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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