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THE ROLE OF CENTRAL GLUCAGON SIGNALING IN THE REGULATION OF
GLUCOSE HOMEOSTASIS
By
Patricia Mighiu
A thesis submitted to the School of Graduate Studies in conformity with the requirements for the
degree of
Master of Science
Graduate Department of Physiology
University of Toronto
© Copyright by Patricia Mighiu (2012)
ii
Title: The role of central glucagon signaling in the regulation of glucose homeostasis
Full Name: Patricia Mighiu
Degree: Master of Science
Year of Convocation: 2012
Department of Physiology
University of Toronto
General Abstract
Circulating glucagon activates hepatic G-protein coupled receptors to stimulate cAMP-
dependent protein kinase A (PKA) signaling and increase glucose production (GP). The
elevation of GP, however, is not sustained even in the presence of a continuous intravenous (i.v.)
glucagon infusion that maintains elevated plasma glucagon levels. We here report that direct
infusion of glucagon into the mediobasal hypothalamus of normal rats surprisingly inhibited GP
during a pancreatic clamp. The GP-lowering effect required the activation of the hypothalamic
glucagon receptor-cAMP-PKA pathway, while activation of the hypothalamic cAMP-PKA
signaling alone was sufficient to lower GP. In a non-clamp setting, inhibition of the MBH
glucagon receptor or cAMP-PKA signaling enhanced the acute ability of an i.v. glucagon
injection to increase plasma glucose levels and GP in rats. Thus, we unveil a novel function of
glucagon in the MBH that inhibits glucose production and counteracts the direct hepatic
stimulatory effect of circulating glucagon in rats.
iii
Acknowledgements
I would first like to thank my supervisor Dr. Tony Lam for the opportunity he has given me, and
for his direction and encouragement over the past two years. Tony, my success would not have
been possible without your helpful suggestions - thank you for challenging me to reach my
fullest potential and for your continued support as I take the next step in my studies. I would also
like to thank the members of my supervisory committee Drs Richard Bazinet and Adria Giacca
for taking the time to share their knowledge and expertise with me.
My time in the lab was made memorable by a few wonderful lab mates and friends who were
always there to provide comedic relief during the stressful times: Danna, Brittany, Clair and
Penny – you all helped to make the lab a great place to be, and long after I’ve forgotten how to
clamp I’ll still remember the laughs we shared. Jessica and Beatrice, I want to extend a special
thank you to the both of you for your hard word and contribution to this project, and for the
personal and professional advice you so willingly shared with me. I am extremely grateful to
have had the opportunity to work alongside and learn from you girls.
Most importantly, I want to express my deepest gratitude to my parents and my sister who have
supported me along every step of this journey. Mom and dad, thanks for your confidence in my
abilities and for always being there to remind me that life has a way of working things out. All of
my accomplishments are a testament to your love and support, and I can only hope to continue to
make you proud in the years to come. Sori, I could not have made it through these past few years
without your sarcastic repertoire to cheer me up after a long day’s work. I will always be your
biggest fan, just as I know you are mine.
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Table of Contents
General Abstract...................................................................................................................................... ii
Acknowledgements ................................................................................................................................ iii
Publications that Contributed to this Thesis ............................................................................................. v
Table of Contents ................................................................................................................................... iv
List of Tables ......................................................................................................................................... vi
List of Figures ....................................................................................................................................... vii
List of Abbreviations .............................................................................................................................viii
1 Introduction ........................................................................................................................................ 1
Diabetes Mellitus .............................................................................................................................. 1
CNS Regulation of Glucose Homeostasis .......................................................................................... 3
Introduction to Glucagon .................................................................................................................. 9
The Hypothalamus: A novel site for glucagon action? ..................................................................... 18
2 Hypothesis and Aims ........................................................................................................................ 20
3 Materials and Methods ..................................................................................................................... 22
4 Results ............................................................................................................................................... 32
Aim 1 ............................................................................................................................................. 32
Figures and Tables ..................................................................................................................... 37
Aim 2 ............................................................................................................................................ 45
Figures and Tables ..................................................................................................................... 47
5 Discussion ......................................................................................................................................... 49
6 Conclusion ........................................................................................................................................ 56
7 Future Directions .............................................................................................................................. 58
8 References ......................................................................................................................................... 61
v
Publications that Contributed to this Thesis
1. Mighiu PI, Filippi BM & Lam TKT. Linking inflammation to the Brain-Liver Axis.
Diabetes 61(6): 1350-1352 (2012)
2. Filippi BM, Mighiu PI & Lam TKT. Is insulin action in the brain clinically relevant?
Diabetes 61(4): 773-775 (2012)
vi
List of Tables
Table 1: Plasma glucose, insulin and glucagon concentrations of the groups during basal and clamp
conditions .......................................................................................................................................... 44
vii
List of Figures
Figure 1. Schematic representation and experimental protocol for Aim 1. ............................................. 37
Figure 2. Intracerebroventricular (ICV) (3
rd) and mediobasal hypothalamic (MBH) glucagon infusion
inhibits glucose production. ................................................................................................................... 38
Figure 3. Tissue distribution of the rat glucagon receptor protein........................................................... 39
Figure 4. Representative image of glucagon receptor immunostaining in the rat brain. .......................... 39
Figure 5. Activation of the glucagon receptor and cAMP-PKA signaling pathway is required for MBH
glucagon to lower glucose production. ................................................................................................... 40
Figure 6. Activation of the cAMP-PKA signaling pathway by Sp-cAMPS is sufficient to lower glucose
production. ............................................................................................................................................ 42
Figure 7. Disruption in hypothalamic glucagon action enhances the ability of intravenous glucagon
injection to increase plasma glucose level and glucose production. ........................................................ 47
Figure 8. Glucagon signaling via the GR-cAMP-PKA pathway in the MBH inhibits glucose production and counteracts the direct hepatic stimulatory effect of circulating glucagon. ......................................... 57
viii
List of Abbreviations
AgRP Agouti-Related Peptide
ARN Arcuate Nucleus
CNS Central Nervous System
DIO Diet-induced Obesity
GIR Glucose Infusion Rate
GLP-1 Glucagon-like peptide 1
GLP1R Glucagon-like peptide 1 Receptor
GP Glucose Production
GR Glucagon Receptor
HFD High-Fat Diet
IL-6 Interleukin-6
IR Insulin Receptor
ICV Intracerebroventricular
i.p. Intraperitoneal
i.v. Intravenous
Jak Janus Kinase
LepRb Leptin receptor
MBH Mediobasal Hypothalamus
NPY Neuropeptide Y
PI3K Phosphatidylinositol 3-kinase
PKA cAMP-Dependent Protein Kinase A
POMC Proopiomelanocortin
ix
PVN Paraventricular Nucleus
STAT Signal Transducer and Activator of Transcription
STZ Streptozotocin
T1DM Type 1 Diabetes Mellitus
T2DM Type 2 Diabetes Mellitus
1
1 Introduction
Diabetes Mellitus
Diabetes mellitus is a metabolic disorder which affects approximately 366 million people
worldwide [1]. It is characterized by an array of dysfunctions secondary to defects in insulin
secretion and/or insulin action, and which predisposes to co-morbidities including microvascular,
macrovascular and neuropathic complications [2]. The disease is divided into two broad
categories: Type 1 (T1DM) and Type 2 diabetes (T2DM). T1DM is caused by an autoimmune-
mediated destruction of the insulin-secreting pancreatic β cells and manifests as an absolute
deficiency in insulin secretion[3]. The more prevalent T2DM comprises 90-95% of all diagnosed
cases[4]. The latter is caused by a combination of insulin resistance at target tissues, as well as
relative insulin deficiency in the face of inappropriately elevated glucagon levels[5;6], the main
counter-regulatory hormone to insulin which acts to stimulate hepatic glucose production
(GP)[7]. As a consequence of glucose underutilization at insulin target tissues such as the
muscle, which is responsible for taking up glucose, and the liver, which is the site of insulin-
mediated suppression of GP, and glucose overproduction due to elevated glucagon levels, a state
of chronic hyperglycemia develops. Since this is an independent risk factor for the development
of metabolic complications[8] and cardiovascular abnormalities[9], the proper regulation of
hepatic glucose production is an important therapeutic goal.
Given the global burden of the disease and the severe health consequences for affected
individuals, large-scale efforts aimed at the prevention, diagnosis and treatment of diabetes have
been developed[10]. While the initial success of these efforts continues to be promising, we
cannot expect to remedy the situation without a thorough understanding of the physiological and
2
pathophysiological mechanisms regulating hepatic glucose production. To this end, tremendous
advancement has been made by research groups around the world which are investigating the
role of the central nervous system (CNS), particularly the hypothalamus, in the regulation of
glucose homeostasis. In this same light, the focus of this thesis is to characterize the role of
glucagon action in the hypothalamus and how this contributes to the regulation of glucose
production and the pathogenesis of diabetes.
3
CNS Regulation of Glucose Homeostasis
The earliest demonstration that the brain is involved in the regulation of peripheral
glucose homeostasis was provided in 1855 by Claude Bernard, who showed that punctures of
the floor of the fourth cerebral ventricle results in hyperglycemia and glucosuria in rabbits [11].
The notion of CNS control of glucose homeostasis has evolved since its introduction over a
century ago, and multiple studies have confirmed that hormones such as insulin, leptin,
glucagon-like peptide-1 (GLP-1) and resistin can signal in the hypothalamus of rodents to
regulate glucose production and homeostasis [12-16]. The observation that many forms of
obesity and diabetes are associated with defects in central hypothalamic signaling cascades
highlights the importance of CNS regulation of glucose homeostasis in normal and disease states
[17;18]. Unfortunately, despite significant advances in the field to unravel the central pathways
by which hormones regulate glucose homeostasis, no study to date has elucidated a similar role
for hypothalamic glucagon action.
The first indication that insulin can act in the brain to regulate peripheral glucose
homeostasis came from studies in neuron-specific insulin receptor knockout mice, which aside
from being obese, consistent with the central anorexigenic role of insulin [19], are also insulin
resistant and have elevated circulating plasma insulin levels [20]. In fact, circulating insulin can
enter the brain via a saturable transporter [21;22] and activate its receptor (IR), found in a
particularly high concentration in the arcuate nucleus (ARN) of the mediobasal hypothalamus
(MBH) [23;24]. Direct activation of central IRs by infusion of insulin or a small-molecule
4
insulin mimetic into the third cerebral ventricle (ICV 3rd
) for 4-6 hours in rats lowers hepatic
glucose production in the presence of basal circulating insulin levels through a
phosphatidylinositol 3-kinase (PI3K)-dependent signaling mechanism [13] and is required for the
physiologic suppression of GP induced by peripheral hyperinsulinemia. Highlighting the
involvement of the ARN is the finding that a 46% reduction in ARN IR protein in this area
impairs insulin action on glucose homeostasis in rats [25]. CNS insulin suppresses GP by
inhibiting gluconeogenesis [26;27] through a neural circuit that requires activation of the
hypothalamic KATP channels, since inhibition of these via ICV or MBH infusion of the KATP
channel blocker glibenclamide blunts central-insulin and hyperinsulinemia-induced suppression
of GP [13;26]. The central insulin effect is also dependent upon intact vagal efferent
communication between the brain and liver, since selective hepatic branch vagotomy blunts the
ability of ICV insulin to suppress GP [26]. At the level of the liver, it appears that CNS insulin
upregulates hepatic interleukin (IL)-6 production to trigger signal transducer and activator of
transcription (STAT)-3 signaling [27]. The observation that streptozotocin (STZ)-induced
diabetic rats [18] and high-fat diet (HFD)-fed rats [17] display blunted hypothalamic insulin
signaling prior to the onset of peripheral metabolic abnormalities suggests that hypothalamic
insulin resistance may lead to hepatic insulin resistance and thus contribute to the increased
glucose production and blood glucose levels in diabetes and obesity. In fact, it has been
demonstrated that one day of HFD feeding is sufficient to induce hypothalamic insulin resistance
which precedes hepatic insulin resistance [17]. Collectively, these findings support a role for
impaired CNS insulin action in the pathogenesis of T2DM.
Leptin is a 16 kDa hormone that circulates in the plasma in proportion to body fat stores,
and which, like insulin, enters the brain via a saturable transport mechanism [28] to regulate the
5
body’s energy status. Mice with a mutation in the obesity (ob) gene, which encodes the leptin
protein, not only display early-onset obesity, but are also insulin resistant and diabetic [29],
eluding to the possibility that leptin plays a role in the regulation of glucose homeostasis. Indeed,
in addition to its well-characterized role in the regulation of food intake and energy expenditure
through modulation of the hypothalamic neuropeptidergic neurons proopiomelanocortin (POMC)
and Neuropeptide Y(NPY)/Agouti-related peptide (AgRP) [30], leptin action in the
hypothalamus can lower glucose production independent of its effects on food intake and body
weight [31]. For example, ICV leptin lowers GP in normal and diet-induced obese (DIO) rodents
[14;32], and normalizes blood glucose levels in STZ-induced diabetic rats [33], by decreasing
both gluconeogenesis and glycogenolysis [14]. As with insulin, the ARN plays a key role, since
adenoviral-mediated restoration of the leptin receptor in the ARN of leptin receptor-null
(Leprneo/neo
) mice, which display a diabetes phenotype, reduces insulinemia and normalizes blood
glucose levels[34]. More specifically, the improvement in insulin sensitivity by leptin appears to
be mediated via the POMC neuronal population, since ablating the negative regulator of leptin
receptor signaling SOCS3in these neurons improves glucose tolerance in mice [35]. The leptin
receptor belongs to the IL-6 family of class 1 cytokine receptors, and the long isoform (LepRb)
activates the intracellular signalling cascades, namely Jak2-STAT3 and PI3K, that mediate the
hormone’s metabolic effects. The observations that inhibiting hypothalamic STAT3 activation
blunts the effect of ICV leptin on GP [14] and that the beneficial effects on insulin sensitivity
obtained by ARN-directed leptin-receptor gene therapy in leptin-receptor deficient Koletsky rats
are reversed by blocking PI3K signaling [36], implicate both of these pathways in the regulation
of glucose homeostasis by leptin. Furthermore, vagal outflow to the liver is required for central
leptin-mediated improvement of insulin sensitivity, as it is for CNS insulin regulation of GP [37].
