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GLUCAGON ACTION IN THE DORSAL VAGAL COMPLEX AND THE
REGULATION OF GLUCOSE HOMEOSTASIS DURING HIGH-PROTEIN FEEDING
by
Mary LaPierre
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Physiology
University of Toronto
© Mary LaPierre (2015)
ii
Glucagon action in the dorsal vagal complex and the regulation of glucose homeostasis
during high-protein feeding
Mary LaPierre
Master of Science
Department of Physiology
University of Toronto
2015
General Abstract
High-protein feeding acutely lowers plasma glucose compared to low-protein feeding, despite a
seemingly dichotomous stimulation of circulating glucagon concentration. The physiological
function of this postprandial glucagon rise has been largely overlooked. Notably, glucagon has
recently been discovered to trigger a negative-feedback system in the brain to reduce glucose
production. Here, we tested the hypothesis that glucagon signals in the dorsal vagal complex
(DVC) to mediate the glucose-lowering effect of high-protein feeding. First, we found that intra-
DVC administration of glucagon suppresses glucose production in vivo. Second, we identified a
Gcgr–PKA–Erk1/2–KATP channel signalling mechanism of DVC glucagon action. Third, we
demonstrated that disruption of Gcgr signalling in the DVC blunts the ability of high-protein
feeding to acutely lower plasma glucose levels compared to low-protein feeding. Collectively,
these data revise the traditional view of how high-protein feeding regulates glucose homeostasis
and introduce a physiological role of postprandial brain glucagon action.
iii
Acknowledgements
Firstly, I thank my supervisor, Dr. Tony Lam, for providing tireless guidance and support over
the past two years. Your scientific enthusiasm and insightful discussions have set a fantastic
example to prepare me for the next steps in my academic career. To the members of my
supervisory committee, Dr. Carol Greenwood and Dr. Jonathan Rocheleau, thank you for your
valuable input and advice during our meetings.
I also thank all the brilliant and motivated members of the Lam lab: Mona Abraham, Jessica
Yue, Beatrice Filippi, Frank Duca, Brittany Rasmussen, Melika Zadeh-Tahmasebi, Beini Wang,
Sophie Hamr, Paige Bauer, Elena Burdett, and Penny Wang. You have all made the lab such a
fun place to be, and I wish you all the best in your future endeavours. A special thanks to Mona,
Jess, and Bea who so eagerly passed on their wisdom and were always a source of
encouragement, commiseration, and entertainment.
Thirdly, I thank my family for their endless support and optimism, and Ben Campbell, who let
me talk his ear off to the point that he can probably clamp as well as I can.
Finally, I acknowledge the financial support for my graduate studies provided by the Canadian
Institutes of Health Research Canada Graduate Scholarships Master’s (CIHR CGS–M) program,
the Ontario Graduate Scholarship (OGS) program, the James F. Crothers Family Fellowship,
and the Unilever/Lipton Graduate Fellowship in Neuroscience. This research was financially
supported by a Canadian Diabetes Association (CDA) research grant to Dr. Tony Lam.
iv
Publications that Contributed to this Thesis
1. LaPierre MP, Abraham MA, Filippi BM, Yue JT, Lam TK. Glucagon and Lipid Signalling
in the Hypothalamus. Mammalian Genome 25(9-10): 434-441 (2014).
2. Abraham MA, Yue JT, LaPierre MP, Rutter GA, Light PE, Filippi BM, Lam TK.
Hypothalamic glucagon signals through the KATP channels to regulate glucose production.
Molecular Metabolism 3(2): 202-208 (2014).
3. Yue JT, Abraham MA, LaPierre MP, Mighiu PI, Light PE, Filippi BM, Lam TK. A fatty
acid-dependent hypothalamic-DVC neurocircuitry that regulates hepatic secretion of
triglyceride-rich lipoproteins. Nature Communications 6: 5970 (2015).
v
Table of Contents
General Abstract ........................................................................................................................... ii
Acknowledgements...................................................................................................................... iii
Publications that contributed to this thesis .................................................................................. iv
List of Tables .............................................................................................................................. vii
List of Figures ............................................................................................................................ viii
List of Abbreviations ................................................................................................................... ix
1 Introduction ............................................................................................................................. 1
1.1 Type 2 Diabetes ................................................................................................................ 1
1.2 Glucagon ........................................................................................................................... 3
1.2.1 Glucagon secretion .................................................................................................. 3
1.2.2 Glucagon action in the liver ..................................................................................... 4
1.2.3 Role of glucagon in diabetic hyperglycemia ........................................................... 6
1.2.4 Extra-hepatic glucagon actions ................................................................................ 7
1.3 Regulation of Glucose Homeostasis by the Brain ............................................................ 9
1.3.1 Hormonal signalling in the mediobasal hypothalamus ............................................ 9
1.3.2 Glucagon action in the brain .................................................................................. 12
1.3.3 The dorsal vagal complex as a novel site of glucagon action................................ 15
1.4 Dietary Protein and Glucose Regulation ........................................................................ 17
1.4.1 Effect of amino acid consumption on metabolic parameters................................. 17
1.4.2 High-protein feeding and postprandial glucose homeostasis ................................ 19
2 Hypothesis and Aims ............................................................................................................. 22
3 Material and Methods ........................................................................................................... 24
3.1 Experimental Animal Model and Surgical Procedures .................................................. 24
3.1.1 Animal Model ........................................................................................................ 24
3.1.2 Stereotaxic Cannulation Surgery ........................................................................... 24
3.1.3 Vascular Catheterization Surgery .......................................................................... 25
3.2 Pancreatic (Basal Insulin) Euglycemic Clamp Procedure .............................................. 26
3.2.1 Experimental Protocol ........................................................................................... 26
3.2.2 Tracer Dilution Methodology Calculations ........................................................... 27
vi
3.3 Fasting-refeeding Procedure ........................................................................................... 28
3.4 Administration of DVC Treatments ............................................................................... 29
3.4.1 Chemical Approach ............................................................................................... 29
3.4.2 Molecular Approach .............................................................................................. 30
3.5 Biochemical Analyses .................................................................................................... 32
3.5.1 Plasma Glucose ...................................................................................................... 32
3.5.2 Plasma Glucose Tracer Specific Activity .............................................................. 32
3.5.3 Plasma Insulin ........................................................................................................ 33
3.5.4 Plasma Glucagon ................................................................................................... 34
3.6 Protein Activity Measurements ...................................................................................... 35
3.7 Statistical Analyses ......................................................................................................... 36
4 Results ..................................................................................................................................... 37
4.1 Aim 1: Does glucagon administration into the DVC regulate glucose homeostasis? .... 37
4.1.1 Figures and Table .................................................................................................. 39
4.2 Aim 2: Does glucagon activate a PKA–Erk1/2–KATP channel signalling cascade in the
DVC to suppress glucose production? .................................................................................. 43
4.2.1 Figures ................................................................................................................... 46
4.3 Aim 3: Does DVC glucagon action contribute to the glucose-lowering effect of high-
protein feeding? .................................................................................................................... 51
4.3.1 Figures ................................................................................................................... 53
5 Discussion ............................................................................................................................... 56
6 Future Directions ................................................................................................................... 62
7 References ............................................................................................................................... 66
vii
List of Tables
Table 1: Plasma glucose, insulin, and glucagon concentrations of groups receiving DVC
infusions during basal and clamp conditions .............................................................................. 42
viii
List of Figures
Figure 1. Distribution of Gcgr protein in rat tissues .................................................................. 39
Figure 2. Schematic representation and experimental protocol of Aim 1 ................................. 40
Figure 3. Glucagon infusion into the DVC activates Gcgr to lower glucose production .......... 41
Figure 4. Schematic representation and experimental protocol of Aim 2 ................................. 46
Figure 5. Activation of PKA is required for DVC glucagon action to suppress glucose
production ................................................................................................................................... 47
Figure 6. Activation of Erk1/2 and KATP channels is necessary for DVC glucagon action to
lower glucose production ............................................................................................................ 49
Figure 7. Schematic representation and experimental protocol of Aim 3 ................................. 53
Figure 8. High-protein feeding lowers plasma glucose levels and elevates plasma glucagon
concentration compared to low protein feeding ......................................................................... 54
Figure 9. DVC glucagon signalling mediates the glucose-lowering effect of high-protein
feeding ........................................................................................................................................ 55
ix
List of Abbreviations
AC Adenylate cyclase
Akt Protein kinase B
ARC Arcuate nucleus
ATP Adenosine triphosphate
Ca2+
Calcium
CaMKII Calcium/calmodulin-dependent protein kinase II
cAMP Cyclic adenosine monophosphate
CNS Central nervous system
CREB cAMP response element-binding protein
CRH Corticotropin-releasing hormone
DAG Diacylglycerol
DMX Dorsal motor nucleus of the vagus
DVC Dorsal vagal complex
Erk1/2 Extracellular signal-regulated kinase ½
FGF21 Fibroblast growth factor 21
G-protein Guanine nucleotide binding protein
G6Pase Glucose 6-phosphatase
Gcgr Glucagon receptor
Gcgr-/- Glucagon receptor-null
GDP Guanosine diphosphate
GFP Green fluorescent protein
GLP-1 Glucagon-like peptide-1
x
GLP-2 Glucagon-like peptide-2
GRA Glucagon receptor antagonist
GTP Guanosine triphosphate
ICV Intracerebroventricular
IP3 Inositol 1,4,5-trisphosphate
KATP channel ATP-sensitive potassium channel
mAb Monoclonal antibody
MBH Mediobasal hypothalamus
MEK1 Extracellular signal-regulated kinase kinase 1/2
MEK1-DN Adenovirus expressing the dominant negative form of MEK1
Na+ Sodium
NMDA N-methyl-D-aspartate
NTS Nucleus of the solitary tract
PEPCK Phosphoenolpyruvate carboxykinase
PI3K Phosphoinositide 3-kinase
PIP2 Phosphatidylinositol 4,5-bisphosphate
PKA Protein kinase A
PLC Phospholipase C
PVN Paraventricular nucleus
STAT-3 Signal transducer and activator of transcription-3
1
1 INTRODUCTION
1.1 Type 2 Diabetes
The body possesses many mechanisms that act in a concerted manner to precisely
regulate glucose homeostasis. Diabetes is a disease brought about by the body’s inability to
maintain glucose homeostasis to an extent that manifests in fasting hyperglycemia1. An
estimated 387 million people worldwide, representing about 8% of the global population, are
currently afflicted with diabetes and this number is expected to rise to nearly 600 million by the
year 20352. The development of therapeutic strategies is decidedly vital for the advancement of
effective treatments against this rapidly mounting disease.
Diabetes has been traditionally divided into two classifications: type 1 diabetes is
characterized by an autoimmune or idiopathic destruction of insulin-producing pancreatic β-
cells ultimately resulting in an absolute deficiency of insulin secretion, whereas type 2 diabetes,
accounting for approximately 90% of diabetes cases, is classically attributed to insulin
resistance eventually leading to secretory defects3. This insulin imbalance impairs glucose
uptake in target tissues such as muscle and adipose tissue and, combined with the actions of
chronically elevated glucagon levels4, increases glucose production from the liver to result in
chronic hyperglycemia. In recent years, it has become clear that diabetes is a heterogeneous
disease with a multitude of causes ranging from genetic predispositions to environmental
factors5 leading to impaired glucose regulation due not only to insulin resistance and inadequate
insulin secretion, but also to insulin-independent defects in glucose tolerance6,7
.
2
Diabetic hyperglycemia strongly associates with components of the metabolic syndrome
and is a risk factor for many vascular complications which can lead to blindness, renal failure,
cardiovascular disease, and stroke8. Thus, it is crucial to identify strategies to restore glucose
homeostasis in diabetes. Given that increased hepatic glucose production is a key contributing
factor to chronic hyperglycemia in diabetes, this thesis aims to dissect physiological
mechanisms regulating glucose production and glucose homeostasis.
3
1.2 Glucagon
Glucagon was first described as a pancreatic contaminant upon its discovery alongside
insulin in 1921, where it was found to elicit a temporary increase in blood glucose levels,
counteracting the effects of insulin9. Forty years after its discovery, the development of a
radioimmunoassay for glucagon10
made it possible to examine its physiological functions.
