretion of metalloproteinases and TIMPs (tissue
inhibitor of metalloproteinases) within the plaque. This is an important question from a clinical
standpoint because (assuming results in mice translate to humans) if plaque stability is
enhanced, this may translate into reduced cardiovascular events from treatment with GLP-1R
agonists. Conversely, if plaques become more unstable from GLP-1R activation, this may lead
to increased plaque rupture and/or thrombosis which translates into increased cardiovascular
events. Thus, in order to assess plaque stability in these mice, stains for collagen, SMCs.
metalloproteinases and TIMPs may aid in answering these questions. Collagen stains would
help determine the thickness of fibrous caps (the thicker the cap, the more stable the plaque) and
an increase in the number of smooth muscle cells may also give an indication of increased
stability. Increased metalloproteinases destabilize the plaque, while increased TIMPs inhibit
metalloproteinases and stabilize the plaque.
Our current studies, as well as studies by others (Arakawa et al. 2010; Matsubara et al. 2012;
Nagashima et al. 2011; Shah et al. 2011; Ta et al. 2011; Terasaki et al. 2012; Vittone et al.
2012), also suggest a small effect in the initiating events of atherosclerosis from GLP-1R
activation. While different pathways in macrophages have been suggested, T cells are also
involved in the initiating events of atherosclerosis (Weber and Noels 2011). Since T cells
express the GLP-1R (Hadjiyanni et al. 2010), it may be important to understand the specific
pathways engaged by activation of the GLP-1R in T cells, especially in the post-prandial state
(when GLP-1 levels rise). First, one must assess whether GLP-1R activation leads to an altered
number of T cells and/or lymphocyte infiltration into the intima. These experiments would be
conducted en face using immunofluorescence methods to stain monocytes, T cells, dendritic
cells and macrophages to assess differences in the ratios of different cells infiltrating the intima.
These experiments would show how GLP-1R activation (either acutely or chronically) in mice
(non-diabetic or diabetic) influences the infiltration of different immune cells, including T cells.
73
6.2 Fatty liver and lipoprotein metabolism
The surprising finding of livers of healthy appearance in mice treated with taspoglutide
compared to the enlarged, granulated and yellow livers of control mice may be worth exploring
further. The results in our study reveal a possible role of the GLP-1R in lipid metabolism as
triglyceride and cholesterol levels in the liver were markedly reduced, while there was a trend
for increased levels in stools (Figures 10 and 11). It may be worth exploring key markers of ER
stress (protein and/or RNA) and inflammation during the course of treatment of mice and
identify the cause of the stress (is it due to the accumulation of triglycerides and/or cholesterol
in the liver?). Thus, an experiment with Apoe-/-
mice on a low-fat diet and mice on a high-fat
diet, both with streptozotocin-induced diabetes, should be first compared at different time points
for liver morphology, intrahepatic triglyceride and cholesterol levels, and ER stress markers
(there is no such data currently in the literature). Then, the effects of activating the GLP-1R in
the liver in low-fat diabetic and high-fat diabetic mice should be contrasted to identify which
signaling pathways may be involved in the liver.
While the acute and chronic effects of GLP-1R activation in fasting and post-prandial
lipoprotein metabolism, triglyceride absorption, triglyceride secretion, and cholesterol
metabolism have been examined in non-diabetic mice and hamsters (see section 1.4.1.1), data is
lacking as to the effect of GLP-1R activation in diabetic mice. Intestinal triglyceride and
cholesterol absorption may be examined by Western blotting of APOB and by colorimetric
methods for triglyceride and cholesterol concentration in plasma during fasting and after an oral
lipid load in diabetic Apoe-/-
mice given tyloxapol to inhibit LPL and the breakdown of
triglycerides. Plasma from these experiments may also be pooled and separated on a gel
filtration column to identify triglyceride and cholesterol concentrations in VLDL, IDL/LDL and
HDL. Triglyceride and cholesterol levels in stools (by colorimetric method) give information
about triglyceride absorption and cholesterol reabsorption from the bile. Lastly, fasting and post-
prandial levels of triglycerides and cholesterol in lipoprotein fractions (VLDL, IDL/LDL and
HDL) during the course of the study (by fractionation through a gel filtration column) may be of
importance to explain the overall cycle and/or liver function.
It is very likely that the reductions in triglycerides and/or cholesterol in the liver in taspoglutide-
treated mice in our studies are mediated indirectly by the brain, or other organs, as no GLP-1R
is expressed in the liver. Thus, further studies may have to examine the effects of
74
intracerobrovascular (icv) injections of a GLP-1R agonist to record acute effects in triglyceride
and cholesterol accumulation and/or changes in signaling and/or expression of genes and
proteins involved in lipoprotein and cholesterol metabolism in the liver. For example, levels of
APOB protein and mRNA transcript levels may be quantified to further examine any role of
GLP-1R activation in the regulation of APOB.
To dissect peripheral and central actions of GLP-1R activation, icv administration of the GLP-
1R antagonist, exendin(9-39) with concurrent i.p. administration of a GLP-1R agonist then
subsequent analysis of livers for triglyceride and cholesterol accumulation, plus genes and
proteins involved in lipoprotein and cholesterol metabolism may give information as to whether
the induced changes in the liver are mediated by the brain.
Upon conclusion of these studies, a much better understanding of the physiological role of the
GLP-1R in the immune system and lipoprotein metabolism could be drawn. This information
could also be applied in clinical practice for treating diabetic patients and/or, perhaps, patients
with non-alcoholic fatty liver disease.
75
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