6
More recently, the enteric peptide glucagon-like peptide-1 (GLP-1), secreted from the
intestinal L cells[38] has been implicated to play a role in glucose homeostasis through its action
in the brain. Traditionally, this incretin is best known for its potentiating effect on glucose-
stimulated insulin secretion, as well as inhibition of glucagon secretion, gastric emptying and
food intake [39]. A central role for GLP-1 in the regulation of glucose homeostasis was first
identified by Knauf and colleagues [40], who showed that infusion of the specific GLP1-receptor
(GLP1R) agonist exendin 4 into the lateral ventricle of mice enhances insulin secretion and
inhibits muscle glucose utilization during a hyperglycemic-hyperinsulinemic clamp. While
GLP1R mRNA has been localized to multiple CNS regions [41] and neurons of the nucleus
tractus solitarius can synthesize GLP-1 [42], it appears that part of the central gluco-regulatory
action of GLP-1 is mediated through arcuate GLP1Rs, since direct administration of GLP-1 into
this brain region, but not the paraventricular nucleus (PVN), reduces hepatic glucose production
in rats [15]. Furthermore, ICV administration of the GLP-1 antagonist des-His1,Glu8-exendin-4
impairs glucose tolerance in these animals, supporting a role for endogenous CNS GLP-1 in the
regulation of glucose homeostasis.
Resistin is an adipokine released by rodent adipocytes [43] and human macrophages [44]
which acts as a negative regulator of insulin action to increase glucose production [45-47]. In
addition to its peripherally-mediated effects, ICV and MBH administration of resistin increases
GP in rats by stimulating glycogenolysis [16]. Additional studies in mice suggest that this effect
may be mediated via NPY, since the effect of centrally administered resistin on GP is abrograted
in NPY knockout mice and in wild-type mice pre-treated with an NPY Y1 receptor antagonist
[48]. Additionally, when the hypothalamic action of resistin is antagonized, peripheral
hyperresistinemia is less able to increase GP in rats, highlighting the necessity of hypothalamic
7
resistin action for the effect of circulating resistin on glucose homeostasis [16]. Therefore,
hypothalamic resistin stimulates GP, which taken together with the studies on the hypothalamic
effects of insulin, leptin and GLP-1, demonstrates the importance of hypothalamic hormonal
signaling pathways in the regulation of hepatic glucose production and glucose homeostasis.
It is worth noting that some controversy still exists as to the physiological relevance of
CNS hormones and the regulation of glucose homeostasis, particularly in regards to the role of
brain insulin signaling in the regulation of hepatic GP[49]. In dogs for example, a 10-fold
increase in brain insulin (induced by intracarotid brain infusion of insulin) reduces net hepatic
glucose output without affecting GP, gluconeogenesis or glycogenolysis, as would be expected
in rodents[50]. Furthermore, whereas the ability of peripheral hyperinsulinemia to suppress GP is
blunted in rats with impaired CNS insulin signaling[13], a four-fold increase in head insulin in
dogs does not enhance the ability of peripheral hyperinsulinemia to suppress GP when insulin
levels within the hepatic sinusoids are decreased[51]. Nevertheless, despite these inconsistencies,
emerging data suggests that at least some components of the CNS gluco-regulatory signaling
pathways described above are not species-specific, since a KATP channel-dependent mechanism
also regulates endogenous glucose production in humans [52]. In this study, oral intake of the
KATP channel activator diazoxide decreased GP by 30% in healthy humans during a euglycemic
pancreatic clamp, independent of changes in plasma insulin and glucagon levels. Parallel studies
performed in rats demonstrated that oral diazoxide suppresses GP by downregulating hepatic
expression of the gluconeogenic enzymes Pepck and G6Pase, and by upregulating hepatic
pSTAT3, but not when the KATP channel is blocked by ICV administration of glibenclamide. Of
note, the suppression of GP in rats was independent of changes in hepatic pAkt, a target of
insulin signaling, thus excluding the possibility that oral diazoxide acts directly on the liver to
8
suppress GP[52]. Additionally, a selective elevation in cerebrospinal fluid insulin levels via
intranasal peptide delivery reduces appetite in humans[53;54], and this is preceded by a
reduction in plasma glucose levels in the absence of changes in serum insulin in postprandial
women[54], highlighting the relevance of CNS insulin action in the regulation of energy and
glucose homeostasis in humans[55]. Interestingly, however, no study to date has examined
whether glucagon signals through a related hypothalamic neurocircuitry to also regulate GP.
Given that CNS signaling mechanisms are critical to the regulation of glycemia and that
glucagon is the main counter-regulatory hormone to insulin in the maintenance of glucose
homeostasis, we propose that glucagon also signals through a hypothalamic pathway to regulate
glucose production.
9
Introduction to Glucagon
Shortly after the landmark discovery of insulin in 1921 by Nobel Laureates Frederick
Banting and John Macleod, Charles Best and James Collip, began the history of glucagon.
Kimball and Murlin at the University of Rochester were the first to observe that intravenous
(i.v.) injection of the crude pancreatic extracts raised blood glucose levels in dogs and rabbits
[56]. In the subsequent decades, this hyperglycemic factor, now called glucagon, was purified
and sequenced, and the development of sensitive assays capable of measuring plasma glucagon
levels in vivo fostered our understanding of the normal and pathophysiological actions of the
hormone [57].
Glucagon and the pancreatic alpha cell
Glucagon is a single-chain 29-amino acid peptide hormone processed from the amino
portion of the pre-proglucagon peptide, which is also the precursor of the glucagon-related
peptides GLP-1 and GLP-2 [57;58]. Glucagon is released from pancreatic alpha cells, as well as
the stomach of some species [59] following tissue-specific processing of proglucagon by the
enzyme prohormone convertase 2, and is simultaneous with the release of glicentin-related
polypeptide, major proglucagon fragment and intervening-peptide 1 [60]. In a series of elegant
cross-circulation experiments in anesthetized dogs, Foà [61] began to outline the physiological
role of glucagon and was the first to identify the stimulatory effect of hypoglycemia on the
hormone’s release. When the pancreatic vein of a donor dog was anastomosed to the femoral
vein of a recipient dog, injection of insulin in the donor dog caused blood glucose levels to rise
10
in the recipient. That is, hypoglycemia stimulated the release of a hyperglycemic substance by
the donor pancreas.
Unger’s group then extended these initial observations on the role of endogenous
glucagon by demonstrating that both phlorizin- and insulin-induced hypoglycemia stimulate
glucagon release, as determined by an increase in pancreatic-duodenal venous glucagon levels in
dogs, and that rapid-onset hyperglycemia suppresses it [62]. When taken together with the
observation that glucagon levels are increased by starvation in healthy humans [63], these studies
implicated glucagon as the main mobilizer of glucose output in the postabsorptive state, acting to
maintain a steady supply of glucose to the periphery [64].
In support of this, Alan Cherrington outlined a role for basal glucagon in the regulation of
postabsorptive glucose levels in normal dogs using tracer and arteriovenous difference
techniques [65]. In the study, somatostatin infusion was used to suppress endogenous insulin and
glucagon secretion, and then the hormones were selectively replaced by intraportal infusion at a
rate equivalent to their basal secretion rates. Glucagon deficiency (from a basal level of
approximately 125 to 31 pg/mL) decreased the rate of GP by 35% as well as plasma glucose
levels; whereas glucagon replacement increased GP by approximately 52% and plasma glucose
levels by 50 mg/dL. From these findings, it was concluded that basal glucagon accounts for one-
third of basal glucose production. Subsequent studies in man corroborated these findings, since
an i.v. somatostatin infusion that reduces plasma glucagon levels to less than 50% of basal but
maintains plasma insulin levels also results in a marked decrease in net splanchnic glucose
production [66].
11
Multiple autonomic neural inputs, metabolic and endocrine mechanisms act in concert to
regulate glucagon secretion in vivo. As introduced previously, hypoglycemia is a potent stimulus
for glucagon release. The mechanisms responsible for this effect include activation of the
sympathetic and parasympathetic branches of the autonomic nervous system [67], as well as
epinephrine release via the sympathoadrenal system [68]. In particular, it appears that the
glucose-sensing neurons of the ventromedial hypothalamus (VMH) are involved in mediating the
autonomic outflow associated with the counter-regulatory response [69;70]. Furthermore,
emerging suggests that low circulating glucose can be sensed by alpha cells themselves, either
directly [71;72] or indirectly in response to a reduction in intra-islet insulin levels, known to
suppress glucagon secretion. Glucagon is also released following exposure to certain amino
acids, and is inhibited by circulating factors including somatostatin and GLP-1 [73;74].
Following its release from α-cells, glucagon is taken up into the hepatic portal vein and
subsequently into the bloodstream. The relatively short half-life of the hormone (10-15 minutes
in man) is due to rapid degradation by proteolysis and enzymatic cleavage within the
hepatocyte[75], as well as loss through biliary and urinary excretion [76]. Degradation by neutral
endopeptidase (NEP) 24.11, an enzyme found in high concentration in the kidney, also plays an
important role in the clearance of glucagon[77].
Glucagon and its target tissues
The cloning of the rat hepatic glucagon receptor (GR) revealed that in order for it to exert
its biological effects in target tissues, glucagon signals through a guanine nucleotide binding
protein-coupled receptor which is structurally similar to the receptors for the glucagon-related
12
peptides [78] and whose primary structure displays 80% sequence homology to the human GR
[79]. Binding of the hormone to its receptor induces a conformational change in the latter, and
activates the associated heterotrimeric G-protein. Subsequently, guanosine diphosphate (GDP) is
exchanged for guanosine tripshosphate (GTP) on Gαs, and the G protein dissociates into its
constituent Gαs and Gβγ subunits, both of which can modulate downstream signaling pathways.
Coupled to Gαs activation is the membrane-associated enzyme adenylate cyclase, which
catalyzes the conversion of adenosine triphosphate (ATP) to cyclic 3’,5’-adenosine
monophosphate (cAMP). In turn, cAMP activates the cAMP-dependent protein kinase A (PKA).
In the liver, activated PKA contributes to GP through the breakdown of glycogen and inhibition
of glycogen synthesis, through a pathway initiated by i) the phosphorylation and activation
phosphorylase kinase, as well as ii) the phosphorylation and inactivation of glycogen synthetase.
The end result is the phosphorylation of glycogen phosphorylase to form phosphorylase A which
initiates the conversion of glycogen to glucose-1-phosphate and the release of approximately 3
million molecules of glucose from the hepatic pool, per molecule of glucagon [7;76]. Upon
cessation of the signal, intracellular phosphatases and phosphodiesterases rapidly inactivate the
enzymes and cAMP to return the system to baseline.
Additionally, glucagon-induced cAMP signaling promotes gluconeogenesis from plasma
precursors including lactate, amino acids, glycerol and pyruvate, which contribute the major
source of carbon that is incorporated into glucose by the liver. Ultimately, control of
gluconeogenesis by glucagon depends upon the regulation of several key enzymes, including
PEPCK and G6Pase, which catalyze the early and terminal steps in the gluconeogenic pathway,
respectively [80;81]. The relative contribution of glycogenolysis vs gluconeogensis to glucose
13
output in response to glucagon stimulation has been heavily investigated, and it appears that both
processes contribute equally to hepatic glucose output in the postabsorptive state [82;83].
Alternatively, activation of the Gq subunit activates phospholipase C (PLC) which
phosphorylates inositol-1,4,5-triphosphate (PIP3) to subsequently increase cytosolic calcium
concentrations [84;85]. It is also possible that the increase in Ca2+
may be a direct consequence
of cAMP, since the activation of PKA is sufficient to increase [Ca2+
] in isolated hepatocytes
[86]. The extent to which this pathway contributes to regulation of GP is still under investigation,
but it has been recently shown that activation of the IP3 receptor in hepatocytes blocks the effect
of glucagon on gluconeogenesis [87].