1.2.1 Glucagon secretion
Glucagon is a 29-amino acid peptide hormone produced primarily in the pancreas. The
gcg gene expressed in pancreatic α-cells encodes the preproglucagon protein, which is cleaved
into proglucagon and subsequently into glucagon11
. Preproglucagon is also expressed in the
intestine and brain, where differential post-translational processing yields varying amounts of
glucagon, glicentin, oxyntomodulin, and the glucagon-like-peptides, GLP-1 and GLP-212,13
.
Glucagon secretion increases during fasting conditions, during which α-cells are
stimulated by neural inputs from glucose-sensing neurons and by direct effects of low blood
glucose levels14,15
. Na+ and Ca
2+ channels on the α-cell generate action potentials which trigger
an influx of Ca2+
into the cell and a subsequent release of glucagon-containing granules. As
glucose levels rise, an elevation of cytosolic ATP inhibits ATP-sensitive potassium (KATP)
channels, leading to inactivation of Na+ and Ca
2+ channels, termination of action potentials and
a cessation of glucagon secretion16
. Exercise is another physiological condition requiring
increased fuel mobilization. As such, the exercise-induced depletion of glucose17
as well as
adrenergic stimulation of α-cells by elevated catecholamine levels 18,19
increase the release of
glucagon to meet increased fuel demands.
4
Beyond its necessity in fuel-deficient conditions, glucagon is also secreted in the post-
prandial period after consumption of dietary proteins. Amino acids potently stimulate α-cells to
secrete glucagon20–22
. Since amino acids also stimulate insulin secretion, it is thought that
glucagon plays a role to prevent hypoglycemia that may occur from insulin-induced glucose
uptake in the absence of an exogenous glucose source23
. Additionally, α-cell function is
modulated by endocrine, paracrine, and autocrine signalling; GLP-1, insulin, and somatostatin
all inhibit glucagon secretion whereas glucagon exerts positive feedback by acting on its
receptors on the α-cell to further promote its own secretion16
.
1.2.2 Glucagon action in the liver
Upon its release from the pancreas, glucagon makes its way to the liver via the portal
vein to stimulate hepatic glucose output. The binding of glucagon to its hepatic receptor, a
guanine nucleotide binding protein (G-protein)-coupled receptor, induces a conformational
change which activates the stimulatory G-protein to liberate the Gαs subunit through the
exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP)4. The freed
subunit then activates adenylate cyclase (AC) to catalyse the conversion of ATP to cyclic
adenosine monophosphate (cAMP), which acts as a second messenger to activate protein kinase
A (PKA). Once activated, PKA acts in the liver to increase the glucose pool available for
hepatic output via two metabolic pathways: glycogenolysis and gluconeogenesis.
The glycogenolytic process begins with the activation of phosphorylase kinase and
inactivation of glycogen synthetase by PKA. Glycogen synthesis is inhibited while glycogen
phosphorylase is synchronously converted to its active form, phosphorylase A, to break down
glycogen molecules into glucose for release into circulation. In the postabsorptive state,
5
glycogenolysis has been documented to provide 46% of glucose for hepatic output, whereas
gluconeogenesis accounts for the remaining 54%24
. Gluconeogenesis consists of the conversion
of precursors such as amino acids, lactate, glycerol, and pyruvate into glucose through an
enzymatic pathway. Phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase
(G6Pase) in particular are key gluconeogenic enzymes. Glucagon-PKA signalling mediates the
transcription of these enzymes through phosphorylation of the transcription factor cAMP
response element-binding protein (CREB).
Aside from glucagon’s ability to activate the classical cAMP-dependent pathway,
Wakelam et al. discovered that a glucagon analogue, which did not activate AC, fully retained
the ability to induce glycogenolysis and gluconeogenesis in hepatocytes while stimulating the
production of inositol phosphates25
. By coupling to the Gq-protein instead of the Gs-protein, the
glucagon receptor alternatively signals via the activation of phospholipase C (PLC) to cleave
phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and
diacylglycerol (DAG), both of which can modulate downstream signalling cascades. IP3
increases intracellular calcium levels which stimulate hepatic glycogenolysis and
gluconeogenesis via calcium/calmodulin-dependent protein kinase II (CaMKII) signalling26
.
The contribution of basal glucagon levels to hepatic glucose production was quantified
by Cherrington et al. through the use of pancreatic clamp experiments in dogs with plasma
insulin, portal insulin, and plasma glucose fixed at basal levels27
. In postabsorptive conditions, a
deficiency of portal glucagon resulted in a 35% reduction of glucose production; thus, basal
glucagon levels account for approximately one-third of glucose output from the liver. However,
despite the ability of glucagon to stimulate glucose production, continuous intravenous infusions
of glucagon only transiently stimulate glucose production, after which a subsequent decline
6
toward baseline is observed after approximately 40 minutes regardless of whether insulin is
maintained at basal levels28
or allowed to fluctuate freely29,30
. The implications of this
phenomenon in the context of this thesis will be discussed later in more detail.
1.2.3 Role of glucagon in diabetic hyperglycemia
Chronic hyperglycemia in diabetes is generally attributed to a lack of insulin action at
the level of the liver and peripheral tissues which results in elevated glucose production coupled
with decreased glucose uptake. Since insulin ordinarily acts as a major inhibitor of glucagon
secretion31
, another metabolic abnormality seen in diabetes is an unbridled secretion of
glucagon. It has recently been argued that this chronic excess of glucagon, rather than the
insufficiency of insulin, is to blame for diabetic hyperglycemia32
. In fact, in conditions of total
insulin deficiency following streptozotocin-induced beta cell destruction, glucagon receptor-null
(Gcgr-/-) mice retain normal glucose levels whereas their wild-type counterparts become
severely hyperglycemic33,34
. Given the relationship between hyperglucagonemia and
hyperglycemia in diabetes, antagonizing glucagon or its receptor becomes a potential
therapeutic strategy to ameliorate glucose levels. The administration of compounds such as
glucagon-neutralizing antibodies35
and glucagon receptor antagonists36
has been shown to
prevent the hyperglycemic effects of glucagon. Furthermore, glucagon analogues which inhibit
glucagon receptor signalling have also been found to suppress glucagon-mediated
hyperglycemia in streptozotocin-diabetic rabbits37
and in high-fat-fed and obese-diabetic mice38
.
Though the deletion of the glucagon receptor protects mice from diabetic
hyperglycemia33,34
, Gcgr-/- mice in non-diabetic conditions display surprisingly mild changes in
glucose metabolism. Gelling et al. found that Gcgr-/- mice exhibited moderately lower blood
7
glucose levels throughout most of the day, but not during feeding hours39
. Glucose tolerance and
insulin sensitivity were also improved in Gcgr-/- mice39,40
. On the other hand, another model of
Gcgr-/- mice generated by Parker et al. did not display the same dysregulation; in fact, the blood
glucose levels of these Gcgr-/- mice fell within the normal range in both fasted and fed
conditions41
. The lack of gross abnormalities in the face of whole-body deletion of glucagon
receptors suggests the existence of compensatory mechanisms, perhaps involving glucagon
actions outside of the liver. The Gcgr-/- mice in both studies displayed pancreatic hyperplasia
and glucagon levels that were approximately two orders of magnitude greater than control
mice39,41
, highlighting the importance of glucagon receptor activity in extra-hepatic organs such
as the pancreas for the maintenance of normal metabolic parameters.
1.2.4 Extra-hepatic glucagon actions
The glucagon receptor is widely expressed in tissues such as the liver, pancreas, kidney,
adipose tissue, stomach, small intestine, ovary, heart, and several brain regions including the
hypothalamus and brainstem42,43
. Accordingly, glucagon exerts a wide range of actions such as
regulating renal blood flow, fetal growth, and cardiac contractility44
. Glucagon receptor
signalling also stimulates pancreatic insulin secretion; in fact, overexpression of the glucagon
receptor on β-cells improved glucose tolerance and hyperglycemia in mice fed a high-fat diet45
.
This finding suggests that glucagon can moderate its own effects with a negative-feedback
system.
Aside from the regulation of glycemia, glucagon additionally regulates energy
homeostasis through its effects on lipid metabolism, energy expenditure, and food intake.
Glucagon has been implicated as a lipid mobilizer during fasting by stimulating lipolysis in
8
adipocytes46
while inhibiting triglyceride synthesis and stimulating fatty acid oxidation in
hepatocytes47
. A hypolipidemic effect of glucagon has been shown in rats which exhibited lower
plasma cholesterol, phospholipid, and triglyceride levels owing to a reduced number of plasma
lipoprotein particles following 21 days of glucagon administration48,49
.
Glucagon also regulates energy balance through a variety of mechanisms. Brown
adipose tissue thermogenesis is a target of glucagon action50
which may underlie the ability of
glucagon to increase metabolic rate in human subjects51
. In concert with its stimulatory effects
on energy expenditure, glucagon acts on the other side of the energy balance equation by
reducing food intake through delayed gastric emptying52
and activation of neural circuits53
. This
neural anorectic effect has been demonstrated to rely on hepatic vagal afferent signals54
but has
also been attributed to the direct actions of glucagon in the brain, since intracerebroventricular
(ICV) injection of glucagon in rats suppressed feeding more potently than an intraperitoneal
administration55
. Studies in chicks56
and sheep57
have corroborated the findings that glucagon
acts directly in the brain to inhibit feeding. Furthermore, chronic ICV glucagon administration
for 7 days reduced body weight gain in rats58
.
Taking together its regulatory effects in lipidemia and energy balance, it appears that
glucagon can play a beneficial role in energy homeostasis though both peripheral and central
mechanisms. In addition to the regulation of energy balance, a novel role of glucagon signalling
in the brain, which will be discussed in more detail later, has recently been discovered to
regulate hepatic glucose metabolism in rodents59
. Thus, a primary goal of this thesis is to
provide further insights into the role of glucagon signalling mechanisms in the brain.
9
1.3 Regulation of Glucose Homeostasis by the Brain
Several decades prior to the discovery of insulin and glucagon, the role of the brain in
regulating glucose homeostasis became apparent when Claude Bernard demonstrated in 1855
that hyperglycemia could be induced in rabbits by puncturing the floor of the fourth cerebral
ventricle in a so-called “piqûre diabétique”60
. Since then, the gluco-regulatory role of the central
nervous system (CNS) has been further described by studies implicating the involvement of
several hormones such as insulin, leptin, GLP-1, and glucagon59,61–65
.
1.3.1 Hormonal signalling in the mediobasal hypothalamus
The hypothalamus is known as an important anatomical site for the regulation of energy
and, more specifically, glucose homeostasis. The arcuate nucleus (ARC) located within the
mediobasal hypothalamus (MBH) has emerged as a key site of hormonal action. It is situated
adjacent to the median eminence, a circumventricular organ which lacks a normal blood brain
barrier and thus which links CNS and peripheral circulation. The MBH appears to have neuronal
and vascular connections into the median eminence milieu66
which suggests that it may integrate
signals from both circulating hormones and those that cross the blood brain barrier, as well as
neural inputs from other brain regions, to aptly regulate peripheral glucose homeostasis.
Beyond insulin’s well-established role in the periphery, this hormone also enters the
brain via saturable transporters67
to act on its central receptors. The first reports of gluco-
regulatory insulin action in the brain were performed in dogs, where intracisternal or ICV
administration of insulin lowered blood glucose levels, but not in vagotomised dogs or in those
with the liver removed68,69
. These initial findings of a brain-liver axis were expanded by studies
10
which found that central insulin increased hepatic glycogen synthesis in dogs70
while in rats,
hepatic glucose output was reduced through a decrease of gluconeogenesis following insulin
infusion into the MBH71
. The idea that central insulin action is indispensable in the maintenance
of glucose homeostasis became evident in mice with a neuron-specific deletion of insulin
receptors which developed insulin resistance and elevated insulin levels72
. Downstream of the
hypothalamic receptor, the insulin signalling cascade also involves the activation of
phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt)61
and stimulation of KATP channels
with a subsequent relay to the liver via the hepatic vagus nerve71
. Notably, the physiological
relevance of insulin and KATP channel signalling in the brain has been demonstrated in human
studies wherein glucose production during a pancreatic clamp was lowered by intranasal
insulin73
and by oral administration of the KATP channel activator, diazoxide74
. This central
action of insulin also appears to be involved in the pathogenesis of impaired glucose tolerance in
obesity and diabetes, as just three days of high-fat diet-feeding blunts the ability of
hypothalamic insulin to suppress glucose production75
. On the other hand, restoration of the
PI3K signalling pathway in this condition is sufficient to recapitulate the lowering of glucose
production76
, highlighting the therapeutic potential of targeting central insulin signalling in
diabetes.
Leptin is secreted from adipocytes and serves as a signal of adiposity. Like insulin,
circulating leptin is also taken up into the brain via saturable transporters77
and activates
signalling mechanisms within the ARC to mediate the glucose-lowering effects of peripheral
leptin administration78
. In both diet-induced obese and streptozotocin-induced diabetic models,
ICV administration of leptin is sufficient to improve glycemia79,80
. Central leptin action has
further been localised to MBH neurons, in which an ARC-specific restoration of leptin receptors
11
in diabetic leptin receptor-null mice normalized blood glucose levels81
. Hypothalamic leptin
signalling is mediated by divergent signalling mechanisms; firstly, inhibition of hypothalamic
signal transducer and activator of transcription-3 (STAT-3) activity was found to negate the
ability of ICV leptin to lower hepatic glucose production63
, suggesting a STAT-3 dependent
mechanism. On the other hand, extracellular signal-regulated kinase 1/2 (Erk1/2) signalling is
also implicated in leptin action, as a hypothalamic blockade of Erk1/2 activation by the leptin
receptor also abolished the glucose-lowering effect of intravenous leptin administration82
.
Finally, leptin has also been shown to signal through the PI3K pathway, since the administration
of a PI3K inhibitor blunted the improvement of insulin sensitivity achieved by restoring ARC
leptin receptors in leptin receptor-deficient rats83
.
Accompanying the hypothalamic sensing of insulin and leptin, the ARC also expresses
receptors for GLP-1, an incretin hormone secreted from intestinal L-cells during meal ingestion
which increases insulin secretion and decreases glucagon secretion while lowering hepatic
glucose production84
. Circulating GLP-1 crosses the blood-brain barrier by passive diffusion85
and a central source of GLP-1 is also synthesized by neurons in the brain12
. The gluco-
regulatory role of central GLP-1 became evident when the glucose response following an
intraperitoneal glucose injection was significantly elevated in the presence of a hypothalamic
blockade of GLP-1 receptor signalling62
. Furthermore, administration of GLP-1 directly into the
ARC lowered hepatic glucose production, an effect which, like that of hypothalamic insulin
action, required the activation of KATP channels62
. The involvement of other intermediate
signalling molecules in hypothalamic GLP-1 action remains to be investigated.
Together, these studies demonstrate the crucial role of hypothalamic hormonal signalling
mechanisms for the maintenance of systemic glucose homeostasis.
12
1.3.2 Glucagon action in the brain
In addition to the aforementioned hormones, glucagon also mediates glucose metabolism
not only through its peripheral actions, but also via signalling mechanisms in the brain. The role
of brain glucagon action to regulate glucose production was first investigated in 1977, where an
ICV administration of 10ng glucagon in dogs transiently lowered blood glucose levels, followed
by a hyperglycemic effect86
. The initial hypoglycemic effect was lost in vagotomised animals,
implicating the parasympathetic innervation of the liver in the glucose-lowering effect of
glucagon. On the other hand, pancreatectomy abolished the hyperglycemic effect of central
glucagon injection. The same group later attributed the hyperglycemic effect of ICV glucagon to
a stimulation of glycogenolysis and gluconeogenesis and a depletion of liver glycogen stores87
.
In line with their previous study86
, this effect was negated by pancreatectomy but not by hepatic
denervation and vagotomy, indicating that the effect of CNS glucagon to elevate blood glucose
was mediated indirectly via the pancreas, as opposed to a direct brain-liver axis which instead
appeared to be involved in the glucose-lowering effect of ICV glucagon administration.
Similarly, other studies in rats88
and chicks89
observed that hyperglycemia following central
glucagon administration was accompanied by reduced plasma insulin levels.
Given the high glucagon doses of these previous studies which were several orders of
magnitude greater than physiological plasma glucagon levels90
and the non-specific nature of
their ICV injection protocols, coupled with the fact that glucagon crosses the blood brain
barrier91
, it is plausible that the initial transient hypoglycemic effect was regulated by brain
glucagon action, and that the hyperglycemic effects only occurred once the administered
glucagon bolus subsequently reached peripheral circulation. This postulation is strengthened by
a study comparing the effects of an intramuscular or intranasal administration of glucagon in
13
humans92
. Intramuscular glucagon doubled the rate of glucose appearance within 5 minutes,
whereas administration of glucagon through the intranasal route, which promotes rapid uptake
of peptide hormones into the CNS93
, did not increase glucose appearance in the same period,
despite an equally rapid rise of plasma glucagon levels92
.
In light of these early studies suggesting a central gluco-regulatory role of glucagon, as
well as the established roles of other gluco-regulatory hormones specifically within the MBH, it
was plausible that this same brain region may have been the site of action of ICV-administered
glucagon. In fact, Mighiu et al. recently demonstrated that a direct infusion of glucagon into the
MBH of rats suppressed hepatic glucose production during a pancreatic euglycemic clamp in the
presence of basal insulin levels59
. The ability of MBH glucagon action to lower glucose
production, a direct opposition of its hepatic effects, introduced a possible negative-feedback
role of glucagon in the brain. This effect required the activation of glucagon receptors and was
also observed in wild-type, but not Gcgr-/- mice. Furthermore, PKA signalling and KATP
channels were shown to be involved in MBH glucagon action, as their inhibition negated the
lowering of glucose production59,94
. Hepatic vagotomy also abolished the ability of MBH
glucagon to suppress glucose production59
, corroborating the findings of the early studies in
dogs86,87
to establish a brain-liver neuronal axis of glucagon action.
Given that glucagon crosses the blood-brain barrier91
and that the anatomical location of
the MBH allows it to sense circulating hormones66
, peripheral glucagon would presumably
reach the MBH to exert its glucose-lowering effect. Accordingly, disruption of the MBH
glucagon signalling pathway enhanced the ability of intravenous glucagon administration to
increase plasma glucose levels59
. These findings may explain why Gcgr-/- mice exhibit only
mild or no disruption of glucose levels39–41
; the hyperglycemic effect of hepatic glucagon action
14
appears to be counteracted by its hypoglycemic effect via the brain, and thus an absence of
glucagon signalling in both systems may result in a relatively minor net change in glucose
metabolism.
The contribution of brain glucagon action is also apparent in the previously mentioned
transiency in the ability of circulating glucagon to stimulate glucose production28–30
. In a
pancreatic clamp setting with fixed plasma insulin and glucose levels, a continuous infusion of
intravenous glucagon to induce sustained hyperglucagonemia resulted in a transient increase in
glucose production that returned to baseline after 40 minutes59
, mimicking the effect seen when
hormones are allowed to fluctuate physiologically29,30
. However, when the MBH glucagon
signalling pathway was blocked by an infusion of glucagon receptor antagonist, the stimulatory
effect of hyperglucagonemia on glucose production was sustained59
, thereby illustrating the
negative-feedback role of MBH glucagon signalling. Interestingly, 3 days of high-fat feeding
induced glucagon resistance in the MBH59
, suggesting that this resistance may also occur in
conditions of diet-induced obesity and diabetes. Since hyperglucagonemia in diabetes is
associated with chronic hyperglycemia95
, it is therefore plausible that MBH glucagon resistance
is partly to blame for the sustained, rather than transient, glucagon-mediated elevation of
glucose production in diabetes. If brain glucagon action is lost in diabetes and only peripheral
glucagon action remains intact, this may explain why ablation of the Gcgr improves glycemia in
diabetes33,34
, whereas in otherwise normal mice it negates both the peripheral and CNS glucagon
systems and therefore only marginally affects glucose metabolism39–41
.
Following the findings of Mighiu et al. which unveiled a role for glucagon signalling
within the MBH59
, it becomes important to fully elucidate whether glucagon acts in the brain to
modulate the effects of circulating glucagon in a physiologically relevant context.
15
Physiologically, glucagon entry into the CNS would not be restricted to the MBH, but would
likely access other highly permeable regions of the brain. Thus, in order to examine whether
widespread brain glucagon action plays a physiological role in peripheral glucose regulation, it
is crucial to examine whether glucagon action in other gluco-regulatory regions of the brain is
analogous to that in the MBH. With this in mind, the goal of this thesis is to reveal novel extra-
hypothalamic glucagon actions that regulate glucose homeostasis.
1.3.3 The dorsal vagal complex as a novel site of glucagon action
The MBH receives much attention as a regulator of energy homeostasis. However, the
dorsal vagal complex (DVC), located in the brainstem, is another key site of hormonal action to
regulate peripheral metabolism. Similarly to the MBH, the DVC contains a circumventricular
organ termed the area postrema. It also encompasses the nucleus of the solitary tract (NTS) and
the dorsal motor nucleus of the vagus (DMX), which synapse with afferent and efferent fibres of
the vagus nerve, respectively. Together, these anatomical features enable the DVC to receive
and integrate neural and hormonal signals to regulate several autonomic functions including
peripheral glucose metabolism. In fact, Claude Bernard’s seminal discovery implicating the
brain in the regulation of glycemia targeted the vagus nerve with his “piqûre diabétique” into the
floor of the 4th
ventricle, presumably abolishing the signalling of DVC neurons which lie just
below the 4th
ventricle60
. Since then, studies have identified the signalling mechanisms of
hormones acting specifically in the DVC to regulate glucose homeostasis.
One such hormone is insulin, which mimics its hypothalamic actions by acting on
insulin receptors in the DVC to lower glucose production64
. However, while insulin activates a
PI3K/Akt signalling cascade in the MBH61
, the inhibition of these signalling molecules in the
16
DVC did not negate insulin’s actions. Instead, it was unveiled that an alternate Erk1/2-
dependent mechanism with subsequent activation of KATP channels mediates the actions of DVC
insulin to suppress glucose production64
. GLP-1 receptors are also expressed in the DVC84
, and
peripherally administered GLP-1 stimulates neuronal activation in this region53
. Furthermore,
the role of DVC GLP-1 signalling in mediating its peripheral actions was highlighted in a study
where an intragastric glucose infusion activated NTS neurons and increased systemic glucose
turnover, whereas this effect was abolished in the presence of an ICV infusion of GLP-1
receptor antagonist65
. The DVC also contributes to the control of energy balance by sensing
insulin96
, GLP-197,98
, and leptin99
to regulate food intake.
Finally, abundant expression of glucagon receptors has been observed in the hindbrain
region43
, and peripheral glucagon administration induces neuronal activity in the DVC and
inhibits food intake53
, suggesting that it may also mediate its peripheral effects on glucose
regulation through the DVC. With this in mind, given that several other gluco-regulatory
hormones act in both the MBH and the DVC, and that a glucagon signalling pathway in the
MBH has recently been revealed, this thesis aims to investigate a whether glucagon similarly
activates a signalling cascade in the DVC to regulate peripheral glucose metabolism.
17
1.4 Dietary Protein and Glucose Regulation
1.4.1 Effect of amino acid consumption on metabolic parameters
Consumption of dietary amino acids regulates glucose homeostasis by stimulating the
secretion of both insulin and glucagon from the pancreas. Aside from insulin’s gluco-regulatory
actions, it is also important during the postprandial period to promote protein synthesis and
inhibit protein breakdown. Glucagon, on the other hand, stimulates the catabolism of ingested
amino acids100
, and is thought to play a role in preventing hypoglycemia which may result from
increased insulin action in the absence of ingested carbohydrates23
.
The ability of amino acids to stimulate pancreatic secretion of insulin was first
demonstrated in 1966, where it was found that individual amino acids vary in their
insulinotropic potency and that arginine was the most effective stimulator of insulin release101
.