Glucagon receptor expression is not restricted to the hepatic membrane, as abundant
mRNA levels have also been detected in adipose tissue, kidney, heart, spleen, ovary, islets,
stomach, thymus and brain [88-90]. The finding that the glucagon receptor is found in the brain
is particularly relevant to this thesis, in light of the important gluco-regulatory role exerted by
CNS hormone signaling mechanisms, and will be discussed further. In vitro, glucagon increases
the release of free fatty acids and stimulates glucose uptake in the adipose tissue of rodents [91],
and its lipolytic effects have been demonstrated in isolated human adipocytes [92], but an in vivo
effect of physiological concentrations of glucagon on fat metabolism has not been clearly
demonstrated. Additional studies using a pancreatic clamp technique to generate physiological
hyperglucagonemia have yielded conflicting results on the role of glucagon in the regulation of
hepatic lipid metabolism, which both support [93;94] and refute [95;96] an in vivo effect in
humans. Activation of glucagon receptors in pancreatic ß-cells triggers an increase in insulin
secretion downstream of adenylate cyclase [89;97], and can exert positive inotropic effects on
14
the heart, as well as regulate gastrointestinal smooth muscle activity [78], although these effects
are more likely to be pharmacological rather than physiological
Glucagon and glucose homeostasis: Studies from Glucagon receptor knockout mice
The development and characterization of whole-body glucagon receptor knockout mice have
made significant contributions to our understanding of glucagon’s role in the regulation of
energy homeostasis and glycemic control. Mice with a targeted deletion within the glucagon
receptor gene display significantly lower fasting and fed blood glucose levels compared to their
non-transgenic littermate controls. This is consistent with defective hepatic GR signaling, as
evidenced by an inability of liver membranes isolated from knockout mice to bind [125
I]-
glucagon [98]. Furthermore, they display lower glucose levels during an insulin tolerance test
and reduced area under the curve during an intraperitoneal (i.p.) glucose tolerance test,
suggesting improved glucose tolerance. Under hyperinsulinemic-euglycemic clamp conditions,
these mice display an increase in the glucose infusion rate, confirming their improved insulin
sensitivity [99]. These changes occur in the context of normal levels of insulin, and elevated
glucagon and GLP-1 levels, which are likely a result of the marked pancreatic (predominantly α
cell) hyperplasia. Similar phenotypes are reported by most groups investigating glucose
homeostasis in receptor knockout mice [99;100]. Interestingly, knockout mice generated by the
Parker et al. have plasma glucose levels within the normal range despite a presumed total
absence of glucagon receptors, confirmed by RT-PCR and [125
I]-glucagon binding studies [100].
This is particularly unexpected, given the critical role of glucagon in the regulation of fasting and
basal glucose production. In part, the effect may be explained by a compensatory increase in
other counterregulatory hormones, although this was not evaluated. However, we cannot exclude
15
the possibility that a disruption in glucagon receptor activation at extra-hepatic sites is also
responsible for the observed phenotype. For example, since the glucagon receptor is also present
in the brain, and the CNS plays a key role in the maintenance of glucose homeostasis, it is
possible that ablating the central GR in these mice contributes to the observed phenotype.
Glucagon and Diabetic Hyperglycemia
Consistent with the aforementioned studies which implicate glucagon in the maintenance
of glucose levels is the hypothesis that chronic hyperglucagonemia contributes to diabetic
hyperglycemia [101;102], as excess glucagon levels are associated with spontaneous diabetes in
man and in experimental animal models of the disease [103;104]. Furthermore, conditions that
normally suppress glucagon release in healthy individuals, including carbohydrate or protein
meals as well as i.v. glucose infusion, fail to do so in T2DM patients [105;106]. Importantly, the
expected rise in plasma insulin levels that normally follows these treatments is also blunted in
the diabetic group [101;105;106]. These observations led to the “bihormonal” hypothesis, which
postulates that the hyperglycemic phenotype of diabetes is the result of the combination of a lack
of insulin action and an excess of glucagon [107].
Supporting the role of glucagon in the pathogenesis of diabetic hyperglycemia are the
results of several studies aimed at antagonizing glucagon or its receptor in vivo. For example,
antagonism of glucagon action in diabetic rats by i.v. injection of the glucagon analog [1-Nα-
trinitrophenylhistidine, 12-homoarginine]glucagon (THG) lowers blood glucose levels to 67% of
baseline within 5 minutes [108]. Following the success of this initial investigation, the
16
development of other peptide and small molecule GR antagonists [109], antibodies [110;111]
and antisense oligonucleotides [112;113] accelerated, and all of these have been demonstrated to
lower glucose levels in animal models of diabetes. It is particularly relevant that these
antagonists can also blunt the increase in blood glucose levels induced by intraperitoneal (i.p.)
glucagon administration in mice engineered to express the human glucagon receptor [114], and
even more promising is the finding that a week-long treatment with an orally-administered
small-molecule GR antagonist blunts glucagon-stimulated glucose production in healthy males
[115]. Nevertheless, given the important role of glucagon in the maintenance of blood glucose
levels, the risk of hypoglycemia posed by prolonged treatment with such antagonists must be
carefully assessed.
The studies highlighted above are inconsistent with the fact that inducing chronic
hyperglucagonemia exerts a rapid, but transient stimulatory effect on glucose production and
blood glucose under normal conditions in animal and human subjects. For example, a continuous
glucagon infusion that raises plasma glucagon levels six-fold in normal dogs rapidly increases
GP within 10-40 min, and consequently raises plasma glucose levels five-to-six fold within 20
minutes. However, despite sustained glucagon administration, both parameters gradually return
to baseline within 3h [116]. Importantly, this observation has been corroborated by studies in
humans [117;118]. Similar to the studies in dogs, an i.v. glucagon infusion that produces a
sustained rise in plasma glucagon levels increases splanchic glucose production 2-3 fold within a
few minutes in healthy men, but this stimulatory effect lasts for less than 30 min despite the
ongoing hyperglucagonemia [118]. Of note, this transience contrasts with the sustained effect of
hyperinsulinemia on suppression of hepatic glucose production [119]. It is unclear why the
stimulatory effect of glucagon is short-lived, although studies have demonstrated that hepatic
17
desensitization to glucagon [120], perhaps mediated by a decrease in the formation of cAMP
[121;122] may contribute to the waning effect. However, this explanation is complicated by the
fact that glucagon can still regulate downstream enzyme activity despite reductions in cAMP
levels[123]. Alternatively, high glucagon and glucose levels may play a direct role in reducing
hepatic glucagon receptor expression [124]. However, these hypotheses have not been evaluated
in vivo, making it difficult to offer a definitive explanation for the cause of this transiency.
Therefore, given the association between chronic hyperglucagonemia and increased
glucose production and glucose levels in diabetes, and the short-lived stimulatory effect of
glucagon on these parameters under normal conditions, it remains imperative to further
characterize the physiological and molecular mechanisms of glucagon action in health and
disease. To this end, the focus of this thesis will be to investigate the role of glucagon action at a
novel site, the hypothalamus, and provide further insight into the metabolic consequences of
glucagon receptor activation.
18
The Hypothalamus: A novel site for glucagon action?
The glucagon receptor was identified in the rat brain almost 3 decades ago by Hoosein &
Gurd [90]. Using 125
I-labeled monoiodoglucagon, the authors showed significant binding of
labeled glucagon to brain membrane fractions of the rat limbic system, hypothalamus, thalamus,
medulla and anterior pituitary. This finding was subsequently confirmed by others showing
glucagon receptor mRNA as well as glucagon and proglucagon gene expression throughout the
brain tissue [125;126]. The presence of glucagon in the brain has been demonstrated by multiple
groups, using both C-terminal specific antibody and an antibody against the N-terminal portion
of glucagon. Detectable levels of glucagon appear to be highest in the thalamus-hypothalamus
and brainstem regions, and increase with starvation or in the context of alloxan-induced insulin
deficiency [127;128]. Importantly, central glucagon-like immunoreactivity has also been
detected in the cerebrospinal fluid [129;130] and brain [131;132] of man, with one study
demonstrating that, similar to rodents, the hormone is present in highest concentration in the
hypothalamus [131]. Furthermore, there is evidence to suggest that glucagon is endogenous to
the CNS, and not simply taken up from the circulation, since a glucagon mRNA transcript is
present in the rat hypothalamus and brainstem, and both of these regions stain with antisera to
glucagon[125]. Also notable is the fact that the prohormone convertase PC2 is expressed in the
brain[133].
Of note, a glucagon-like extract isolated from the canine hypothalamus displaced 125
I-
glucagon from the rat liver cell membrane and activated adenylate cyclase [134], which was also
activated by adding glucagon to rat brain homogenates [90], suggesting that similar glucagon
signaling pathways exist in the brain and peripheral tissues. Indeed, application of glucagon to
intact pigeon retina in vitro stimulates cAMP production [135], and given that both cAMP and
19
PKA are present in the hypothalamus [136], it is plausible that glucagon acts through its classic
signaling cascade in the brain.
Taken together, the findings that glucagon mRNA and the glucagon receptor, as well as
key components of its signaling pathway are expressed in the brain suggest a potential central
role for glucagon action. Particularly intriguing is the observation that glucagon appears to be
concentrated in the hypothalamus, an area known to play a role in the regulation of energy
homeostasis.
20
2 Hypothesis and Aims
In light of the previously described observations that the brain, particularly the MBH, is able to
detect hormones to regulate glucose production and glucose levels [12], we propose that
glucagon is a likely candidate to signal via a hypothalamic pathway to regulate hepatic glucose
production and glucose homeostasis. Given that circulating glucagon and resistin both increase
glucose production, and that central administration of resistin has been shown previously to
contribute to this effect [16], we initially hypothesized that glucagon signaling in the
hypothalamus increases glucose production and blood glucose levels. To test this hypothesis, we
focused on two experimental aims:
Aim 1: Does activation of CNS glucagon signaling regulate hepatic glucose production and
glucose kinetics?
Glucagon signals through a GR-cAMP-PKA pathway to regulate hepatic glucose output[137].
Since glucagon and its receptor, as well as components of the glucagon signaling pathway have
been identified throughout brain regions [90;125;127;136], as well as in the hypothalamus, we
hypothesize that glucagon signals through a hypothalamic GR-cAMP-PKA pathway to regulate
glucose production.
21
Aim 2: Does MBH glucagon signaling via the GR-cAMP-PKA pathway mediate the effect
of circulating glucagon to regulate glucose production and blood glucose levels?
Although multiple lines of evidence support the claim that hyperglucagonemia contributes to the
increased hepatic glucose output in diabetes [101;102], the observation that in both animal and
human subjects, sustained hyperglucagonemia induces only a transient rise in blood glucose
levels and glucose production is paradoxical [116-118]. The ability of glucagon to cross the
blood brain barrier [138] makes plausible the assumption that central glucagon signaling
contributes to the regulation of glucagon-stimulated glucose homeostasis. Thus, we hypothesize
that glucagon signaling via the GR-cAMP-PKA pathway in the hypothalamus alters the ability of
circulating glucagon to increase glucose levels and hepatic glucose production.
These aims have been addressed using the methodology described below.
22
3 Materials and Methods
Animal Preparation and Surgical Procedures
Animal Preparation
Adult 8-week-old male Sprague-Dawley rats (280-300g) obtained from Charles River
Laboratories (Montreal, QC) were subjected to a standard light-dark cycle and maintained on
regular rat chow (Teklad 6% Mouse/Rat Diet) with ad-libitum access to drinking water. For all
surgeries, rats were anethesized with a cocktail of ketamine (90 mg/kg Vetalar; Bioniche) and
xylazine (10 mg/kg Rompun; Bayer) administered intraperitoneally. Recovery from surgical
procedures was monitored by daily food intake and weight-gain in the days preceding the clamp
and injection protocols, and only rats which recovered to within 10% of pre-surgery body weight
underwent experimentation. To ensure comparable post-absorptive nutritional status and mimic
the post-absorptive state, all animals were restricted to 15g of food the day prior to all in vivo
experiments. All study protocols were reviewed and approved by the Institutional Animal Care
and Use Committee of the University Health Network (Toronto, ON).
Ten days before the in vivo study, rats were implanted with a chronic catheter placed into the
third ventricle (i.c.v.)[139] or bilateral catheters into the mediobasal hypothalamus (MBH)[140].
The stereotaxic coordinates used for the placement of the 22-gauge stainless steel single guide
cannula (C313G; Plastics One Inc., Roanoke, VA) i.c.v. were 2.5 mm posterior to bregma and
8.0 mm below the skull surface at the midsaggital suture. For MBH infusions, a 26-gauge
stainless steel bilateral guide cannula (C235G; Plastics One Inc.) was implanted 3.1 mm
posterior to bregma and 9.6 mm below the skull surface at the midsaggital suture. The cannula
23
was secured to the skull with instant adhesive (Loctite) and dental cement. A bilateral dummy
cannula (C235DC; Plastics One Inc.) was inserted into the guide cannula to prevent clogging. At
the end of all in vivo experiments, cannula placement was verified by injecting 3 µL of diluted
bromophenol blue on each side of the bilateral cannula.
Vascular Catheterization Surgery
Six days after implantation of the brain cannulae, indwelling catheters were placed in the right
internal jugular vein and left carotid artery for infusion and blood sampling during the in vivo
studies[141;142]. Using a scalpel, a small skin incision was made near the middle of the neck
with the rat placed ventral side up, and the subcutaneous connective tissue was cleared away by
blunt dissection. The left carotid artery was exposed and a small section was isolated using
hemostat clamps. The artery was ligated rostrally using 4-0 silk sutures and a loose ligature was
placed at the caudal end. Micro-dissecting scissors were used to make a small incision in the
vessel in between the ligatures, and a catheter made using 15 cm of polyethylene (PE-50) tubing
capped with a 1.5 cm tip of silastic tubing, was inserted and tunneled into the vessel toward the
aortic arch. Sampling of arterial blood was verified, and the catheter was secured in place by
tying the loose ligature around the catheterized vessel. The right jugular vein was isolated using
similar methods, cannulated with the catheter advanced toward the superior vena cava, and
secured after proper placement was verified by venous sampling. A subcutaneous skin pocket
was made between the shoulder blades, and the catheters were externalized and flushed with a
10% heparin solution to facilitate patency. The catheters were plugged with blunted pins and
secured to the animals using masking tape. Finally, the midline neck incision was closed using 4-
0 tapered silk sutures.