The mechanism of amino-acid induced insulin secretion has been shown to rely on inhibition of
KATP channels and influx of Ca2+
within β-cells, as the presence of the KATP channel activator,
diazoxide, or the Ca2+
channel blocker, verapamil, reduced the secretory response to amino
acids102
. However, the insulinotropic property of arginine is lost in low-glucose conditions, and
rather acts to potentiate glucose-stimulated insulin secretion by mediating membrane
depolarization103
. In fact, the co-ingestion of amino acids with carbohydrates results in a
synergistic effect, where the plasma insulin response may be several times greater than the
consumption of carbohydrates alone104,105
.
The glucagonotropic role of amino acids was described a few years after the discovery of
their role in insulin secretion22
. Like in the β-cells, arginine potently stimulates glucagon release
18
from α-cells, but conversely to its actions in insulin secretion, this process can occur in the
absence of glucose and is inhibited by elevated concentrations of glucose22,106
. Though the
cellular mechanisms activated by amino acids in β-cells have been established, those within the
α-cells remain rather enigmatic. Glutamate20
and glycine107
have both been shown to activate
their respective receptors on the α-cell, resulting in membrane depolarization and Ca2+
influx to
trigger glucagon release. The exact mechanisms triggered by other amino acids remain
unknown. Furthermore, the relative effectiveness of individual amino acids has been studied by
several groups, but with no apparent consensus. In an isolated rat pancreas, the most potent
secretory action was seen with arginine, followed by glycine, homoserine, valine, alanine,
glutamic acid, and serine, with 10 other amino acids having progressively minor effects106
. On
the other hand, intravenous infusions of alanine, glycine, and serine had the strongest
glucagonotropic effect in sheep, while arginine, asparagine, and 12 others stimulated glucagon
to a lesser extent21
. Differing effects were again observed in the case of humans consuming
various amino acid mixtures. In this case, methionine and tyrosine levels were the best
predictors of glucagon release108
. Nevertheless, regardless of the relative contribution of each
amino acid, it is clear that dietary protein sources which contain heterogeneous compositions of
amino acids can potently stimulate glucagon secretion.
Upon its secretion, a major role of glucagon is to stimulate hepatic gluconeogenesis from
precursors such as amino acids. Indeed, perfusion of amino acids directly into the liver almost
instantaneously triggers gluconeogenesis and the liver subsequently releases a quantity of
glucose that is directly related to the amount of amino acids infused101
. However, the use of
tracer methodology to assess the fate of ingested amino acids after consumption of a protein
meal determined that only 43% of metabolized proteins appear as glucose in circulation109
.
19
Furthermore, total splanchnic glucose output does not increase after protein ingestion110
,
suggesting that dietary amino acids simply replace other gluconeogenic substrates and that the
total rate of glucose production is not stimulated by glucagon in this condition. In fact, it has
been known for over a century that the consumption of dietary protein does not increase blood
glucose concentration, despite the gluconeogenic potential of the ingested amino acids111,112
. It
has been proposed that the unchanged rate of glucose production and concentration of plasma
glucose after high protein is due to the opposing actions of insulin and glucagon, which are both
stimulated by amino acids113
. However, their asynchronous postprandial secretion patterns114
suggest that a precise counterbalance of their hepatic effects is unlikely.
1.4.2 High-protein feeding and postprandial glucose homeostasis
The complexity of glucose regulation following dietary protein consumption becomes
apparent when comparing the effects of mixed-nutrient diets containing varying amounts of
protein. Though the ability of a high-protein meal to acutely lower postprandial glucose
compared to a low-protein meal has been well established in humans104,108,115
and rodents116–118
,
the precise mechanism underlying this response remains elusive. The importance in
understanding the mechanism whereby dietary proteins lower glucose levels lies in the fact that
increasing dietary protein content from 15% to 30% for 5 weeks reduces postprandial plasma
glucose concentration even in people with uncontrolled type 2 diabetes119,120
. As such, the
underlying mechanisms of this effect may possess therapeutic implications for the management
of glucose homeostasis in type 2 diabetes.
Multiple factors have been proposed to explain the ability high-protein meals to lower
plasma glucose compared to low-protein meals. One such factor is the fact that increasing
20
relative dietary protein content can result in a reciprocal reduction of carbohydrates in the diet,
such as in a study comparing the effects of healthy men consuming a low-protein meal with 24
grams of protein plus 77 grams of carbohydrates versus a high-protein meal consisting of 75
grams of protein plus 25 grams of carbohydrates115
. In this case, the lower plasma glucose
response after the high-protein meal corresponded with a reduction of digested carbohydrates
available to enter the system as glucose. However, another study in healthy men demonstrated
that the ingestion of 0.2 g/kg of malodextrin plus 0.2 g/kg of protein hydrolysates effectively
reduced the postprandial glucose rise compared to 0.2 g/kg of malodextrin alone104
, thereby
ascertaining that, in this case, the lowering of plasma glucose was a consequence of the ingested
protein per se as it occurred despite an equal amount of carbohydrates provided by the diet.
Increased insulin levels have also been thought to mediate the glucose lowering effects
of high-protein feeding, since adding protein to ingested glucose has a positive synergistic effect
on insulin secretion and increased insulin levels have been found to correspond with reduced
plasma glucose concentration after high-protein feeding104,121,122
. However, increasing dietary
protein content at the expense of carbohydrates actually reduces the post-feeding increase in
insulin levels while still lowering glucose115
, and protein intake alone only marginally stimulates
insulin117,121
, indicating that the ability of high-protein feeding to reduce the plasma glucose
response does not rely entirely on the contribution of insulin.
Though the acute, potent elevation of circulating glucagon concentration after a high-
protein meal has been established in both rats116
and humans115
, this factor has been largely
overlooked when considering the mechanism by which dietary protein lowers plasma glucose
concentration. Given that glucagon is classically known to stimulate glucose production from
the liver, increased glucagon secretion would therefore be expected to correlate with increased
21
glucose levels. This apparent discrepancy therefore suggests the existence of another mechanism
to counteract the effect of peripheral glucagon action during high-protein feeding to maintain
glucose homeostasis. Considering the recently discovered ability of glucagon to stimulate a
CNS-mediated negative-feedback system which inhibits hepatic glucose production59
, it is
possible that this central system is involved in counteracting peripheral glucagon action to
reduce postprandial glucose levels. Thus, this thesis aims to investigate whether glucagon
signalling in the brain contributes to the regulation of glucose homeostasis after high-protein
feeding.
22
2 HYPOTHESIS AND AIMS
In light of the recent findings that glucagon signalling in the MBH suppresses glucose
production59
and that other hormones such as insulin and GLP-1 regulate glucose homeostasis
through their actions in both the MBH62,71
and the DVC64,65
, we hypothesize that glucagon
activates a signalling cascade in the DVC to similarly regulate hepatic glucose production.
Given that high-protein feeding stimulates plasma glucagon secretion and reduces plasma
glucose levels compared to low-protein feeding104,108,115,116
, we further propose that glucagon
signalling in the DVC contributes to the glucose-lowering effect of high-protein feeding. To
address these hypotheses, we investigated three experimental aims:
Aim 1: Does glucagon administration into the DVC regulate glucose homeostasis?
Glucagon acts through Gcgr-mediated signalling cascades4. Given that Gcgr expression
is found in several brain regions including the brainstem42,43,59
, we hypothesize that glucagon
triggers Gcgr signalling in the DVC to regulate glucose production.
Aim 2: Does glucagon activate a PKA–Erk1/2–KATP channel signalling cascade in the DVC
to suppress glucose production?
The Gcgr couples to a Gαs protein which classically activates PKA signalling4. Gcgr-
PKA signalling, in turn, has been shown to activate Erk1/2 in cell lines and pancreatic
islets123,124
. Furthermore, DVC insulin activates Erk1/2–KATP channel signalling to lower
glucose production64
while MBH glucagon also requires KATP channel activation to suppress
glucose production59
. With these findings in mind, we propose that glucagon activates a Gcgr-
23
mediated PKA–Erk1/2–KATP channel signalling mechanism in the DVC to lower glucose
production.
Aim 3: Does DVC glucagon action contribute to the glucose-lowering effect of high-protein
feeding?
High-protein feeding reduces postprandial glucose levels compared to low-protein
feeding, while concurrently stimulating an increase in circulating glucagon levels104,108,115,116
.
Knowing that glucagon crosses the blood-brain barrier91
and that glucagon action in the brain
counteracts the effects of circulating glucagon on glucose homeostasis59
, we hypothesize that the
DVC glucagon signalling pathway mediates the ability of high-protein feeding to lower
postprandial plasma glucose levels.
These aims have been investigated using the methodology outlined below.
24
3 MATERIALS AND METHODS
3.1 Experimental Animal Model and Surgical Procedures
3.1.1 Animal Model
Male Sprague Dawley rats (260-280g upon arrival) were obtained from Charles River
Laboratories (Montreal, Quebec) and individually housed in a room with a 12h light:12h dark
cycle. Rats had ad libitum access to drinking water and regular rat chow with a kilocalorie
distribution of 49% carbohydrate, 33% protein, and 18% fat and a caloric content of 3.1 kcal/g
(#7002, Harlan Laboratories). All animal protocols were approved by the Institutional Animal
Care and Use Committee of the University Health Network (Toronto, Ontario).
3.1.2 Stereotaxic Cannulation Surgery
Rats were stereotaxically implanted with a chronic bilateral catheter into the DVC. Rats
were anaesthetised for surgeries with an intraperitoneal injection of a cocktail of ketamine (60
mg/kg body weight Vetalar, Bioniche) and xylazine (8 mg/kg body weight Rompun, Bayer) and
mounted onto the stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) by securing ear
bars into each ear canal and inserting the nose into the anterior nose piece. An anterior-posterior
incision was made on the midline of the scalp. To ensure the skull was level, the position of the
head was adjusted until the bregma and lambda were at equal dorso-ventral coordinates. A hole
was drilled on the midline of the occipital crest and a 26-gauge stainless steel bilateral guide
cannula (C235G, Plastics One Inc.) was implanted into the DVC, targeting the NTS, 7.9 mm
below skull surface directly under the occipital crest, 0.4 mm on either side of the mid-sagittal
25
suture. The cannula was secured onto the skull with an instant adhesive (Loctite) followed by
dental cement. A dummy cannula was inserted into the guide cannula to prevent backflow or
entry of foreign substances into the brain. Recovery from surgery was monitored by daily food
intake and weight gain. Cannula placement was verified at the end of experiments by injecting 3
μl of diluted bromophenol blue in each side of the cannula and visually confirming the correct
localization.
3.1.3 Vascular Catheterization Surgery
Six days following stereotaxic cannulation, rats underwent intravenous and intra-
arterial catheterization surgery to allow infusion and blood sampling. Rats were anaesthetised
again with an intraperitoneal injection of ketamine (60 mg/kg body weight Vetalar, Bioniche)
and xylazine (8 mg/kg body weight Rompun, Bayer). An incision was made across the midline
of the neck. The left carotid artery and the right internal jugular vein were each isolated and
ligated rostrally with silk sutures. Indwelling catheters composed of PE50 tubing (Clay Adams,
0.023ʺ I.D. x 0.038ʺ O.D.) capped with a tip of silastic tubing (Corning, 0.020ʺ I.D. x 0.037ʺ
O.D.) were inserted and advanced into the vessels, and then secured in place with sutures once
proper placement was verified by blood sampling. The catheters were tunneled subcutaneously
and exteriorized between the shoulder blades, then flushed with a 10% heparin solution to
maintain patency and sealed with a blunted metal pin. The neck incision was then closed using
4-0 gauge tapered silk sutures. Recovery was monitored by daily food intake and weight gain;
rats that recovered to at least 90% of their pre-surgical weight were selected for experiments.