24
Pancreatic (Basal Insulin) Euglycemic Clamp Procedure
The in vivo clamp experiments lasted a total of 210 min. A primed-continuous infusion of [3-
3H]-glucose (Perkin Elmer; 40 µCi bolus; 0.4 µCi/min) was initiated at the start of the
experiment (t = 0 min) and maintained at a constant rate until t = 210 min to assess glucose
kinetics through the tracer-dilution methodology. Glucose turnover [rate of appearance (Ra) of
glucose determined with [3-3H]-glucose] was calculated using steady-state formulae. In the basal
period (t = 60-90 min), the total Ra corresponds to the rate of endogenous glucose production and
is equivalent to the rate of glucose disappearance (Rd) during steady state:
Ra = Rd = Constant tracer infusion rate (µCi/min)/ Specific Activity (µCi/mg)
A pancreatic (basal insulin)-euglycemic clamp was started from t = 90-210 min, during which
insulin (1.2 mU kg-1
min-1
) and somatostatin (SRIF; 3 µg kg-1
min-1
) were continuously infused,
to maintain insulin levels at basal and to inhibit endogenous insulin and glucagon secretions,
respectively, as well as a variable infusion of 25% glucose solution to maintain plasma glucose
concentrations at levels similar to basal state (Table 1). Under this condition, there is an
additional source of Ra from the exogenous glucose infusion (glucose infusion rate; GIR), and
thus the glucose production during the clamp can be calculated by subtracting the GIR from the
Ra value calculated above:
Rd = Ra – Glucose infusion rate
In these experimental conditions, plasma insulin and glucagon levels were maintained at basal
level during the clamps as well (Table 1). Plasma samples were obtained at 10-minute intervals
for determination of [3-3H]-glucose specific activity, plasma insulin and glucagon levels. The
25
Harvard Apparatus PHD 2000 infusion pumps were used for all infusions during the clamp. At
the end of the experiment, rats were anesthetized, and hypothalamic tissue wedges were
collected, frozen in liquid nitrogen and stored at -80°C for later analysis. For sample collection, a
section of tissue was dissected which included the mediolateral and dorsoventral extent of the
arcuate nuclei while minimizing ventromedial nucleus tissue.
Intracerebroventricular and MBH infusions
For ICV infusion experiments, glucagon (Sigma, 100, 50, 5 ng/µL and 5 pg/µL) was
continuously infused into the 3rd
ventricle from t = 90-210 min. For MBH infusion experiments,
the following substances were continuously infused at a rate of 5 µL/hr: (1) glucagon (5 pg/ µL)
or (2) Sp-cAMPS – PKA activator (Tocris Bioscience, 20 µM) at t = 90-210 min with or without
(3) monoclonal anti-glucagon antibody (glucagon mAb): clone K79bB10 (Sigma-Aldrich, 0.02
µg/µL); (4) glucagon receptor (GR)-antagonist - Des-His1 [Glu
9]glucagon amide (Tocris
Bioscience, 0.005 µg/µL), (5) H-89 – PKA inhibitor (Tocris Bioscience, 12 µM) or (6) Rp-
cAMPS – PKA inhibitor (Tocris Bioscience, 40 µM) at t = 0-210 min. All infusions were
performed using the CMA/400 syringe microdialysis infusion pumps. Rp-cAMPS inhibits PKA
activation by binding to the regulatory subunit of the PKA tetramer and preventing it from
releasing its active, catalytic subunits[143]. H-89 is a competitive inhibitor of PKA which
prevents ATP from binding to the active site of the catalytic subunit, and thus from mediating
downstream phosphorylation events[144].
26
Intravenous glucagon injection procedure
At the beginning of the experiment, a primed-continuous infusion of [3-3H]-glucose was initiated
and maintained until t=150 min to assess glucose kinetics via the tracer-dilution methodology.
Rats were injected with a bolus of 100 μg of glucagon[145] in 200 μL of saline administered i.v.
at t=90 min, and blood samples were collected at 10-min intervals for determination of plasma
glucose levels and glucose specific activity. Rats were pre-treated with either MBH GR-
antagonist, Rp-cAMPS or saline from t=0–90 min.
Biochemical Analysis
Plasma glucose concentrations were measured by the glucose oxidase method (Glucose
Analyzer GM9, Analox Instruments, Lunenburg, MA). Blood samples were collected and
centrifuged at 6000 rpm. 10 µL of plasma sample was pipetted into a solution containing oxygen
and glucose oxidase, which catalyzes the oxidation of glucose to gluconic acid:
D-glucose + O2 + H2O gluconic acid + H2O2
A polarographic oxygen sensor monitors the change in oxygen due to oxygen consumption by
the above reaction, which is directly proportional to the glucose concentration. Prior to use, the
analyzer was calibrated using 8.0 mM glucose standard in saturated benzoic acid.
Plasma glucose tracer ([3-3H]-glucose) specific activity was measured at each time point using
50 µL of plasma sample, which was deproteinized using ZnSO4 and Ba(OH)2 and then
centrifuged for 5 min (6000 rpm at 4°C). Then, a 75 µL aliquot of the protein-free supernatant
27
containing 3-3H-glucose was obtained and evaporated to dryness to remove any tritiated water
present from the glycolysis of 3-3H-glucose. 7.0 mL of scintillation fluid (Budget-Solve;
Research Products International, Mount Prospect, IL) was added to each vial, and samples were
counted with a scintillation counter (Beckman Coulter LS6500) to determine the radioactivity of
3-3H-glucose in the plasma samples.
Radioimmunoassays (RIA) were used to determine the plasma concentrations of insulin and
glucagon (Linco Research, St. Charles, MO). A radioimmunoassay is based on an
antibody/antigen reaction in which a fixed concentration of radiolabeled (125
I) tracer antigen
competes with unlabelled endogenous antigen for a limited number of antigen binding sites on
the antibody. Therefore, as the concentration of unlabelled antigen increases, the amount of
tracer bound to antibody will decrease. This can be measured by separating antibody-bound
tracer from unbound tracer and counting radioactivity. To calculate the amount of antigen in an
unknown sample, a standard curve is used which includes increasing concentrations of standard
unlabelled antigen.
For measurement of plasma insulin levels (ng/µL), a two-day protocol was utilized. First, a
standard curve was generated using the provided insulin standards at 2, 5, 10, 20, 50, 100 and
200 µU/mL in duplicate. In a separate set of tubes, 100 µL of each sample was added in
duplicate. To all tubes, 100 µL each of 125I-Insulin and porcine insulin antibody were added,
then samples were vortexed and incubated overnight at 4°C. On day 2, 1.0 mL of precipitating
reagent was added to each tube, which was subsequently vortexed and incubated at 4°C for 20
min. The samples were then centrifuged to obtain a firm pellet, and the tubes were counted in a
gamma counter for one minute (Perkin Elmer 1470). The counts (B) for the samples and
28
standards were expressed as a percentage of the mean counts of the total binding reference tubes
(B0):
% Activity Bound = B/B0 x 100%
This value was plotted against the known concentration of standard, while the unknown
concentration of samples was determined by interpolation of the standard curve.
Measurement of plasma glucagon levels (pg/mL) involved an additional overnight incubation
period of the samples and standards at 4°C with the glucagon antibody alone, before the 125
I-
Glucagon was added. For the glucagon assay, the standards provided were 20, 50, 100, 200 and
400 pg/mL.
Calculations and Statistical Analysis
For the pancreatic clamp studies, 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. Statistical
analysis was performed by unpaired Student’s t-test. A P value <0.05 was considered statistically
significant.
29
Molecular Analysis
Protein kinase activity assay
Immediately after the clamp, animals were anesthetized and decapitated, and MBH wedges were
isolated, immediately frozen and stored at -80°C until use. PKA activity was measured with the
PepTag ® Assay Kit for nonradioactive detection of PKA (Promega) according to
manufacturer’s instructions. Briefly, MBH wedges were homogenized on ice in PKA
homogenization buffer (25 mM Tris·HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM β-
mercaptoethanol and protease inhibitor cocktail EDTA-free (Roche Diagnostics)). The
homogenate was centrifuged at 14,000 x g for 5 min at 4°C, and protein concentration of the
supernatant was determined using Pierce’s 660 nm Protein Assay. All reaction components were
added on ice to a final volume of 25 µL: 5 µL of PepTag PKA reaction buffer, 5 µL of PepTag
A1 Peptide, 5 µL of PKA Activator 5X solution and 2.5 µg of sample homogenate. The mixture
was incubated for 30 min at 37°C, and the reaction was stopped by incubation in a 100°C water
bath for 10 min. Before loading samples into the electrophoretic apparatus, 1 µL of 80% glycerol
was added to ensure the sample remained in the well. The PKA-specific peptide substrate used
was PepTag A1 Peptide, L-R-R-A-S-L-G (kemptide), which changes charge from +1 to -1
following phosphorylation by PKA and allows phosphorylated and nonphosphorylated versions
of the substrate to be readily separated on agarose gel. Data was analyzed using ImageJ (MIH
Software).
30
Western blot analysis
Tissues and HEK293 cells were collected and homogenized in lysis buffer containing 50 mM
Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1% (w/v) Non-idet P40, 1 mM sodium
orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM
DTT and Tablet protease inhibitors (Roche). MBH, PVN and liver total protein lysates, as well
as HEK 293 cells were subjected to SDS-PAGE and electrotransferred onto a nitrocellulose
membrane (Pall). The membrane was incubated with blocking solution (5% milk in Tris-
buffered saline containing 0.2% Tween-20) for 1h and then with rabbit glucagon receptor
antibody (1:1000 in blocking solution, Novus) overnight at 4°C. Protein expression was detected
using an HRP-linked anti-rabbit secondary antibody and an enhanced chemoluminescence
commercial kit (Pierce).
Immunohistochemistry
MBH tissues were frozen in liquid nitrogen and stored at -80°C until sectioning using a cryostat
(Leica CM1950; Leica Microsystems, Nussloch, Germany) at -20°C. Prior to
immunohistochemical staining, a heat-mediated antigen retrieval step using Tris/EDTA pH 9.0
buffer was performed and slides were incubated in buffer in a 60°C water bath overnight. The
following morning, slides were washed in TBS plus 0.025% Triton X-100 with gentle agitation,
and blocked in 10% normal serum with 1% BSA in TBS for 2 hours at room temperature. Slides
were incubated with rabbit glucagon receptor antibody (1:200 in blocking solution, Abcam)
overnight at 4°C. The slides were then washed as described, and incubated with 488-labelled
anti-rabbit antibody (1:200) for one hour at room temperature. To detect for background
immunoreactivity, slides were incubated with 488-labelled anti-rabbit antibody (1:200) only. The
31
sections were then washed, mounted and coversliped using ProLong Gold Antifade reagent with
DAPI. Immunostaining was detected with the use of a fluorescence microscope.
32
4 Results
Aim 1: Does activation of brain glucagon signaling regulate hepatic
glucose production and glucose kinetics?
To begin evaluating whether a central gluco-regulatory role of glucagon exists, we first infused
glucagon into the third ventricle and evaluated changes in glucose kinetics during the pancreatic
clamp (Fig. 1A,B). A previous study demonstrated that ICV (3rd
) infusion for 60 min of a
concentration of glucagon (100 ng) suppresses short-term feeding in Wistar rats[146]. Based on
this observation, this dose was progressively lowered in an attempt to identify the most
physiologically relevant glucagon dose for central regulation of glucose production. To much of
our surprise and contrary to our initial hypothesis, direct, short-term administration of glucagon
(100 ng/μL; 5 μL/hr) into the 3rd
ventricle increased the exogenous glucose infusion rate (GIR)
required to maintain euglycemia from 1.8+0.5 mg kg-1
min-1
(saline) to 10.9+1.9 mg kg-1
min-1
(Fig. 2A). The increase in GIR corresponded to suppression in glucose production from 10.2+0.7
mg kg-1
min-1
to 2.5+0.1 mg kg-1
min-1
(Fig. 2B) rather than an increase in glucose uptake (Fig.
2C.) To begin generating a dose-response curve, we lowered the dose to 50 ng/μL and found that
the extent of elevation in glucose infusion rate and the reduction in glucose production were
comparable to the higher dose, at 12. 5 mg kg-1
min-1
and 1.2 mg kg-1
min-1
, respectively
(Figure 2A,B). Finally, when the dose of ICV glucagon was reduced to 5 pg/μL (n=3), glucagon
ICV increased glucose infusion rate to 6.8+0.8 mg kg-1
min-1
(Fig. 2A) and lowered glucose
production to 6.5+0.3 mg kg-1
min-1
(Fig. 2B).
While the ICV route of administration has been used previously to activate ARN IR
signaling to regulate GP [13;26], we wanted to exclude the confounding effects that could arise
33
due to distribution of the hormone throughout the brain ventricular system and extra-
hypothalamic sites. Thus, we infused glucagon directly into the MBH at a dose 15-fold lower
than that used for ICV infusion. This dose was chosen because a 15-fold reduction of the ICV
insulin dose, when administered directly into the MBH, reproduced the inhibitory effects of ICV
administration on GIR and glucose production[13;26]. As shown in Figure 2B, in the pre-clamp
and pre-brain treatment period (60-90 min), the rate of basal glucose production is comparable in
both the MBH glucagon (5 pg/μL; 0.33 μL/hr) and MBH saline-treated groups. Surprisingly,
during the final 30 min of clamps, when circulating plasma insulin and glucagon levels were
maintained at near basal levels (Table 1), MBH administration of glucagon significantly
increased the exogenous GIR (Fig. 2B) required to maintain euglycemia in comparison to MBH
saline (6.5 ± 0.5 mg kg-1
min-1
vs. 1.3 ± 0.6 mg kg-1
min-1
, p < 0.05)(Fig. 2A). Based on the
steady-state tracer data, this was attributed to an inhibition of GP from 13.6 ± 0.8 mg kg-1
min-1
to 4.8 ± 1.0 mg kg-1
min-1
(Fig. 2B) rather than to an increase in glucose uptake (Fig. 2C).
Therefore, these data show that direct administration of glucagon into the MBH lowers glucose
production in rats in vivo independent of changes in circulating glucagon or insulin levels.
Previous studies have localized glucagon binding sites to multiple brain regions including
the hippocampus, pituitary and hypothalamus [90] and glucagon receptor mRNA has been
detected in the brain of the mouse [147] and rat [88;89]. We first confirmed the presence of the
G-protein coupled receptor protein in the MBH (Fig. 3). Immunohistochemistry staining further
confirmed the presence of the glucagon receptor in mediobasal hypothalamic regions, adjacent to
the third ventricle (Fig. 4). To examine whether glucagon is signaling through its receptor in the
MBH to lower glucose production, we tested the ability of glucagon to regulate glucose
homeostasis when the glucagon receptor was blocked via pre-infusion of a glucagon mAb or a
34
GR-antagonist (Fig. 5A). To our knowledge, no study has directly tested the ability of this
blocking antibody to neutralize glucagon signaling in vivo, although it has been shown that
intravenous injection of another monoclonal glucagon antibody abolished the hyperglycemic
effect of glucagon injection in normal rats when it was given at a dose approximately 4000-fold
higher than the glucagon dose[110]. It also reduced BG levels in rats made diabetic by STZ
injection, as well as type-1 and type-2 diabetic rabbits[111]. Based on these observations, we
tested the ability of MBH glucagon to lower GP in the presence of increasing doses of mAb. Pre-
infusion of the antibody (0.02 µg/µL, 4000-fold higher dose than MBH glucagon dose) negated
the ability of MBH glucagon infusion to suppress glucose production (Fig. 5C), without altering
glucose uptake (Fig. 5D). Des-His1 [Glu9]glucagon amide is a pure glucagon receptor antagonist
which does not activate adenylate cyclase in rat liver membranes and has been shown to reduce
hyperglycemia in glucagon-injected rabbits and STZ rats[148]. Consistent with the results
observed with mAB, pre-infusion of the GR-antagonist (0.005 µg/µL) negated the ability of
MBH glucagon to lower GP (Fig. 5C), without affecting glucose uptake (Fig. 5D). Infusion of
mAb or GR-antagonist alone did not affect whole-body glucose metabolism (Fig. 5C,D). These
results confirm that glucagon signals through its G-protein coupled receptor in the MBH to lower
glucose production.
Next, we co-administered the PKA-specific inhibitors H-89 (12 µM) or Rp-cAMPS (20
µM) with glucagon into the MBH, to test the hypothesis that activation of the cAMP-PKA
pathway downstream of the GR is required for central glucagon to regulate hepatic glucose
production and glucose kinetics. In the presence of either inhibitor, the effects of MBH glucagon
administration on glucose infusion rate (Fig. 5B) and glucose production (Fig. 5C) were
abolished, with no difference observed on glucose uptake between treatment groups (Fig. 5D).
35
Administration of either H-89 or Rp-cAMPS alone had no effect on glucose infusion rate,
glucose production, and glucose uptake (Fig. 5B-D). We next assessed PKA activity in MBH
wedges taken from rats after the clamps. A1 peptide is phosphorylated by PKA; thus, a greater
ratio of phospho(P)-A1/A1 reflects a higher degree of PKA activation. MBH glucagon infusion
increased MBH PKA activity (ratio of P-A1/A1; Fig. 5E), and this stimulatory effect by MBH
glucagon on PKA was abrogated by pre-infusion of GR-antagonist and Rp-cAMPS (Fig. 5E).
Collectively, these findings suggest that activation of the GR-cAMP-PKA pathway is required
for hypothalamic glucagon to suppress glucose production.
Since we have established that hypothalamic PKA is required for central glucagon to
lower glucose production, it follows that activation of PKA per se should be sufficient to lower
glucose production. To test this hypothesis, we administered the PKA-specific activator Sp-
cAMPS (40 µM) into the MBH. As shown in Fig. 6C, in the pre-clamp and pre-brain treatment
period (60-90 min), the rate of basal glucose production is comparable in the MBH Sp-cAMPS
and MBH saline treated groups (12.2 ± 0.9 mg kg-1
min-1
vs 12.1 ± 0.6 mg kg-1
min-1
). However,
in the final 30 min of clamps, MBH administration of Sp-cAMPS significantly increased the GIR
compared to MBH saline-treated groups (Fig. 6B; 5.6 ± 1.4 mg kg-1
min-1
vs 1.8 ± 0. mg kg-1
min-1
). The steady-state tracer data indicated that this was due to an inhibition in glucose
production (from 12.2 ± 0.9 mg kg-1
min-1
to 5.2 ± 0.7 mg kg-1
min-1
; Fig. 6C) rather than to an
increase in glucose uptake (Fig. 6D). Importantly, the effects of Sp-cAMPS on lowering glucose
production were independent of any difference in plasma levels of glucagon or insulin (Table 1).
Thus, direct activation of PKA in the MBH by the agonist Sp-cAMPS reproduced the effect
observed with MBH glucagon infusion to lower GP.
36
Finally, in order to confirm the specificity of Sp-cAMPS, hypothalamic PKA was
inhibited in the presence of MBH PKA activation. As shown in Fig. 6C, when rats were pre-
treated with 20 µM Rp-cAMPS for 90 min, the ability of 40 µM Sp-cAMPS to lower glucose
production was impaired. This finding confirms that activation of cAMP-PKA signaling is
required for Sp-cAMPS to regulate hepatic glucose production and glucose kinetics. Activation
of PKA in hypothalamic wedges was confirmed through the use of a PepTag Non-Radioactive
Protein Kinase assay. Fig. 5E shows that MBH glucagon increases PKA activity (represented as
the ratio of phosphorylated:nonphosphorylated substrate), and that this effect is abolished when
Rp-cAMPS is co-administered. Similarly, results from the PKA activity assay confirm that the
ability of Sp-cAMPS to activate PKA is inhibited in the presence of Rp-cAMPS (Fig. 6E).
37
Figures and Tables
A)
B)
Figure 1. Schematic representation and experimental protocol for Aim 1.
A: Schematic representation of the working hypothesis: Activation of cAMP-PKA signaling in
the mediobasal hypothalamus (MBH) by glucagon or Sp-cAMPS, a PKA-specific activator,
lowers glucose production. Inhibition of this pathway via glucagon mAb, GR-antagonist or the
PKA-specific antagonists H-89 or Rp-cAMPS negates the ability of MBH glucagon or Sp-
cAMPS to lower glucose production. B) Experimental procedure and clamp protocol. Chronic
catheters were placed icv (3rd
) or into the MBH on day 0. Venous and arterial cannulations were
performed on Day 6, and the pancreatic clamp protocol was performed on Day 10. Control
animals received MBH saline infusions. SRIF, somatostatin.
38
A) B)
C)
Figure 2. Intracerebroventricular (ICV) (3rd
) and mediobasal hypothalamic (MBH)
glucagon infusion inhibits glucose production.
During the pancreatic clamps, ICV (100 ng/µL, n=3; 50 ng/µL, n=3 or; 5 pg/µL, n=3) or MBH
glucagon infusions (5 pg/µL; n=6) vs. control saline (n=5) led: (A) to an increase in glucose
infusion rate (*P < 0.05 versus saline) and (B) a decrease in glucose production (*P < 0.05
versus saline). C: Glucose uptake. Values are means + SEM.
39
Figure 3. Tissue distribution of the rat glucagon receptor protein.
Representative Western blot showing glucagon receptor protein expression in rat MBH and
paraventricular hypothalamus (PVN) as compared with rat muscle (negative control), liver and
HEK293 cell line (positive control). The molecular weights of the protein marker bands are
shown to the left of the blot.
Figure 4. Representative image of glucagon receptor immunostaining in the rat brain.
Shown is the expression of immunocytochemically detectable glucagon receptor in the medial
region of the arcuate nucleus, adjacent to the third ventricle (3V). A: Immunofluorescence
staining for glucagon receptor in the mediobasal hypothalamus. B: Nuclei are stained with 4,6-
diamidino-2-phenylindole (DAPI). C: Merged images (A and B)
40
A) B)
C) D)
E)
Figure 5. Activation of the glucagon receptor and cAMP-PKA signaling pathway is
required for MBH glucagon to lower glucose production.
A: Clamp protocol. Starting at t=0 min, MBH glucagon mAb (n=4), GR-antagonist (n=8), Rp-
cAMPS (n=5) or H-89 (n=5) were pre-infused, followed by co-infusion with MBH glucagon
(t=90 min). A separate group of rats received co-infusion of glucagon mAb (n=5), GR-antagonist
41
(n=5), Rp-cAMPS (n=5) or H-89 (n=6) with saline at t=90 min. Control rats received continuous
saline infusions (n=5) from t=0-210 min. B,C: MBH glucagon (n=6) increased the glucose
infusion rate (*P < 0.05 versus other groups) and lowered glucose production (*P < 0.05 versus
other groups), but not in the presence of glucagon mAb, GR-antagonist, Rp-cAMPS or H-89 pre-
infusion. Glucagon mAb, GR-antagonist, Rp-cAMPS or H-89 alone did not affect the glucose
infusion rate or glucose production. D: Glucose uptake. E: PKA activity was assayed in MBH
wedges isolated and frozen immediately after the clamp protocol. Rats were pre-treated with
MBH GR-antagonist, Rp-cAMPS or vehicle (VEH) from t = 0-90 min followed by co-infusion
with MBH glucagon from t= 90-210 min. Representative blot showing three samples per
treatment group, as well as negative and positive controls. Rats infused with MBH saline (n=2),
GR-antagonist (n=4) or Rp-cAMPS (n=4) alone were combined as one VEH group. Relative
level of phosphorylated:nonphosphorylated peptide substrate (index of PKA activity) was
increased (*P < 0.01 vs. other groups) following MBH glucagon (n=5) versus MBH glucagon +
GR-antagonist (n=5) or Rp-cAMPS (n=5), or MBH vehicle (n= 6) administration. Values are
means + SEM.
42
A) B)
C) D)
E)
Figure 6. Activation of the cAMP-PKA signaling pathway by Sp-cAMPS is sufficient to
lower glucose production.
A: Clamp protocol. Starting at t=0 min, MBH Rp-cAMPS (n=5) or saline (n=6) were pre-
infused, followed by co-infusion with the Sp-cAMPS (t=90 min). A group of rats received co-
infusion of Rp-cAMPS with saline (n=5) at t=90 min, and control rats received continuous MBH
saline infusions (n=5) from t=0-210min. B,C: During the clamps, MBH Sp-cAMPS (n=6)
43
increased glucose infusion rate (*P < 0.01 versus other groups) and decreased glucose production
(*P < 0.001 versus other groups). When Rp-cAMPS was pre-infused, MBH Sp-cAMPS failed to
increase glucose infusion rate and lower glucose production. Rp-cAMPS alone did not affect
glucose kinetics. D: Glucose uptake. E: PKA activity. Rats were pre-treated with MBH Rp-
cAMPS or saline from t = 0-90 min followed by co-infusion with MBH Sp-cAMPS from t= 90-
210 min. Another group received MBH saline (SAL) only from t=0-210 min. Relative level of
phosphorylated:nonphosphorylated peptide substrate (index of PKA activity) was increased (*P
< 0.05 versus other groups) following MBH Sp-cAMPS (n=5) versus Sp-cAMPS + Rp-cAMPS
(n=5) or MBH saline (n=6). Values are means + SEM.
44
Table 1: Plasma glucose, insulin and glucagon concentrations of the groups during basal
and clamp conditions
45
Aim 2: Does MBH glucagon signaling through the GR-cAMP-PKA
pathway mediate the effect of circulating glucagon to regulate
glucose production?
To address the physiological relevance of hypothalamic glucagon action and to explore
whether the hypothalamus is able to sense a rise in circulating glucagon levels to regulate
glucose homeostasis, we inhibited MBH GR-cAMP-PKA signaling via MBH infusion of GR-
antagonist and Rp-cAMPS in the presence of an i.v. glucagon injection and evaluated changes in
plasma glucose levels and glucose production (Fig. 7A). Consistent with previous
studies[116;145], i.v. injection of glucagon increased plasma glucose levels by approx. 23%
within 10 min (Fig. 7B) and this was paralleled by a 27% increase in glucose production (Fig.
7C). However, despite sustained hyperglucagonemia of approximately 1200 pg/ml (20-fold
elevation over basal), and consistent with previous findings[116], blood glucose levels and GP
returned to baseline values one hour after injection (Fig. 7B,C). Interestingly, when MBH GR-
cAMP-PKA signaling was inhibited by MBH infusion of GR-antagonist and Rp-cAMPS, i.v.
glucagon injection increased plasma glucose levels and GP to a greater extent at 10 and 20 min
compared to rats with intact MBH GR-cAMP-PKA signaling within the same timeframe (Fig.
7B,C). These findings are in line with previous studies which have suggested that circulating
glucagon can cross the blood-brain-barrier [138], and subsequently activate the hypothalamic
signaling pathway to counteract the direct stimulation on hepatic glucose production induced by
systemic administration of the hormone. To confirm that circulating glucagon can modulate
hypothalamic PKA activity under our experimental conditions, we performed a PKA activity
assay in MBH wedges isolated after the glucagon injection, during the 10 to 20 min timeframe
within which we see a difference in GP and blood glucose levels between rats with normal and
impaired MBH GR-cAMP-PKA signaling. As shown in Fig. 7D, compared to saline-injected
46
rats, i.v. glucagon injection increases MBH PKA activity, and this is partially reversed by pre-
treatment with either GR-antagonist or Rp-cAMPS. Collectively, these results demonstrate that
GP and plasma glucose levels are not affected by basal MBH glucagon signaling, but MBH
glucagon action antagonizes the stimulatory effects of circulating glucagon on glucose
production and blood glucose levels via a GR-cAMP-PKA pathway.