26
3.2 Pancreatic (Basal Insulin) Euglycemic Clamp Procedure
3.2.1 Experimental Protocol
Rats were restricted to 15g of chow the night before experiments to ensure comparable
nutritional status. The procedure began between 1030 and 1130 and was conducted in
conscious, unrestrained rats for a total duration of 210 min. At t = 0 min, a primed, continuous
intravenous infusion of [3-3H]-glucose tracer (40 μCi bolus, 0.4 μCi/min; Perkin Elmer) was
initiated with the use of infusion pumps (Harvard Apparatus PHD 2000) and maintained
throughout the experiment to assess glucose kinetics. Infusion of compounds into the DVC
began at either t = 0 min or t = 90 min as specified below (see Administration of DVC
Treatments). DVC infusions were maintained until the end of the experiment at a rate of 0.33
μl/h using CMA/400 syringe microdialysis infusion pumps. At t = 90 min, the pancreatic clamp
was initiated with a continuous infusion of somatostatin (3 μg/kg/min) and insulin (1.2
μg/kg/min) to inhibit secretion of insulin and to replace basal plasma insulin levels, respectively.
A 25% glucose solution was infused intravenously at a variable rate as necessary to maintain
euglycemia.
After allowing sufficient time for the [3-3H]-glucose to equilibrate, plasma samples were
collected from t = 60 onwards at 10-min intervals for determination of plasma glucose levels,
[3-3H]-glucose specific activity, plasma insulin, and plasma glucagon concentrations. Plasma
glucose levels were measured throughout the experiment to determine whether adjustments to
the rate of exogenous glucose infusion were required. At the end of experiments, rats were
anaesthetised and DVC, MBH, liver, and gastrocnemius muscle tissues were collected, frozen in
liquid nitrogen, and stored at -80◦C for later analysis.
27
3.2.2 Tracer Dilution Methodology Calculations
Rate of appearance (Ra) and rate of disappearance (Rd) of glucose were determined using
[3-3H]-glucose dilution methodology and the steady state formula. Ra represents the rate of
endogenous glucose production. During the basal period (t = 60-90 min), Ra is equivalent to Rd,
which corresponds to glucose uptake.
Ra = Rd = Constant tracer infusion rate (μCi/min)/ Specific activity (μCi/mg)
During the clamped condition (t = 180-210 min), the exogenous glucose infusion rate
(GIR) provides an additional source of glucose. Thus, the Rd is equivalent to the combined rates
of Ra and GIR. Glucose production (Ra) during the clamp can therefore be calculated by
subtracting GIR from Rd.
Ra = Rd – GIR
28
3.3 Fasting-refeeding Procedure
Rats were maintained on regular chow and fasted for 24 hours prior to refeeding. The
procedure was conducted in conscious, unrestrained rats. Infusion of compounds into the DVC
began at t = -90 min and continued until the end of the experiment (t = 60 min) at a rate of 0.33
μl/h using CMA/400 syringe microdialysis infusion pumps. The refeeding experiment began at
1100 (t = 0 min) by presenting 5 g of high-protein diet (Harlan Laboratories, TD.91352) or low-
protein diet (Harlan Laboratories, TD.06220) to the rats. These purified diets were isocaloric
with casein as the protein source. The kilocalorie distribution of the high-protein diet was 65.4%
protein, 21.3% carbohydrate, and 13.4% fat, while the low-protein diet consisted of 21.5%
protein, 65.3% carbohydrate, and 13.1% fat. Plasma samples were obtained at 15-min intervals
for determination of plasma glucose, insulin, and glucagon levels. Cumulative food intake was
assessed by weighing food remaining in the cage at 15-min intervals. Food was promptly
returned to the cage after each weighing.
29
3.4 Administration of DVC Treatments
3.4.1 Chemical Approach
All compounds were administered into the DVC at a rate of 0.33 μl/h using CMA/400
syringe microdialysis infusion pumps. For fasting-refeeding experiments, either saline (0.9%) or
the Gcgr antagonist, des-His1 [Glu
9] glucagon amide (Tocris Bioscience, 5 ng/μl) were infused
from t = -90–60 min. For pancreatic clamp experiments, either saline (0.9%) or glucagon
(Sigma, 5 pg/μl) were infused from t = 90-210 min. In a previous in vivo study administering
glucagon into the MBH, this was the lowest dose of glucagon which suppressed GP59
. The
following substances were pre-infused from t = 0-90 min and infused alone or with glucagon
during the clamp (t = 90-210):
(i) Glucagon-specific monoclonal antibody, K79bB10 (Sigma-Aldrich, 0.02 μg/μl).
K79bB10 binds to glucagon, thus preventing the binding of glucagon to its receptor. The
dose was selected based on previous findings demonstrating that a similar antibody
abolished the hyperglycemic effect of glucagon in vivo when given at a dose 4000-fold
higher than glucagon35
. The selected dose has been demonstrated to inhibit the actions of
5 pg/ul glucagon in the MBH59
.
(ii) Gcgr antagonist, des-His1 [Glu
9] glucagon amide (Tocris Bioscience, 5 ng/μl). This
glucagon analog competitively inhibits Gcgr activation and almost completely
suppresses the metabolic effects of glucagon at when given at a dose 100-fold higher
30
than glucagon37
. The selected dose previously negated the actions of 5 pg/ul glucagon in
the MBH59
.
(iii) PKA inhibitor, Rp-cAMPS (Tocris Bioscience, 40 μM). Rp-cAMPS is a cAMP analog
that binds onto the regulatory subunit of PKA to prevent the release and activation of its
catalytic subunits125
. The selected dose has been shown to inhibit PKA activation by 5
pg/ul glucagon in the MBH59
.
(iv) Extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitor, PD98059 (Sigma-
Aldrich, 100 μM). This allosteric inhibitor binds near the Mg-ATP binding site of
MEK1/2, preventing its catalytic activity. Administration of this dose into the DVC has
been shown to block the activation of ERK1/2 by insulin64
.
(v) KATP channel inhibitor, glibenclamide (Sigma-Aldrich, 100 μM). This sulphonylurea
binds to the SUR1 subunit to selectively inhibit KATP channels and, at the selected dose,
has been previously reported to inhibit the metabolic actions of MBH glucagon and
DVC insulin infusions64,94
.
3.4.2 Molecular Approach
Two groups of rats received adenoviral injections into the DVC. An adenovirus
expressing the dominant-negative form of MEK1 (MEK1-DN) was prepared by Filippi et al. by
mutating the magnesium binding site (D208 mutated to A) to disable its catalytic activity64
.
Purified adenoviruses with pfu of 1.8 x 108 (MEK1-DN) or 3 x 10
8 (GFP) were injected
31
immediately following DVC cannulation surgery with a volume of 3 μl into each cannula.
MEK1-DN has been previously shown to abolish the ability of DVC insulin to lower GP64
.
32
3.5 Biochemical Analyses
3.5.1 Plasma Glucose
Plasma glucose concentration was measured by glucose oxidase method (Glucose
Analyser GM9, Analox Instruments, Lunenbertg, MA). The glucose analyser was calibrated
with 8.0 mM glucose standard prior to each experiment. Blood samples were centrifuged
immediately after collection to separate plasma, of which 10 μl was pipetted into a solution of
glucose oxidase and oxygen in the glucose analyser. The glucose oxidase catalyses the oxidation
of glucose to gluconic acid.
D-glucose + O2 + H2O gluconic acid + H2O2
A polarographic oxygen sensor detects oxygen consumption by the above reaction, which is
directly proportional to the glucose concentration of the plasma sample.
3.5.2 Plasma Glucose Tracer Specific Activity
Two 50 μl samples of [3-3H]-glucose standards as well as plasma samples (50 μl) from
each experimental time point were deproteinised using 100 μl of each ZnSO4 and Ba(OH)2, and
then were centrifuged for 5 minutes at 13200 rpm at 4◦C. Then, 75 μl of the protein-free
supernatant was pipetted into plastic vials and evaporated to remove all tritiated water occurring
from the glycolysis of the [3-3H]-glucose to ensure that any radioactivity represented only the
presence of [3-3H]-glucose. Next, 7.0 ml of scintillation fluid (Bio-Safe II, Research Products
International, Mount Prospect, IL) was added to each of the vials which were subsequently
counted in a scintillation counter (Beckman Coulter LS6500) to determine radioactivity of [3-
33
3H]-glucose present in the samples. The dilution of [3-
3H]-glucose in the samples in comparison
to the average of the two standards was calculated.
3.5.3 Plasma Insulin
Plasma insulin concentration was determined by radioimmunoassay (RIA) (Millipore,
St. Charles, MO). In this assay, a known concentration of radiolabelled tracer 125
I-Insulin and
the unknown amount of unlabelled insulin compete for binding sites on an antibody. A higher
concentration of insulin in the plasma sample will result in a greater amount of unbound tracer
which can then be separated from antibody-bound tracer and counted to measure radioactivity.
Thus, the radioactivity of the unbound tracer is directly proportional with the concentration of
insulin in the sample. A standard curve created from samples of unlabelled insulin standard is
used to calculate the amount of insulin in the unknown samples.
The insulin radioimmunoassay was conducted over two days. First, 50 μl of unlabelled
insulin standard samples with known concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 ng/ml)
were added to tubes in duplicate in order to generate a standard curve. Then, 50 μl of each
experimental sample was added to subsequent tubes, and 50 μl of each 125
I-Insulin and rat
insulin antibody were added to all tubes. Two tubes received only 125
I-Insulin and antibody
without unlabelled insulin in order to measure total binding capacity. Samples were vortexed
and incubated at 4◦C overnight. The following day, 1.0 ml of precipitating reagent was added to
all tubes, which were vortexed and incubated at 4◦C for 20 minutes. The samples were then
centrifuged at 2500 xg for 20 minutes to obtain a firm pellet, the supernatant was decanted, and
tubes were counted in a gamma counter (Perkin Elmer 1470) for 1 minute.
34
The sample and standard counts (B) were expressed as a percentage of the total binding
reference tube counts (B0).
% Activity Bound = B/B0 x 100%
The % activity bound of the standards were plotted against their known concentrations.
The unknown concentrations of the samples were determined by interpolation of the standard
curve.
3.5.4 Plasma Glucagon
Plasma glucagon concentration was measured by RIA (Millipore, St. Charles, MO). The
principle and protocol of the glucagon RIA was similar to the insulin RIA as described above,
with the following modifications: samples and standards were incubated overnight at 4◦C with
the glucagon antibody alone before addition of the 125
I-Glucagon, and the standard samples had
concentrations of 20, 50, 100, 200, and 400 pg/ml glucagon.
35
3.6 Protein Activity Measurements
Tissues were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM
EGTA, 1 mM EDTA, 1% (w/v) Nonidet P40, 1 mM sodium orthovanadate, 50 mM sodium
fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM Dithiotritolo (DTT), and protease
inhibitor cocktail (Roche). Protein concentration of each homogenized tissue was determined
with the Pierce 660 nm protein assay (Thermo Scientific). Tissue lysates were subjected to
electrophoresis on a polyacrylamide gel and transferred onto nitrocellulose membranes. For
glucagon receptor detection, 60 μg of protein was run in an 8% gel; for CREB detection, 60 μg
of protein was run in a 10% gel; for Erk1/2 detection, 20 μg of protein was run in a 10% gel.
The membranes were incubated for 1h with 5% (w/v) BSA in TBS-T and then immunoblotted
with the indicated primary antibodies (diluted 1/1000 in 5% BSA) for 16h at 4⁰C. The blots
were then washed 3 times with TBS-T and incubated with the secondary antibody diluted
1/4000 in 5% skimmed milk for 1 hour. Blots were washed 5 times with TBS-T; then, the signal
was detected with an enhanced chemiluminescence commercial kit (Clarity western ECL, Bio-
Rad). Blots were imaged with GelCapture (DNR) and the phosphorylation level of CREB and
Erk1/2 was quantified GelQuant software (DNR) and normalized for the corresponding total
protein level. The following primary antibodies were used: glucagon receptor-specific antibody
from Novus (Littleton, CO) and antibodies to CREB (total and p-S133) and Erk1/2 (total and p-
T202/Y204) from Cell Signaling Technology (Danvers, MA).
36
3.7 Statistical Analyses
In clamp experiments, the time period 60-90 min was averaged for the basal condition
and the time period 180-210 min was averaged for the clamp condition. Unpaired Student’s t
tests were performed in statistical analyses between two groups. Where comparisons were made
across more than two groups, ANOVA was performed and, if differences between groups were
found significant, Tukey’s Multiple Comparison post-hoc test was used to compare each group
against all other groups. Measurements that were taken repeatedly over time were compared
using repeated measures ANOVA and, if significant, this was followed by a Bonferroni post-
hoc test to determine the statistical significance between groups. These statistical analyses were
performed for all figures unless otherwise stated. P<0.05 was considered statistically significant.