47
Figures and Tables
A)
B) C)
D)
Figure 7. Disruption in hypothalamic glucagon action enhances the ability of intravenous
glucagon injection to increase plasma glucose level and glucose production.
A: Rats fed regular chow were pre-treated with MBH GR-antagonist (n=6), Rp-cAMPS (n =12)
or saline (n=7) starting at t=0 min. All rats received a primed-continuous infusion of [3-3H]-
glucose from t=0-210 min and an intravenous (i.v.) glucagon injection (100 µg) at t=90 min. B:
Plasma glucose level during a non-clamp physiological setting. *P < 0.05 MBH saline versus
other groups. C: Glucose production during a non-clamp physiological setting. *P < 0.05 MBH
saline versus other groups. D: PKA activity. Glucagon injection increased the relative level of
phosphorylated:nonphosphorylated peptide substrate compared to iv saline (*P < 0.05 versus
48
other groups), and this effect was reversed by pre-treatment with MBH GR-antagonist or Rp-
cAMPS. Values are means + SEM.
49
5 Discussion
Proper control of glycemia depends upon the coordinated release of insulin and glucagon
from the pancreatic β and α cell, respectively. Abnormal function of these cells as well as an
excess of plasma glucagon:insulin levels contributes to increased hepatic glucose output and the
development of diabetes. The parallel observations that i) glucagon levels are elevated in diabetic
patients, ii) antagonizing of glucagon action improves glycemia in animal models of disease and
iii) inhibiting endogenous glucagon secretion reduces glucose output in diabetic patients [149] all
support a pathogenic role for glucagon in disease progression. However, the demonstration that
sustained hyperglucagonemia transiently stimulates glucose production and blood glucose levels
in dogs and humans underscores the need to more thoroughly evaluate glucagon action in normal
and disease states. Given that glucagon binding sites and glucagon receptor mRNA have been
identified in multiple tissues, a valid assumption is that investigating glucagon action at extra-
hepatic sites can contribute to a more thorough understanding of how this hormone regulates
blood glucose profiles. Notably, glucagon and its receptor have been identified in the
hypothalamus of animals [90;127] and glucagon-like immunoreactivity has been identified in the
human hypothalamus [127]. Since multiple hormones signal via this brain region to regulate
glucose metabolism, we sought to examine whether the hypothalamus is also a site of glucagon
action.
Circulating hormones such as insulin and leptin can cross the blood-brain barrier to enter
the MBH and trigger PI3K and JAK2-STAT3 dependent signaling pathways to regulate energy
and glucose homeostasis in rodents [12-14;31;150-152]. Similarly, resistin acts in the MBH of
rodents to regulate glucose homeostasis, but in contrast to the aforementioned, acts to increase
50
hepatic glucose production[16;48] via a pathway likely involving modulation of NPY
expression. It is worth noting that all of these hormones exert their gluco-regulatory effects at the
level of the arcuate nucleus – Norsted E. et al [153] have previously proposed the lack of a
blood-brain barrier in this region, which permits the passage of these blood-borne substances into
the MBH where they can contribute to the regulation of energy and glucose homeostasis. Given
that glucagon behaves similarly as resistin in the circulation to stimulate hepatic glucose
production [154], we initially hypothesized that glucagon triggers a signaling cascade within the
MBH to increase glucose production. In direct contrast to our expectation, the results highlighted
in this thesis indicate that glucagon action in the MBH inhibits glucose production.
First, in an attempt to extend previous in vitro findings [90;136], we investigated whether
the components of the glucagon signaling cascade known to mediate the hormone’s peripheral
effects are present in the MBH. Indeed, we demonstrated that glucagon receptor and PKA are
expressed in the hypothalamus and that MBH PKA is activated by the glucagon-GR complex in
vivo. Importantly, the stimulation of glucagon receptor and PKA by glucagon in the MBH
inhibited glucose production, and this occurred independent of changes in circulating insulin and
glucagon levels during a pancreatic clamp in normal rats. In a non-clamp setting, direct
inhibition of GR-cAMP-PKA signaling in the MBH enhanced the acute stimulatory effect of
intravenous injection of glucagon on glucose production and plasma glucose levels. These
findings illustrate that glucagon signaling within the MBH counteracts the direct effect of
circulating glucagon on the liver to maintain glucose homeostasis.
Unfortunately, the results of this thesis do not account for the transiency observed with
peripheral glucagon injection, since the change in GP and plasma glucose levels displayed the
51
same transient effect in rats with either intact or disrupted MBH glucagon signaling. That is, they
both returned to near-normal values despite a sustained elevation in plasma glucagon levels. It is
worth noting that the elevation in blood glucagon levels (approx. 20-fold increase over basal at
60 min) observed with this injection is substantially higher than would be obtained under normal
physiological conditions such as fasting[155] or strenuous exercise[156], or even under
pathological conditions such as diabetic hyperglucagonemia[7]. Nevertheless, we do show that
MBH glucagon action antagonizes the acute stimulatory effect of a rise in circulating glucagon
levels on GP and plasma glucose levels, since the elevation in both variables was greater when
MBH GR-cAMP-PKA signaling was inhibited. It is possible that GR desensitization and/or
cAMP depletion in both the liver and MBH are mediating this effect, although this remains to be
demonstrated.
Ours is not the first study to implicate CNS glucagon in the regulation of peripheral
glucose levels. The finding that both glucagon and its receptor are expressed in the rodent brain,
and the intriguing observation that alloxan-induced diabetes in dogs increases central glucagon-
like immunoreactivity [128] collectively suggest that brain glucagon plays a role in the
regulation of glucose homeostasis. In fact, it has been shown that lateral ventricle injection of
100 ng of glucagon in Wistar rats, a dose much higher than our MBH dose (3.3 pg total dose),
produces a sustained rise in plasma glucose levels which is prevented by pharmacological
autonomic nerve blockade or adrenalectomy [157]. Another group localized the hyperglycemic
effect of the same amount of central glucagon to the paraventricular hypothalamus[158]. It is
worth noting that the same group reported that administration of insulin into the PVN dose-
dependently increases blood glucose levels, with an increase of 49.6% observed after PVN
injection of 0.1 mU insulin[159]. This finding is in contrast with the GP-lowering effect of
52
insulin infusion (2 μU) in the MBH [26], and the more general role of insulin as a hormone that
maintains homeostasis by limiting exogenous and endogenous energy availability. Therefore,
numerous studies support the anorexigenic and GP-lowering effects of MBH insulin action in
animals and humans, and have demonstrated a role for endogenous MBH insulin action in the
regulation of peripheral glucose homeostasis. Nevertheless, we cannot exclude the possibility
that glucose homeostasis is differentially regulated within differing CNS sites. In particular, the
hyperglycemia observed upon insulin injection into the PVN may have been a consequence of
corticosteroid release from the adrenal cortex downstream hypothalamic–pituitary–adrenal
(HPA) axis activation, since this is controlled by the neuroendocrine division of the PVN [160].
Finally, since the arcuate region of the MBH is in close proximity to the median eminence, a
circumventricular organ with an incomplete BBB, it is therefore accessible to blood-borne
substances such as insulin and glucagon, making it a more likely target of endogenously derived
peptides [153;161].
The same argument as cited above comes into play when evaluating the results of a study
which found that ICV administration of glucagon in fed mice raises blood glucose levels when
sympathetic nerve transmission is intact [162]. In this study, the authors show that the
hyperglycemic effect of glucagon is maintained even in mice pre-treated with somatostatin, and
conclude that the effect is due to direct neurally-mediated stimulation of hepatic glucose
production, and not indirectly through mobilization of pancreatic glucagon, which is inhibited by
somatostatin treatment [162]. Unfortunately, the authors did not specify the exact
neuroanatomical site of glucagon injection.
53
In our study, glucagon was administered into the MBH, and we show that glucagon
signaling in this region lowers glucose production, suggesting that, like insulin, MBH glucagon
action limits endogenous nutrient availability to maintain energy homeostasis. In fact, central
glucagon action can also limit exogenous nutrient availability, as ICV (3rd
) injection of glucagon
inhibits food intake in rodents [146;163;164], and similar effects have been observed in chicks
[165], sheep [166] and following i.v. infusion in humans [167], although the underlying
mechanisms remain largely unknown. However, it has been demonstrated that perifornicular
hypothalamic administration of the PKA activator Sp-cAMPS increases hypothalamic PKA
activity and reduces schedule- and NPY-induced feeding in rats [168], highlighting the
possibility that a cAMP-PKA pathway is also responsible for the glucagon-induced suppression
of feeding. Of note, central insulin administration also inhibits fasting-induced NPY gene
expression [169]. This finding is particularly relevant to the results of this thesis, as it supports
our claim that in the CNS, glucagon activity does not oppose the metabolic effects of insulin as it
does in the periphery [7], and the two hormones actually act in concert to maintain energy
homeostasis by limiting exogenous (ie. by suppressing the orexigenic effect of NPY[168;169])
and, as we show here, endogenous nutrient availability (ie. by lowering GP[26]).
Further substantiating the claim that activation of glucagon activity regulates energy and
nutrient homeostasis are the results of two independent studies which reported that dual
activation of glucagon and GLP-1 receptors led to greater weight loss reduction than selective
GLP-1 receptor activation in rodents with diet-induced obesity [170;171]. Interestingly, one
study reports that dual activation of glucagon and GLP-1 receptors, using a modified
oxyntomodulin (OXM) analog which is equipotent at the GCGR and GLP1Rs as native OXM,
improves glucose tolerance to the same extent as selective GLP-1 receptor activation despite
54
inducing increased expression of hepatic gluconeogenic genes [171]. In a related study, Day et al
[170] demonstrated that dual glucagon and GLP-1 receptor activation for seven days reduced
blood glucose levels to a greater extent compared to selective GLP-1 receptor activation, which
again is unexpected given the stimulatory effect on hepatic gluconeogenesis and glycogenolysis
by circulating glucagon. When taken together with the findings of this thesis and the potential of
native oxyntomodulin to cross the blood-brain-barrier and control appetite by modulating arcuate
GLP1R activity [172], the surprising results described above may be attributed to an activation
of central GR signaling that counteracts the stimulation of hepatic gluconeogensis by the
peripheral action of the agonist, although this clearly remains to be demonstrated. This is a
particularly important issue to address given that dual agonists are already being developed by
certain pharmaceutical companies as therapies for diabetic patients [173].
Prior to the development of dual agonists, other groups have tested the hypothesis that
interfering with glucagon action is beneficial to mediate the hyperglycemic insult of diabetes,
either by pharmacologic targeting of glucagon or its receptor [108;110;111;113;148;174], or by
gene targeting to knock out the receptor completely. What is particularly intriguing about the
previous studies is that despite the complete ablation of GR expression in the knockout mice
[100], the animals display improvements in blood glucose regulation without the consequence of
severe hypoglycemia expected to occur in the absence of glucagon stimulation of hepatic glucose
production. In part, this may be attributed to the effects of redundant counterregulatory signals
mediated by catecholamines or corticosteroids [116;175;176], but the results of our study
highlight another possibility. Since the glucagon receptor gene is also expressed in the mouse
brain [147], it is plausible that knockout of the central, as well as the peripheral glucagon
55
receptors would ablate the GP-lowering effect of MBH GR-cAMP-PKA signaling, and therefore
maintain glucose levels within the normal range.
The results of the current study do not offer an explanation as to how the glucagon signal
generated in the MBH is conveyed to peripheral target organs, namely the liver. However, the
ATP-sensitive potassium (KATP) channel represents a potential mediator, since this channel is a
primary target whereby CNS insulin [13;26] and perhaps even GLP-1 [15] regulate glucose
homeostasis. The likelihood that this channel is involved in glucagon-mediated suppression of
GP is further supported by the observation that PKA phosphorylates and activates the
Kir6.2/Sur1 subunits of the channel in vitro [177], and that activation of the KATP channels in the
MBH of rodents inhibits glucose production in vivo[26] and may play a role in suppression of
GP in humans in vivo[52]. Furthermore, whether the lowering of GP is due to a suppression of
glycogenolysis or gluconeogenesis, or both, is not currently known. Since MBH insulin and
leptin suppress GP by regulating the expression of the gluconeogenic enzymes G6Pase and
PEPCK[14;27], these represent potential hepatic targets of MBH glucagon action as well,
although this remains to be confirmed.
56
6 Conclusion
Extraordinary progress in recent years has improved our understanding of the vital role
that hypothalamic hormone signaling cascades exert upon glucose homeostasis. Insulin and
leptin, as well as the more recently implicated enteric peptide GLP-1, have received the most
attention as important regulators of the central pathways involved in whole-body glucose
metabolism. In this study, we unveil a novel role of glucagon action in the MBH that, in contrast
to our initial hypothesis and to the hormone’s systemic effect on glucose homeostasis, actually
lowers hepatic glucose production. We have shown that under euglycemic clamp conditions and
in the absence of changes in circulating gluco-regulatory hormones, direct administration of
glucagon into the MBH activates the GR-cAMP-PKA pathway to lower glucose production.
Additionally, in a non-clamp setting, inhibition of the GR-cAMP-PKA signaling pathway
enhanced the stimulatory effect of an i.v. glucagon injection on plasma glucose levels and
glucose production, supporting the hypothesis that glucagon signaling in the MBH counteracts
the direct effect of circulating glucagon on hepatic glucose production (Fig. 8). Collectively,
these results introduce the potential to develop novel pharmacological molecules targeting the
brain to alter glucagon action and consequently lead to an improvement in regulation of blood
glucose levels in diabetes and obesity.