37
4 RESULTS
4.1 Aim 1: Does glucagon administration into the DVC regulate glucose homeostasis?
Glucagon receptors are located in several brain regions including the brainstem42,43,59
,
though their localisation specifically in the DVC has not yet been documented. Western blot
analyses revealed the presence of Gcgr in DVC rat tissue (Figure 1). We also confirmed the
previous findings of Gcgr localization in the MBH59
and the liver and muscle were used as
positive and negative controls, respectively. Circulating glucagon accesses the MBH, where it
triggers Gcgr-mediated mechanisms to lower glucose production59,94
. To address the hypothesis
that glucagon also acts on its receptors within the DVC to regulate glucose metabolism (Figure
2A), rats underwent a pancreatic (basal insulin)-euglycemic clamp procedure (Figure 2B). A
previous study identified that 5 pg/μl of glucagon administered at a rate of 0.33 μl/h was the
lowest effective dose in the MBH to regulate glucose production59
. The same dose was therefore
administered into the DVC in order to evaluate whether glucagon acts similarly in both regions.
Prior to initiation of the clamp and of brain treatments (t=60-90 min), the rates of basal glucose
production between the DVC saline and DVC glucagon-treated were comparable (Figure 3B).
In the clamped period (t=180-210 min), DVC glucagon infusion increased the glucose infusion
rate required to maintain euglycemia (Figure 3A) compared to DVC saline infusion. Tracer
dilution calculations during this steady-state condition attributed this to a suppression of glucose
production (Figure 3B; 3C) while there was no change in glucose uptake (Figure 3D). This
effect of DVC glucagon administration to regulate glucose kinetics occurred independently from
any changes in plasma glucose, insulin, or glucagon levels (Table 1).
38
To next address whether DVC glucagon signals through the Gcgr, we repeated the clamp
protocol with a co-administration of glucagon and the glucagon-specific monoclonal antibody
(mAb), K79bB10, to inhibit Gcgr signalling by preventing the binding of glucagon to its
receptor (Figure 2A; 2B). At the same concentration (0.02 μg/μl) that blocked MBH glucagon
action59
, the mAb alone did not affect glucose metabolism but effectively negated the ability of
DVC glucagon to increase the glucose infusion rate (Figure 3A) and inhibit glucose production
(Figure 3B, 3C) without altering glucose uptake (Figure 3D). We alternatively blocked Gcgr
signalling via infusion of the Gcgr antagonist (GRA), des-His1 [Glu
9]glucagon amide, at the
same dose (5 ng/μl) that inhibited MBH glucagon signalling59
(Figure 2A; 2B). This antagonist
also attenuated the effect of DVC glucagon administration to inhibit glucose production without
independently affecting glucose metabolism (Figure 3A, 3B, 3C, 3D). Therefore, administration
of glucagon into the DVC activates Gcgr signalling to lower glucose production.
39
4.1.1 Figures and Table
Figure 1. Distribution of Gcgr protein in rat tissues.
Representative immunoblot showing Gcgr protein expression in rat DVC and MBH as
compared to rat liver (positive control) and muscle (negative control). The molecular weight of
the protein marker band is shown on the left of the blot.
40
Figure 2. Schematic representation and experimental protocol of Aim 1.
(A): Schematic representation of the working hypothesis and experimental approach. Glucagon
administration into the DVC activates Gcgr to lower glucose production. Inhibition of glucagon
action via infusion of glucagon-specific monoclonal antibody (mAb) or Gcgr antagonist negates
the ability of glucagon to lower glucose production. (B): Experimental procedure and clamp
protocol. Stereotaxic DVC cannulation on day 0 was followed by vascular catheterization
surgery on day 6, and the pancreatic clamp experiment was performed on day 10. Saline (sal) or
glucagon (gcg) was administered into the DVC during the clamp with or without a pre- and co-
infusion of mAb and Gcgr antagonist (GRA).
41
Figure 3. Glucagon infusion into the DVC activates Gcgr to lower glucose production.
(A): Direct infusion of glucagon (n=6) into the DVC increased glucose infusion rate and
lowered glucose production (B) compared to saline infusion (n=5), but failed to do so when co-
infused with mAb (n=5) or GRA (n=6). Infusion of mAb (n=5) or GRA (n=4) alone did not
affect glucose kinetics. (C): Suppression of glucose production during the clamp state (180-210
min) expressed as the percent change from basal state (60-90 min). (D): Glucose uptake was
comparable in all groups. Values are shown as mean + SEM. **p<0.01, ***p<0.001 compared
to all other groups as determined by ANOVA and Tukey’s post-hoc test.
42
Table 1. Plasma glucose, insulin, and glucagon concentrations of groups receiving DVC
infusions during basal and clamp conditions.
Values are shown as mean ± SEM. Basal, t=60-90 min; Clamp, t=180-210 min.
43
4.2 Aim 2: Does glucagon activate a PKA–Erk1/2–KATP channel signalling cascade in
the DVC to suppress glucose production?
Following our discovery that glucagon action in the DVC regulates glucose production,
we next sought to identify the downstream Gcgr-mediated signalling mechanisms through
which it does so (Figure 4A) via the use of pancreatic clamp procedures coupled with chemical
(Figure 4B) and molecular (Figure 4C) loss-of-function approaches. Given that the Gcgr couples
to the Gαs protein which signals through AC activation, increased cAMP levels, and PKA
activation4, we first investigated whether DVC glucagon signalling activates PKA. We assessed
PKA activity by measuring the relative phosphorylation of CREB, a downstream target of PKA,
in the DVC tissues collected after clamp experiments. We observed via western blot analysis
that infusion of glucagon into the DVC increased the level of phosphorylated CREB (p-CREB)
over total CREB (tot-CREB) compared to a saline infusion (Figure 5A), whereas the co-infusion
of GRA negated the glucagon-induced increase in the p-CREB/tot-CREB ratio, indicating that
the PKA activity induced by DVC glucagon infusion was specific to a Gcgr-mediated
mechanism.
Next, to investigate whether the observed PKA activation mediates the ability of DVC
glucagon infusion to reduce glucose production, we performed a pancreatic clamp in which
glucagon was co-infused with Rp-cAMPS, a cAMP analog that acts as a specific, competitive
inhibitor of PKA, at the same concentration (40 μM) that inhibited MBH glucagon-induced
PKA activity59
(Figure 4A, 4B). While Rp-cAMPS alone had no effect on glucose metabolism,
it negated the ability of a DVC glucagon infusion to increase the glucose infusion rate (Figure
5B) and lower glucose production (Figure 5C, 5D) without affecting glucose uptake (Figure
5E). We confirmed by Western analysis that PKA activity was negated in the DVC tissues of
44
rats receiving co-infusion of glucagon with Rp-cAMPS during the clamp (Figure 5F). Thus,
PKA is a downstream effector of glucagon-Gcgr signalling in the DVC to lower glucose
production.
Gcgr-PKA signalling activates Erk1/2 in pancreatic islets as well as MIN6123
and
HEK293124
cell lines via phosphorylation of MEK1. Furthermore, insulin administration into the
DVC activates Erk1/2 and subsequent KATP channel signalling to lower glucose production64
,
while MBH glucagon infusion also requires KATP channel activation to inhibit glucose
production94
. We therefore investigated whether DVC Gcgr-PKA signalling lowers glucose
production through an Erk1/2–KATP channel dependent mechanism (Figure 4A). We first used
Western analyses to quantify Erk1/2 phosphorylation, reflecting its activation, in DVC tissues
collected from clamp experiments. DVC glucagon treatment promoted the phosphorylation of
Erk1/2 compared to a saline infusion, whereas this effect was nullified when Rp-cAMPS was
co-administered with glucagon (Figure 6A), indicating that glucagon activates Erk1/2 via a
PKA-dependent mechanism.
To evaluate the involvement of the observed Erk1/2 activation in the gluco-regulatory
effects of DVC glucagon administration, we first chemically inhibited Erk1/2 activity during a
pancreatic clamp via co-infusion of glucagon with the inhibitor of the Erk1/2 activator MEK1,
PD98059, at the same dose (100 μM) which negated the ability of a DVC insulin infusion to
activate Erk1/2 without independently affecting glucose kinetics64
(Figure 4A; 4B). In the
presence of PD98059, the effect of glucagon to increase the glucose infusion rate (Figure 6B)
and inhibit glucose production (Figure 6C, 6D) were abolished, while no differences were seen
in glucose uptake (Figure 6E). To alternatively evaluate the necessity of Erk1/2 signalling using
a molecular approach, a group of rats received DVC injection of either an adenovirus expressing
45
the dominant-negative form of MEK1 (MEK1-DN) or a GFP-tagged adenovirus immediately
following DVC cannulation surgery (Figure 4C). We have previously published findings using
this same viral construct and injection protocol in which the direct DVC injection of this MEK1-
DN adenovirus blocks the ability of insulin to activate Erk1/2 in the DVC in vivo compared to
the GFP control virus64
. We therefore used the same procedure to evaluate the necessity of
Erk1/2 in DVC glucagon action. In a pancreatic clamp in GFP-injected rats, glucagon
maintained its potency to increase the glucose infusion rate (Figure 6B) and to reduce glucose
production (Figure 6C, 6D), whereas this effect was abolished in rats injected with MEK1-DN.
No changes were observed in glucose uptake (Figure 6E). Together, these data indicate that
DVC glucagon infusion triggers PKA-dependent Erk1/2 signalling to suppress glucose
production.
To continue dissecting the downstream signalling cascade of DVC glucagon action, we
next tested the involvement of KATP channels by performing pancreatic clamps in which
glucagon was co-administered with of the KATP channel inhibitor, glibenclamide, at the same
dose which previously blocked the metabolic effects of MBH glucagon59
and DVC insulin
signalling64
(Figure 4A; 4B). Glibenclamide negated the suppression of glucose production by
DVC glucagon without independently affecting glucose kinetics (Figure 6B, 6C, 6D, 6E),
indicating that KATP channel activation is required for the effects of DVC glucagon signalling.
Knowing that DVC Gcgr signalling activates PKA (Figure 5A), that PKA mediates the
activation of Erk1/2 by glucagon (Figure 6A), and that Erk1/2 triggers KATP channels64
, these
findings collectively indicate that glucagon activates a Gcgr–PKA–Erk1/2–KATP channel
signalling pathway in the DVC to lower glucose production (Figure 4A).
46
4.2.1 Figures
Figure 4. Schematic representation and experimental protocol of Aim 2.
(A): Schematic representation of the working hypothesis and experimental approach. Gcgr
signalling in the DVC activates a PKA–Erk1/2–KATP channel cascade to lower glucose
production. Inhibition of these signalling molecules via Rp-cAMPS, PD98059, MEK1-DN, or
Glibenclamide negates the glucose production-lowering effect of DVC glucagon administration.
(B): Experimental procedure and clamp protocol for chemical inhibition approaches. A bilateral
DVC cannula was implanted on day 0. Vascular catheterization was performed on day 6, and
the clamp experiment was conducted on day 10. Saline or glucagon was administered with or
without a pre- and co-infusion of Rp-cAMPS, PD98059 (PD), or glibenclamide (Glib). (C):
Experimental procedure and clamp protocol for molecular inhibition approach. DVC
cannulation on day 0 was immediately followed by injection of adenovirus tagged with GFP or
an adenovirus expressing MEK1-DN. Vascular catheterization (day 6) was followed by the
clamp protocol (day 10) during which glucagon was administered into the DVC.
47
48
Figure 5. Activation of PKA is necessary for DVC glucagon action to suppress glucose
production.