57
Figure 8. Glucagon signaling via the GR-cAMP-PKA pathway in the MBH inhibits glucose
production and counteracts the direct hepatic stimulatory effect of circulating glucagon.
Glucagon signals through its hepatic receptor to increase glucose production (GP) by regulating
glycogenolysis and gluconeogenesis. Furthermore, the hormone can cross the blood-brain barrier
(BBB) to activate a similar pathway in the mediobasal hypothalamus (MBH). Activation of the
central GR-cAMP-PKA pathway opposes the stimulatory effect of circulating glucagon on
glucose output, and actually suppresses GP.
58
7 Future Directions
This MSc thesis provides novel insight into the role of glucagon and hypothalamic signaling on
the regulation of peripheral glucose homeostasis, but important questions remain to be addressed
with future experiments.
First, to more fully investigate and to confirm the role of central glucagon receptors in the
regulation of glucose homeostasis, experiments can be performed in glucagon receptor knockout
mice which lack functional glucagon receptors. Previous studies have demonstrated that infusion
of hormones such as insulin, resistin and GLP-1 into the lateral ventricle of mice regulates
hepatic glucose production[48;178;179], thus validating this brain region as a site whereby
hormones can regulate peripheral glucose homeostasis. Can lateral ventricular infusion of
glucagon in wild-type mice also regulate GP? In GR knockout mice? A significant advantage
would be to ascertain the relative contribution of glucagon signaling in the hypothalamus vs the
liver in the regulation of peripheral glucose fluxes. Since brain- and liver-specific glucagon
receptor knockout mice do not exist, this aim could be partly addressed by performing clamp
studies in mice with a targeted reduction in the hepatic glucagon receptor, mediated via systemic
administration of a glucagon receptor antisense oligonucleotides[112].
Second, given the high degree of homology between the glucagon and GLP1R [180], and
the fact that both glucagon and GLP1R signaling involves activation of adenylate cyclase, cAMP
and PKA via the stimulatory G protein Gs, another important question is whether the observed
GP-lowering effect of MBH glucagon administration is mediated, in part, via activation of
central GLP1R signaling. Given that i) glucagon is capable of activating GLP1Rs[181], ii)
GLP1Rs are present in the hypothalamus [41] and iii) activation of these has been demonstrated
59
to lower GP [15], we can test this possibility by pre-infusing the specific GLP1R antagonist
Exendin 9-39 into the MBH, followed by MBH glucagon administration during the pancreatic
clamp. Alternatively, this issue could be addressed using GLP1R-/-
mice, which do not express
functional GLP1Rs in the hypothalamus [182].
Third, it is well established that a brain-liver circuit exists which regulates the
suppression of glucose output in response to central hormone sensing. For example, the ability of
insulin and leptin to suppress GP is blunted in rats with selective hepatic branch
vagotomy[26;37]. To determine whether intact descending efferent fibers to the liver are
required for MBH glucagon to suppress GP, the ability of MBH glucagon to lower GP will be
evaluated in vagotomized rats.
Fourth, it is already well established that excess nutrient intake contributes to central
insulin and leptin resistance, and thus to the pathogenesis of hyperglycemia and type 2 diabetes.
In fact, even one day of high-fat feeding is sufficient to induce hypothalamic insulin resistance in
rodents [17], so perhaps it is also the case that exposure to nutrient excess impairs hypothalamic
glucagon signal transduction and contributes to the hyperglycemic phenotype of diabetes and
obesity. Furthermore, HFD-feeding upregulates hypothalamic expression of the inflammatory
cytokines TNFα and IL-1β in rats and consequently impairs the ability of insulin to lower GP via
the brain-liver axis[183;184]; since rhTNFα blunts glucagon-induced gene regulation
downstream of cAMP in cultured rat hepatocytes[185], the possibility remains that HFD-feeding
impairs MBH glucagon action by upregulating hypothalamic cytokine expression. To address
this, we can place rats on a HFD and examine whether MBH glucagon administration can still
regulate GP and glucose homeostasis.
60
Fifth, the results of the current study do not identify the specific neuronal population
involved in glucose regulation by glucagon. Since the POMC and AgRP/NPY expressing
neurons of the ARC have been implicated to play a role in the regulation of glucose homeostasis
by other gluco-regulatory hormones [186], it is likely that they mediate the effect of glucagon in
the MBH.
61
8 References
1. Unwin N, Guariguata L, Whiting D et al. Complementary approaches to estimation of the global burden of diabetes. Lancet 2012; 379:1487-8.
2. Diagnosis and classification of diabetes mellitus. Diabetes Care 2012; 35 Suppl 1:S64-S71.
3. Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature 2010; 464:1293-300.
4. Saltiel AR. New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell 2001;104:517-29.
5. Raskin P, Unger RH. Hyperglucagonemia and its suppression. Importance in the metabolic control of diabetes. N Engl J Med 1978;299:433-6.
6. Unger RH. Glucagon physiology and pathophysiology. N Engl J Med 1971;285:443-9.
7. Jiang G, Zhang BB. Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab 2003;284:E671-E678.
8. Bonora E, Muggeo M. Postprandial blood glucose as a risk factor for cardiovascular disease in Type II diabetes: the epidemiological evidence. Diabetologia 2001;44:2107-14.
9. Klein R. Hyperglycemia and microvascular and macrovascular disease in diabetes. Diabetes Care 1995;18:258-68.
10. Standards of medical care in diabetes--2012. Diabetes Care 2012;35 Suppl 1:S11-S63.
11. Bernard C. Leçons de physiologie expérimentale appliquée à la médecine, faites au collége de France. Leçons de physiologie expérimentale appliquée à la médecine, faites au collége de France. 1855. Paris, J.-B. Baillière et fils
12. Lam CK, Chari M, Lam TK. CNS regulation of glucose homeostasis. Physiology (Bethesda)
2009;24:159-70.
13. Obici S, Zhang BB, Karkanias G et al. Hypothalamic insulin signaling is required for inhibition of glucose production. Nat Med 2002;8:1376-82.
14. Buettner C, Pocai A, Muse ED et al. Critical role of STAT3 in leptin's metabolic actions. Cell Metab 2006;4:49-60.
15. Sandoval DA, Bagnol D, Woods SC et al. Arcuate glucagon-like peptide 1 receptors regulate glucose homeostasis but not food intake. Diabetes 2008;57:2046-54.
62
16. Muse ED, Lam TK, Scherer PE et al. Hypothalamic resistin induces hepatic insulin resistance. J Clin Invest 2007;117:1670-8.
17. Ono H, Pocai A, Wang Y et al. Activation of hypothalamic S6 kinase mediates diet-induced hepatic insulin resistance in rats. J Clin Invest 2008;118:2959-68.
18. Gelling RW, Morton GJ, Morrison CD et al. Insulin action in the brain contributes to glucose lowering during insulin treatment of diabetes. Cell Metab 2006;3:67-73.
19. Gerozissis K. Brain insulin and feeding: a bi-directional communication. Eur J Pharmacol 2004;490:59-70.
20. Bruning JC, Gautam D, Burks DJ et al. Role of brain insulin receptor in control of body weight and reproduction. Science 2000;289:2122-5.
21. Banks WA, Jaspan JB, Kastin AJ. Selective, physiological transport of insulin across the blood-brain barrier: novel demonstration by species-specific radioimmunoassays. Peptides 1997;18:1257-62.
22. Baura GD, Foster DM, Porte D, Jr. et al. Saturable transport of insulin from plasma into the central nervous system of dogs in vivo. A mechanism for regulated insulin delivery to the brain. J Clin Invest 1993;92:1824-30.
23. van HM, Posner BI, Kopriwa BM et al. Insulin-binding sites in the rat brain: in vivo localization to the circumventricular organs by quantitative radioautography. Endocrinology 1979;105:666-73.
24. van HM, Posner BI, Kopriwa BM et al. Insulin binding sites localized to nerve terminals in rat median eminence and arcuate nucleus. Science 1980;207:1081-3.
25. Obici S, Feng Z, Karkanias G et al. Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats. Nat Neurosci 2002;5:566-72.
26. Pocai A, Lam TK, Gutierrez-Juarez R et al. Hypothalamic K(ATP) channels control hepatic glucose production. Nature 2005;434:1026-31.
27. Inoue H, Ogawa W, Asakawa A et al. Role of hepatic STAT3 in brain-insulin action on hepatic glucose production. Cell Metab 2006;3:267-75.
28. Banks WA, Kastin AJ, Huang W et al. Leptin enters the brain by a saturable system independent of insulin. Peptides 1996;17:305-11.
29. Ahima RS, Flier JS. Leptin. Annu Rev Physiol 2000;62:413-37.
30. Schwartz MW, Woods SC, Porte D Jr. et al. Central nervous system control of food intake. Nature 2000;404:661-71.
31. Morton GJ, Schwartz MW. Leptin and the central nervous system control of glucose metabolism. Physiol Rev 2011;91:389-411.
63
32. Pocai A, Morgan K, Buettner C et al. Central leptin acutely reverses diet-induced hepatic insulin resistance. Diabetes 2005;54:3182-9.
33. Lin CY, Higginbotham DA, Judd RL et al. Central leptin increases insulin sensitivity in streptozotocin-induced diabetic rats. Am J Physiol Endocrinol Metab 2002;282:E1084-E1091.
34. Coppari R, Ichinose M, Lee CE et al. The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity. Cell Metab 2005;1:63-72.
35. Kievit P, Howard JK, Badman MK et al. Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMC-expressing cells. Cell Metab 2006;4:123-32.
36. Morton GJ, Gelling RW, Niswender KD et al. Leptin regulates insulin sensitivity via phosphatidylinositol-3-OH kinase signaling in mediobasal hypothalamic neurons. Cell Metab 2005;2:411-20.
37. German J, Kim F, Schwartz GJ et al. Hypothalamic leptin signaling regulates hepatic insulin sensitivity via a neurocircuit involving the vagus nerve. Endocrinology 2009;150:4502-11.
38. Varndell IM, Bishop AE, Sikri KL et al. Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry. J Histochem Cytochem 1985;33:1080-6.
39. Drucker DJ. Minireview: the glucagon-like peptides. Endocrinology 2001;142:521-7.
40. Knauf C, Cani PD, Perrin C et al. Brain glucagon-like peptide-1 increases insulin secretion and muscle insulin resistance to favor hepatic glycogen storage. J Clin Invest 2005;115:3554-63.
41. Alvarez E, Martinez MD, Roncero I et al. The expression of GLP-1 receptor mRNA and protein allows the effect of GLP-1 on glucose metabolism in the human hypothalamus and brainstem. J Neurochem 2005;92:798-806.
42. Merchenthaler I, Lane M, Shughrue P. Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system. J Comp Neurol 1999;403:261-80.
43. Kim KH, Lee K, Moon YS et al. A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. J Biol Chem 2001;276:11252-6.
44. Patel L, Buckels AC, Kinghorn IJ et al. Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators. Biochem Biophys Res Commun 2003;300:472-6.
45. Rangwala SM, Rich AS, Rhoades B et al. Abnormal glucose homeostasis due to chronic hyperresistinemia. Diabetes 2004;53:1937-41.
46. Banerjee RR, Rangwala SM, Shapiro JS et al. Regulation of fasted blood glucose by resistin. Science 2004;303:1195-8.
64
47. Qi Y, Nie Z, Lee YS et al. Loss of resistin improves glucose homeostasis in leptin deficiency. Diabetes 2006;55:3083-90.
48. Singhal NS, Lazar MA, Ahima RS. Central resistin induces hepatic insulin resistance via neuropeptide Y. J Neurosci 2007;27:12924-32.
49. Ramnanan CJ, Edgerton DS, Cherrington AD. Evidence against a physiologic role for acute changes in CNS insulin action in the rapid regulation of hepatic glucose production. Cell Metab 2012;15:656-64.
50. Ramnanan CJ, Saraswathi V, Smith MS et al. Brain insulin action augments hepatic glycogen synthesis without suppressing glucose production or gluconeogenesis in dogs. J Clin Invest 2011;121:3713-23.
51. Edgerton DS, Lautz M, Scott M et al. Insulin's direct effects on the liver dominate the control of hepatic glucose production. J Clin Invest 2006;116:521-7.
52. Kishore P, Boucai L, Zhang K et al. Activation of K(ATP) channels suppresses glucose production in humans. J Clin Invest 2011;121:4916-20.
53. Benedict C, Kern W, Schultes B et al. Differential sensitivity of men and women to anorexigenic and memory-improving effects of intranasal insulin. J Clin Endocrinol Metab 2008;93:1339-44.
54. Hallschmid M, Higgs S, Thienel M et al. Postprandial administration of intranasal insulin intensifies satiety and reduces intake of palatable snacks in women. Diabetes 2012;61:782-9.
55. Filippi BM, Mighiu PI, Lam TK. Is insulin action in the brain clinically relevant? Diabetes 2012;61:773-5.
56. Kimball CP, Murlin JR. Aqueous extracts of pancreas. III. Some precipitation reactions of insulin. J.Biol.Chem. 58[1], 337-46. 1923.
57. Lefebvre PJ. Glucagon and its family revisited. Diabetes Care 1995;18:715-30.
58. Lopez LC, Frazier ML, Su CJ et al. Mammalian pancreatic preproglucagon contains three glucagon-related peptides. Proc Natl Acad Sci U S A 1983;80:5485-9.
59. Unger RH, Ketterer H, Eisentraut AM. Distribution of immunoassayable glucagon in gastrointestinal tissues. Metabolism 1966;15:865-7.