(A): In DVC tissues collected immediately after the clamp protocol, PKA activity was assessed
by relative levels of phosphorylated CREB as quantified by immunoblot and expressed as fold
increase over saline. Direct infusion of glucagon into the DVC (n=5) increased phosphorylation
of CREB compared to saline infusion (n=6), but not in the presence of GRA (n=5). A
representative blot is shown. (B): During a pancreatic clamp, DVC glucagon infusion (n=7)
increased glucose infusion rate and lowered glucose production (C) compared to saline infusion
(n=5), but not in the presence of Rp-cAMPS (n=4). Infusion of Rp-cAMPS alone did not affect
glucose kinetics (n=4). (D): Suppression of glucose production during the clamp state (180-210
min) expressed as the percent change from basal state (60-90 min). (E): Glucose uptake was
comparable in all groups. (F): In DVC tissues collected immediately after the clamp protocol,
PKA activity, as assessed by CREB phosphorylation, was increased after DVC infusion of
glucagon (n=5) compared to saline (n=6). Infusion of DVC glucagon + Rp-cAMPS negated
PKA activation (n=4). A representative blot is shown. Values are shown as mean + SEM.
*p<0.05, **p<0.01, ***p<0.001 compared to all other groups as determined by ANOVA and
Tukey’s post-hoc test.
49
50
Figure 6. Activation of Erk1/2 and KATP channels is required for DVC glucagon action to
lower glucose production.
(A): In DVC tissues collected immediately after the clamp protocol, Erk1/2 activity was
measured by relative levels of phosphorylation as quantified by immunoblot and expressed as
fold increase over saline. Direct infusion of glucagon into the DVC (n=5) increased Erk1/2
phosphorylation compared to saline infusion (n=5), but not when co-administered with Rp-
cAMPS (n=4). A representative blot is shown. (B): During a pancreatic clamp, direct infusion of
glucagon into the DVC of normal (n=7) or GFP-injected rats (n=4), but not MEK1-DN-injected
rats (n=5), increased glucose infusion rate and lowered glucose production (C) compared to
saline infusion (n=6). Co-administration of glucagon with PD (n=4) or Glib (n=7) negated the
effects of DVC glucagon infusion. Infusion of Glib alone did not affect glucose kinetics (n=4).
(D): Suppression of glucose production during the clamp state (180-210 min) expressed as the
percent change from basal state (60-90 min). (E): Glucose uptake was comparable in all groups.
Values are shown as mean + SEM. *p<0.05, **p<0.01 compared to all other groups as
determined by ANOVA and Tukey’s post-hoc test.
51
4.3 Aim 3: Does DVC glucagon action contribute to the glucose-lowering effect of high-
protein feeding?
After establishing that the administration of exogenous glucagon into the DVC initiates a
Gcgr-mediated PKA–Erk1/2–KATP channel signalling cascade to reduce glucose production, it
next became important to examine the relevance of DVC Gcgr signalling in a physiological
setting. High-protein feeding acutely reduces plasma glucose levels and increases glucagon
secretion compared to low-protein feeding in both rats116
and humans115
. We therefore
investigated whether the increased concentration of circulating glucagon observed after high-
protein feeding acts in the DVC to trigger the Gcgr signalling cascade and contribute to the
regulation of glucose homeostasis after high-protein feeding (Figure 7A).
To first verify the effect of high-protein feeding in the context of an unclamped fasting-
refeeding protocol, rats were maintained on a regular chow diet, fasted for 24 hours, and then re-
fed with isocaloric high-protein or low-protein purified diets (Figure 7B). Rats also underwent
DVC cannulation surgery and DVC saline infusion during the experiment to ensure a
comparable status to those in later experiments in which the role of glucagon was to be
examined via DVC infusions of compounds. As expected, intake of the low-protein diet from
t=0 min onwards (Figure 8B) stimulated an increase of plasma glucose (Figure 8A) and insulin
levels (Figure 8C) compared to baseline. Consistent with previous literature, we found that,
compared to low-protein feeding, high-protein feeding lowered plasma glucose levels at 30
minutes onwards and reduced the area under the curve (AUC) of plasma glucose (Figure 8A).
This effect was independent of changes in cumulative food intake (Figure 8B) and plasma
insulin (Figure 8C) but correlated with a rise in plasma glucagon (Figure 8D) compared to low-
protein feeding. Though previous studies report a lowering of insulin after consumption of high-
52
versus low-protein diets with similar compositions as ours115
, we here observed no change,
albeit with a non-significant trend of high-protein feeding to reduce insulin levels at 30 minutes
post-refeeding (Figure 8C).
To test whether the high-protein-induced elevation of circulating glucagon levels triggers
Gcgr signalling in the DVC to reduce the post-refeeding glucose response in this unclamped
setting (Figure 7A), we administered GRA into the DVC at the same concentration as in clamp
experiments to inhibit Gcgr signalling (Figure 7B). While DVC GRA infusion had no effect on
the glucose response in low-protein-fed rats (Figure 9A), it increased the glucose AUC and
reversed the glucose-lowering effect of high-protein feeding, to the extent that plasma glucose
concentration was significantly elevated compared to high-protein-fed rats receiving DVC saline
infusion and was comparable to both low-protein-fed groups at t=60 min post-refeeding (Figure
9A). This elevation of glucose occurred independently of any changes in cumulative food intake
(Figure 9B), plasma insulin (Figure 9C), or plasma glucagon concentration (Figure 9D) at 60
minutes post-refeeding, arguing that the increased plasma glucagon concentration seen at earlier
time points during the high-protein refeeding (Figure 8D) had, by this point, reached the brain to
trigger DVC glucagon signalling to lower plasma glucose and maintain glucose homeostasis.
This effect was presumably attained through a lowering of glucose production as demonstrated
earlier in pancreatic clamp experiments. In summary, these data indicate that DVC glucagon
signalling mediates the acute glucose-lowering effect of high-protein feeding in healthy,
physiological conditions.
53
4.3.1 Figures
Figure 7. Schematic representation and experimental protocol of Aim 3.
(A): Schematic representation of the working hypothesis and experimental approach. Ingestion
of a high-protein diet increases the concentration of circulating glucagon which initiates the
glucagon signalling pathway in the DVC to lower glucose production and regulate postprandial
glucose homeostasis. Inhibition of DVC glucagon signalling via GRA infusion negates the
gluco-regulatory contribution of DVC glucagon action during high-protein feeding. (B):
Experimental procedure and fasting-refeeding protocol. Stereotaxic implantation of a DVC
cannula on day 0 was followed by vascular catheterization surgery on day 6, and the fasting-
refeeding experiment was performed on day 10. Saline or GRA was infused into the DVC of
rats fed either a low-protein (LP) or high-protein (HP) diet.
54
Figure 8. High-protein feeding lowers plasma glucose levels and elevates plasma glucagon
concentration compared to low protein feeding.
(A): High-protein feeding (HP/saline; n=7) lowered post-refeeding plasma glucose
concentration and area under the curve (AUC) compared to low-protein feeding (LP/saline; n=6)
without affecting cumulative food intake (B) throughout the experiment. (C): Plasma insulin
concentration was comparable in both groups, while plasma glucagon concentration (D) was
increased by HP feeding. Values are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001
compared to LP/saline determined by repeated measures ANOVA and Bonferonni post-hoc test.
†p<0.05 compared to LP/saline determined by unpaired Student’s t-test.
55
Figure 9. DVC glucagon signalling mediates the glucose-lowering effect of high-protein
feeding.
(A): Administration of GRA into the DVC during LP feeding (LP/GRA; n=5) did not affect
plasma glucose levels compared to DVC saline infusion during LP feeding (LP/saline; n=6).
DVC infusion of GRA during HP feeding (HP/GRA; n=5) increased the glucose AUC and
glucose concentration at 60 minutes post-refeeding compared to HP feeding with DVC saline
infusion (HP/saline; n=7). At 60 minutes post-refeeding, cumulative food intake (B), plasma
insulin concentration (C), and plasma glucagon concentration (D) were not different between
HP/saline and HP/GRA groups. Values are shown as mean ± SEM. *p<0.05 compared to
HP/saline determined by two-way ANOVA and Bonferonni post-hoc test. †p<0.05 compared to
HP/saline determined by unpaired Student’s t-test.
56
5 DISCUSSION
Increasing the protein content of a meal acutely lowers the postprandial glucose response
while stimulating a rise in circulating glucagon levels104,108,115,116
. Given that glucagon is
classically known to stimulate glucose production from the liver, increased glucagon would be
expected to correlate with increased glucose levels. This apparent discrepancy therefore
suggests the existence of another mechanism to counteract the effect of peripheral glucagon
action during high-protein feeding to maintain glucose homeostasis. In fact, a negative-feedback
system of glucagon action in the hypothalamus has recently been discovered, in which
administration of glucagon into the MBH suppressed hepatic glucose production and improved
glucose tolerance, while a disruption of MBH glucagon signalling enhanced the ability of
intravenous glucagon administration to increase plasma glucose levels59
. Physiologically,
glucagon entry into the CNS would not be restricted to the MBH and would likely access other
highly permeable regions of the brain.
Beyond the MBH, the DVC is another region of the brain which integrates hormonal
signals to regulate glucose homeostasis64,65
. Glucagon crosses the blood-brain barrier91
and the
Gcgr is expressed in the brainstem43
, though a gluco-regulatory role of glucagon signalling in
this region has not yet been identified. A Gcgr–PKA–Erk1/2 signalling cascade has been
observed in vitro123,124
. Furthermore, DVC insulin activates Erk1/2–KATP channel signalling to
lower glucose production64
and MBH glucagon also requires KATP channel activation to
suppress glucose production59
.
With these previous findings in mind, we therefore sought to examine whether, in
addition to its role in the MBH, glucagon acts in the DVC via a Gcgr–PKA–Erk1/2–KATP
57
channel cascade to suppress glucose production. Furthermore we hypothesized that glucagon
signalling in the DVC would contribute to the regulation of plasma glucose levels after high-
protein feeding.
In the current study, we discovered that glucagon indeed acts in the DVC to lower
glucose production in vivo during pancreatic clamp experiments in healthy rats. With the use of
chemical and molecular loss-of-function approaches, we demonstrated that inhibiting the
activation of the Gcgr, PKA, Erk1/2, or KATP channels negated the ability of a DVC glucagon
infusion to lower glucose production, therefore implicating each of these as necessary
components of the DVC glucagon signalling mechanism. Furthermore, we demonstrated that
activation of PKA required intact Gcgr signalling, and that Erk1/2 activation was dependent
upon PKA activity. Combined with previous findings that KATP channel activation in the DVC
lies downstream of Erk1/2 signalling64
, we thus established the directionality of these
components and identified a Gcgr–PKA–Erk1/2–KATP channel signalling cascade in the DVC.
Given that glucagon and insulin are classically known to oppose each other’s gluco-
regulatory actions, it is intriguing that, upon their entry into the DVC, these two hormones act in
a concerted manner to suppress glucose production through complementary mechanisms that
converge at the level of Erk1/2 and KATP channel activation64
. The DVC emerges as a site of
hormonal integration to curb hyperglycemia that may ensue in conditions with an elevated
glucagon:insulin ratio. On the other hand, the DVC also contains glucose-sensing neurons which
are activated during low glucose conditions to stimulate vagal firing and increase glucagon
secretion to prevent hypoglycemia14
. In addition to regulating glycemia, the DVC is also
responsive to glycine and neural transmissions from the hypothalamus to regulate peripheral
lipid homeostasis via redundant mechanisms that converge at the activation of N-methyl-D-
58
aspartate (NMDA) receptors126,127
. Considering these diverse roles, the DVC clearly holds an
important function in the precise maintenance of energy homeostasis in the face of dynamic
physiological conditions.
The present study expands the list of extra-hepatic actions whereby glucagon plays a
beneficial role in the regulation of energy homeostasis. In addition to its hypolipidemic
effects48,49
, its ability to increase metabolic rate50,51
, reduce food intake52,53
, and lower body
weight58
, as well as its gluco-regulatory actions in the MBH59
, this newfound role of glucagon
signalling in the DVC highlights the potential of activating localized glucagon signalling
mechanisms to improve metabolic parameters. This therapeutic potential is highlighted a study
by Kishore et al. which found that activation of central KATP channels by an oral administration
of diazoxide lowered glucose production in humans74
. Recently, intranasal insulin
administration has also been shown to lower glucose production in humans73
; it is therefore
plausible that the same effect could be elicited by oral or intranasal treatments to activate
components of the glucagon signalling pathway. The brain-specific actions of glucagon may
also explain why Gcgr-/- mice with a complete ablation of glucagon receptors exhibit
surprisingly moderate changes in blood glucose regulation39–41
. Since Gcgr signalling in the
brain counteracts the effect of hepatic Gcgr during normal physiological conditions, the loss of
both systems may therefore result in an overall minor net change in glucose kinetics.
A limitation of the pancreatic clamp studies performed in this thesis is that the infusion
of exogenous glucagon into the DVC does not necessarily mimic the hormonal milieu seen in
physiological conditions. Despite the fact that our glucagon dosage was at least 4 orders of
magnitude lower than previous investigations of brain glucagon action56,86,88
, it remained
59
important to examine the functional relevance of DVC glucagon action in a truly physiological
setting.
Since circulating glucagon levels are increased after high-protein feeding, it seemed
plausible that an increased amount of glucagon may access the DVC in this condition to activate
its Gcgr-mediated signalling cascade. In the context of a fasting-refeeding protocol, we indeed
discovered that while high-protein feeding acutely lowered plasma glucose levels compared to
low-protein feeding, this effect was blunted when DVC glucagon signalling was disrupted.
Therefore, DVC glucagon action contributes to the glucose-lowering ability of dietary protein
intake, presumably through a suppression of glucose production as demonstrated in pancreatic
clamp experiments. Since we observed elevated plasma glucagon levels in this condition, we
believe that circulating glucagon crossed the blood-brain barrier to act on glucagon receptors
within the DVC. However, glucagon can also be synthesized within the brainstem12
and so the
possibility that brain-derived glucagon triggers the Gcgr signalling cascade cannot be excluded.
The effect of increased dietary protein content to reduce the postprandial glucose rise has
been traditionally attributed to a reduction in the amount of carbohydrates provided by the diet
or to the stimulation of insulin secretion that is synergistically increased when dietary proteins
are combined with carbohydrates. However, dietary protein is effective in lowering the glucose
response even when dietary carbohydrates are not reduced104
and when circulating insulin levels
are lowered115
. The findings of this thesis revise the current state of knowledge on the
mechanism whereby high-protein feeding acutely lowers plasma glucose compared to low-
protein feeding by implicating a novel physiological role of brain glucagon action in post-
prandial conditions. However, our findings also indicate that DVC glucagon action is clearly not
the sole mediator of this phenomenon. The fact that disrupting Gcgr signalling did not
60
significantly blunt the effect of high-protein feeding in the early time points of the fasting-
refeeding experiment suggests the involvement of other factors prior to the onset of DVC
glucagon action at later time points. It is likely that the reduction of dietary carbohydrates (from
65.3% to 21.3%) to account for increased protein levels in our experimental diet was involved in
preventing the high glucose peak seen in the low-protein feeding condition. Other factors such
as nutrient sensing mechanisms in the gastrointestinal tract or direct sensing of amino acids in
the brain may have also contributed to the effect. Insulin does not appear to contribute to the
glucose-lowering effect in this context since we did not observe an elevation of plasma insulin
levels after high-protein feeding. However, this does not exclude the possible role of insulin in
other conditions of elevated protein which do not have a reciprocal reduction of glucose, and
which have been shown to synergistically stimulate insulin secretion104
.
Dietary protein ingestion is not the only physiological condition to stimulate glucagon
secretion. Whether DVC glucagon action is relevant in other conditions with elevated glucagon
levels, such as extended fasting and exercise, has not yet been investigated. However, the
metabolic changes that occur in these situations are such that the contribution of DVC glucagon
signalling is likely negligible. Exercise requires the stimulation of glucose production to meet
increased energy requirements. Though glucagon secretion is stimulated during exercise, the
liver becomes very sensitive to glucagon in this condition, and the increased hepatic extraction
of glucagon from the portal vein before it reaches systemic circulation results in a dampened
increase in peripheral glucagon levels128
. This lack of elevation in circulating glucagon during
exercise would conceivably curb any counter-regulatory actions of brain glucagon action,
ensuring sufficient levels of glucose to meet the increased fuel demand during exercise.
Prolonged fasting is another condition that requires the stimulation of glucagon secretion to
61
mobilize energy stores due to a lack of incoming nutrients. Though elevated circulating
glucagon would be expected to act in the brain, we have made preliminary observations (data
not shown) that the ability of MBH glucagon action to suppress glucose production in rats is lost
after a 40 hour fast, mimicking the effect of high-fat feeding to induce MBH glucagon
resistance59
. It is not uncommon to see similar metabolic defects in both conditions of nutrient
excess and nutrient deficiency; in fact, the effects of prolonged fasting to increase plasma free
fatty acids and to shift to lipid utilization are similar to the effects of high-fat feeding on the
same parameters129
, and are associated with peripheral insulin resistance130
. Hypothalamic
sensing of fatty acids also becomes impaired during fasting131
despite the effect of fasting to
increase plasma free fatty acids. Therefore, it is likely that the brain glucagon resistance
observed during fasting occurs in a similar mechanism as high-fat diet-induced glucagon
resistance, which manifests in the inability of the Gcgr to activate PKA59
. We therefore believe
that DVC glucagon signalling is primarily relevant in postprandial conditions as demonstrated in
this thesis, whereas it is unlikely to play a major role during extended fasting or exercise.
In addition to the gluco-regulatory effect of dietary proteins in healthy physiology, high-
protein diets can also acutely lower blood glucose in people with untreated type 2 diabetes132
.
Furthermore, a chronic high-protein/low carbohydrate diet in obese, type 2 diabetic men
dramatically reduces both fasting and post-meal plasma glucose levels as well as 24-h integrated
plasma insulin concentration, while increasing 24-h integrated plasma glucagon levels120
.
Therefore, the findings of this thesis, which describe the mechanism whereby DVC glucagon
action suppresses glucose production and lowers plasma glucose levels during high-protein
feeding, may hold therapeutic potential for the management of glucose homeostasis in diabetes.
62
6 FUTURE DIRECTIONS
This thesis identified a novel role of glucagon action in the DVC to regulate glucose
homeostasis during high-protein feeding. The current findings have brought to light several
more areas of investigation which may be addressed in future studies.
1) We here identified a Gcgr–PKA–Erk1/2–KATP channel signalling cascade in the DVC
which suppresses glucose production. Whether other signalling effectors are involved in
DVC glucagon action remains to be investigated. In rat mesangial cells, concurrent
activation of PKA and PLC is required for glucagon to stimulate Erk1/2133
. Glucagon-
PKA signalling activates MEK/Erk in a Rap-, Ras-, and Raf-independent manner124
. On
the other hand, glucagon acts via PLC-IP3 signalling to increase [Ca2+
]i133
, and [Ca2+
]i
promotes Erk1/2 phosphorylation via a Ras/Raf/MEK cascade134
. We speculate that
glucagon activates these converging PKA- and PLC-dependent mechanisms in the DVC
to regulate glucose homeostasis. This could be investigated by concurrent activation of
PKA and PLC by co-infusion of their respective chemical activators, Sp-cAMPS and m-
3M3FBS, to establish whether their co-activation per se induces Erk1/2 phosphorylation
and lowers glucose production during pancreatic clamp experiments. These would be
followed by experiments where the activator of one is co-infused with the inhibitor of
the other (i.e. m-3M3FBS with Rp-cAMPS, or Sp-cAMPS with the PLC inhibitor,
U73122) to evaluate whether the lack of concurrent PKA/PLC activation abolishes the
effect. Furthermore, the MBH glucagon signalling mechanism requires intact hepatic
vagal innervation to suppress glucose production, and so the role of the hepatic vagus in
DVC glucagon action warrants investigation. This could be verified by performing
63
hepatic vagotomy surgery to ablate vagal signalling in rats prior to pancreatic clamps
with DVC glucagon infusions.
2) Recently, fibroblast growth factor 21 (FGF21) has been shown to mediate several key
metabolic effects of glucagon such as increasing energy expenditure, lowering plasma
cholesterol, stimulating lipolysis, and increasing blood glucose levels135
. Since FGF21
crosses the blood-brain barrier and activates Erk1/2 in the brain136
, it is therefore
possible that FGF21 signalling is also involved in the Erk1/2-mediated actions of DVC
glucagon. Chronic ICV infusion of FGF21 has been shown to improve glucose tolerance
in lean rats and increase insulin sensitivity in diet-induced obese rats137
. On the other
hand, an ICV injection of FGF21 was demonstrated to increase blood glucose levels in
FGF21 knockout mice136
. However, this effect required the involvement of corticotropin
releasing hormone (CRH) in the paraventricular nucleus (PVN) of the hypothalamus,
leaving the possibility that FGF21 action specifically within the MBH or the DVC may
have the opposite effects on glucose homeostasis as that in the PVN. Another study
found that, although FGF21 acts centrally to reduce plasma glucose levels in high-fat
diet-fed mice, a specific disruption of FGF21 receptor signalling in the DVC did not
abolish these effects, suggesting that the glucose-lowering actions of FGF21 are not
mediated via the DVC138
. Nevertheless, the possible role of FGF21 in DVC glucagon
signalling could be examined by co-infusing glucagon with a neutralizing antibody
against the FGF21 receptor as done by Liang et al.136
and measuring subsequent PKA
and Erk1/2 activation as well as the rate of glucose production during a pancreatic
clamp.
64
3) In obesity and diabetes, chronic hyperglucagonemia is thought to contribute to
hyperglycemia through sustained stimulation of glucose production32,95
, suggesting that
the counter-regulatory actions of glucagon in the brain may be impaired in these
conditions. This supposed impairment is corroborated by the recent finding that 3 days
of high-fat diet feeding induces MBH glucagon resistance in rats59
. Whether glucagon
resistance occurs equally in the DVC remains to be examined. To address this, rats could
be fed ad libitum with a high-fat diet for 3 days and subjected to pancreatic clamps to
examine whether DVC glucagon administration still lowers glucose production. Notably,
MBH glucagon resistance is manifested in the inability of the Gcgr to activate PKA,
since administration of the PKA activator Sp-cAMPS was able to recapitulate the
glucose-lowering effect during a pancreatic clamp in high-fat-fed rats59
. This may
therefore also be the case for DVC glucagon resistance.
4) Given the parallel actions of glucagon in the DVC and MBH59
, another important
question remaining to be addressed is the relative role of each brain region and whether
concurrent glucagon action in both MBH and DVC would elicit redundant, additive, or
synergistic mechanisms to lower glucose production. This may begin to address whether
the reversal of glucagon resistance in the brain requires specific targeting of MBH,
DVC, or both. By performing a double cannulation of both the MBH and DVC, it would
be possible to concurrently infuse glucagon into both sites, or rather to administer
glucagon into one location while inhibiting glucagon signalling in the other to assess
their respective effects. It is interesting to note that the chronic inhibition of DVC
glucagon signalling via injection of MEK1-DN virus did not affect the basal rate of
65
glucose production, hinting at a compensatory role of MBH glucagon signalling in the
absence of DVC glucagon action.
5) Assuming that the DVC becomes resistant to glucagon in high-fat feeding and diabetes
(as postulated in point 3), the mechanism whereby high-protein feeding lowers glucose
in diabetic subjects119,120
is called into question. A possible explanation would be that
dietary proteins reverse brain glucagon resistance in diabetes by sensitizing the brain to
glucagon. In support of this hypothesis, administration of the amino acid leucine into the
DVC at a dose comparable to postprandial cerebrospinal leucine levels activates Erk1/2
signalling139
, implying that dietary leucine and other amino acids may interact with the
Erk1/2-dependent DVC glucagon mechanism. Leucine ingestion has specifically been
shown to stimulate glucagon secretion and lower the postprandial glucose response when
ingested with glucose, compared to glucose ingestion alone140
. Dietary amino acids may
therefore play a dual role by increasing glucagon secretion while concurrently
sensitizing the brain to glucagon. This could be investigated by first establishing the
presence of DVC glucagon resistance (through the experiments outlined in point 3), and
then feeding rats ad libitum with a high-fat for 3 days prior to a high-protein fasting-
refeeding study. If high-protein feeding is still able to lower plasma glucose levels, but
not in the presence of a DVC glucagon signalling blockade, then it would be clear that
DVC glucagon signalling is restored during high-protein refeeding.
66
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