60. Holst JJ, Bersani M, Johnsen AH et al. Proglucagon processing in porcine and human pancreas. J Biol Chem 1994;269:18827-33.
61. Foa PP. The Chicago Medical School Quaterly 14, 145. 1953. 62. Unger RH, Eisentraut AM, McCall MS et al. Measurements of endogenous glucagon in plasma and
the influence of blood glucose concentration upon its secretion. J Clin Invest 1962;41:682-9.
65
63. Unger RH, Eisentraut AM, Madison LL. The effects of total starvation upon the levels of circulating glucagon and insulin in man. J Clin Invest 1963;42:1031-9.
64. Unger RH, Eisentraut AM. Studies of the physiologic role of glucagon. Diabetes 1964;13:563-8.
65. Cherrington AD, Chiasson JL, Liljenquist JE et al. The role of insulin and glucagon in the regulation of basal glucose production in the postabsorptive dog. J Clin Invest 1976;58:1407-18.
66. Liljenquist JE, Mueller GL, Cherrington AD et al. Evidence for an important role of glucagon in the regulation of hepatic glucose production in normal man. J Clin Invest 1977;59:369-74.
67. Taborsky GJ Jr., Mundinger TO. Minireview: The role of the autonomic nervous system in mediating the glucagon response to hypoglycemia. Endocrinology 2012;153:1055-62.
68. Yamaguchi N. Sympathoadrenal system in neuroendocrine control of glucose: mechanisms involved in the liver, pancreas, and adrenal gland under hemorrhagic and hypoglycemic stress. Can J Physiol Pharmacol 1992;70:167-206.
69. Frizzell RT, Jones EM, Davis SN et al. Counterregulation during hypoglycemia is directed by widespread brain regions. Diabetes 1993;42:1253-61.
70. Borg WP, During MJ, Sherwin RS et al. Ventromedial hypothalamic lesions in rats suppress counterregulatory responses to hypoglycemia. J Clin Invest 1994;93:1677-82.
71. Heimberg H, De VA, Moens K et al. The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells. Proc Natl Acad Sci U S A 1996;93:7036-41.
72. Suzuki M, Fujikura K, Kotake K et al. Immuno-localization of sulphonylurea receptor 1 in rat pancreas. Diabetologia 1999;42:1204-11.
73. Quesada I, Tuduri E, Ripoll C et al. Physiology of the pancreatic alpha-cell and glucagon secretion: role in glucose homeostasis and diabetes. J Endocrinol 2008;199:5-19.
74. Dunning BE, Foley JE, Ahren B. Alpha cell function in health and disease: influence of glucagon-like peptide-1. Diabetologia 2005;48:1700-13.
75. Barazzone P, Gorden P, Carpentier JL et al. Binding, internalization, and lysosomal association of 125I-glucagon in isolated rat hepatocytes. A quantitative electron microscope autoradiographic study. J Clin Invest 1980;66:1081-93.
76. Foa PP. Glucagon: An Incomplete and Biased Review with Selected References. American Zoologist 13[3], 613-23. 1973.
77. Trebbien R, Klarskov L, Olesen M et al. Neutral endopeptidase 24.11 is important for the
degradation of both endogenous and exogenous glucagon in anesthetized pigs. Am J Physiol Endocrinol Metab 2004;287:E431-E438.
66
78. Mayo KE, Miller LJ, Bataille D et al. International Union of Pharmacology. XXXV. The glucagon receptor family. Pharmacol Rev 2003;55:167-94.
79. MacNeil DJ, Occi JL, Hey PJ et al. Cloning and expression of a human glucagon receptor. Biochem Biophys Res Commun 1994;198:328-34.
80. Gustavson SM, Chu CA, Nishizawa M et al. Glucagon's actions are modified by the combination of epinephrine and gluconeogenic precursor infusion. Am J Physiol Endocrinol Metab 2003;285:E534-E544.
81. Liang H, Ward WF. PGC-1alpha: a key regulator of energy metabolism. Adv Physiol Educ 2006;30:145-51.
82. Wahren J, Ekberg K. Splanchnic regulation of glucose production. Annu Rev Nutr 2007;27:329-45.
83. Gerich JE. Control of glycaemia. Baillieres Clin Endocrinol Metab 1993;7:551-86.
84. Wakelam MJ, Murphy GJ, Hruby VJ et al. Activation of two signal-transduction systems in hepatocytes by glucagon. Nature 1986;323:68-71.
85. Hansen LH, Gromada J, Bouchelouche P et al. Glucagon-mediated Ca2+ signaling in BHK cells expressing cloned human glucagon receptors. Am J Physiol 1998;274:C1552-C1562.
86. Staddon JM, Hansford RG. Evidence indicating that the glucagon-induced increase in cytoplasmic free Ca2+ concentration in hepatocytes is mediated by an increase in cyclic AMP concentration. Eur J Biochem 1989;179:47-52.
87. Wang Y, Li G, Goode J et al. Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes. Nature 2012;485:128-32.
88. Svoboda M, Tastenoy M, Vertongen P et al. Relative quantitative analysis of glucagon receptor mRNA in rat tissues. Mol Cell Endocrinol 1994;105:131-7.
89. Dunphy JL, Taylor RG, Fuller PJ. Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression. Mol Cell Endocrinol 1998;141:179-86.
90. Hoosein NM, Gurd RS. Identification of glucagon receptors in rat brain. Proc Natl Acad Sci U S A 1984;81:4368-72.
91. Hagen JH. Effect of glucagon on the metabolism of adipose tissue. J Biol Chem 1961;236:1023-7.
92. Perea A, Clemente F, Martinell J et al. Physiological effect of glucagon in human isolated adipocytes. Horm Metab Res 1995;27:372-5.
93. Carlson MG, Snead WL, Campbell PJ. Regulation of free fatty acid metabolism by glucagon. J Clin Endocrinol Metab 1993;77:11-5.
67
94. Xiao C, Pavlic M, Szeto L et al. Effects of acute hyperglucagonemia on hepatic and intestinal lipoprotein production and clearance in healthy humans. Diabetes 2011;60:383-90.
95. Jensen MD, Heiling VJ, Miles JM. Effects of glucagon on free fatty acid metabolism in humans. J Clin Endocrinol Metab 1991;72:308-15.
96. Gravholt CH, Moller N, Jensen MD et al. Physiological levels of glucagon do not influence lipolysis in abdominal adipose tissue as assessed by microdialysis. J Clin Endocrinol Metab 2001;86:2085-9.
97. Kawai K, Yokota C, Ohashi S et al. Evidence that glucagon stimulates insulin secretion through its own receptor in rats. Diabetologia 1995;38:274-6.
98. Gelling RW, Du XQ, Dichmann DS et al. Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice. Proc Natl Acad Sci U S A 2003;100:1438-43.
99. Sorensen H, Winzell MS, Brand CL et al. Glucagon receptor knockout mice display increased insulin sensitivity and impaired beta-cell function. Diabetes 2006;55:3463-9.
100. Parker JC, Andrews KM, Allen MR et al. Glycemic control in mice with targeted disruption of the glucagon receptor gene. Biochem Biophys Res Commun 2002;290:839-43.
101. Muller WA, Faloona GR, Aguilar-Parada E et al. Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion. N Engl J Med 1970;283:109-15.
102. Unger RH. Role of glucagon in the pathogenesis of diabetes: the status of the controversy. Metabolism 1978;27:1691-709.
103. Aguilar-Parada E, Eisentraut AM, Unger RH. Pancreatic glucagon secretion in normal and diabetic subjects. Am J Med Sci 1969;257:415-9.
104. Muller WA, Faloona GR, Unger RH. The effect of experimental insulin deficiency on glucagon secretion. J Clin Invest 1971;50:1992-9.
105. Raskin P, Aydin I, Yamamoto T et al. Abnormal alpha cell function in human diabetes: the response to oral protein. Am J Med 1978;64:988-97.
106. Aronoff SL, Bennett PH, Unger RH. Immunoreactive glucagon (IRG) responses to intravenous glucose in prediabetes and diabetes among Pima Indians and normal Caucasians. J Clin Endocrinol Metab 1977;44:968-72.
107. Unger RH, Orci L. The essential role of glucagon in the pathogenesis of diabetes mellitus. Lancet 1975;1:14-6.
108. Johnson DG, Goebel CU, Hruby VJ et al. Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist. Science 1982;215:1115-6.
68
109. Djuric SW, Grihalde N, Lin CW. Glucagon receptor antagonists for the treatment of type II diabetes: current prospects. Curr Opin Investig Drugs 2002;3:1617-23.
110. Brand CL, Rolin B, Jorgensen PN et al. Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic rats. Diabetologia 1994;37:985-93.
111. Brand CL, Jorgensen PN, Svendsen I et al. Evidence for a major role for glucagon in regulation of plasma glucose in conscious, nondiabetic, and alloxan-induced diabetic rabbits. Diabetes 1996;45:1076-83.
112. Liang Y, Osborne MC, Monia BP et al. Reduction in glucagon receptor expression by an antisense oligonucleotide ameliorates diabetic syndrome in db/db mice. Diabetes 2004;53:410-7.
113. Sloop KW, Cao JX, Siesky AM et al. Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors. J Clin Invest 2004;113:1571-81.
114. Dallas-Yang Q, Shen X, Strowski M et al. Hepatic glucagon receptor binding and glucose-lowering in vivo by peptidyl and non-peptidyl glucagon receptor antagonists. Eur J Pharmacol 2004;501:225-34.
115. Petersen KF, Sullivan JT. Effects of a novel glucagon receptor antagonist (Bay 27-9955) on glucagon-stimulated glucose production in humans. Diabetologia 2001;44:2018-24.
116. Eigler N, Sacca L, Sherwin RS. Synergistic interactions of physiologic increments of glucagon, epinephrine, and cortisol in the dog: a model for stress-induced hyperglycemia. J Clin Invest 1979;63:114-23.
117. Bomboy JD, Jr., Lewis SB, Lacy WW et al. Transient stimulatory effect of sustained hyperglucagonemia on splanchnic glucose production in normal and diabetic man. Diabetes 1977;26:177-4.
118. Felig P, Wahren J, Hendler R. Influence of physiologic hyperglucagonemia on basal and insulin-inhibited splanchnic glucose output in normal man. J Clin Invest 1976;58:761-5.
119. Insel PA, Liljenquist JE, Tobin JD et al. Insulin control of glucose metabolism in man: a new kinetic analysis. J Clin Invest 1975;55:1057-66.
120. Plas C, Nunez J. Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon. J Biol Chem 1975;250:5304-11.
121. Komjati M, Breitenecker F, Bratusch-Marrain P et al. Contribution by the glycogen pool and adenosine 3',5'-monophosphate release to the evanescent effect of glucagon on hepatic glucose production in vitro. Endocrinology 1985;116:978-86.
122. Liljenquist JE, Bomboy JD, Lewis SB et al. Effect of glucagon on net splanchnic cyclic AMP production in normal and diabetic men. J Clin Invest 1974;53:198-204.
69
123. Ramnanan CJ, Edgerton DS, Kraft G et al. Physiologic action of glucagon on liver glucose metabolism. Diabetes Obes Metab 2011;13 Suppl 1:118-25.
124. Nishimura E, Abrahamsen N, Hansen LH et al. Regulation of glucagon receptor expression. Acta Physiol Scand 1996;157:329-32.
125. Drucker DJ, Asa S. Glucagon gene expression in vertebrate brain. J Biol Chem 1988;263:13475-8.
126. Lui EY, Asa SL, Drucker DJ et al. Glucagon and related peptides in fetal rat hypothalamus in vivo and in vitro. Endocrinology 1990;126:110-7.
127. Tominaga M, Ebitani I, Marubashi S et al. Species difference of glucagon-like materials in the brain. Life Sci 1981;29:1577-81.
128. Tominaga M, Marubashi S, Kamimura K,YK et al. Effects of starvation and alloxan treatment of glucagon-like materials in the dog brain. 7th Asia and Oceania Congress if Endocrinology Suppl. 1982.
129. Tominaga M Msktyksh. Glucagon-like materials in the cerebrospinal fluid. Biomedical Research
3[3], 261-4. 1982. 130. Graner JL, Abraira C. Glucagon in the cerebrospinal fluid. N Engl J Med 1985;312:994-5.
131. Dorn A, Rinne A, Bernstein HG et al. The glucagon/glucagon-like immunoreactivities in neurons of the human brain. Exp Clin Endocrinol 1983;81:24-32.
132. Dorn A, Rinne A, Bernstein HG et al. Immunoreactive glucagon in neurons of various parts of the human brain. Demonstration by immunofluorescence technique. Acta Histochem 1981;69:243-7.
133. Zhou A, Webb G, Zhu X et al. Proteolytic processing in the secretory pathway. J Biol Chem 1999;274:20745-8.
134. Sasaki H, Ebitani I, Tominaga M et al. Glucagon-like substance in the canine brain. Endocrinol Jpn 1980;27 Suppl 1:135-40.
135. Schorderet M, Sovilla JY, Magistretti PJ. VIP-and glucagon-induced formation of cyclic AMP in intact retinae in vitro. Eur J Pharmacol 1981;71:131-3.
136. Wetsel WC, Eraly SA, Whyte DB et al. Regulation of gonadotropin-releasing hormone by protein kinase-A and -C in immortalized hypothalamic neurons. Endocrinology 1993;132:2360-70.
137. Jiang G, Zhang BB. Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab 2003;284:E671-E678.
138. Banks WA, Kastin AJ. Peptides and the blood-brain barrier: lipophilicity as a predictor of permeability. Brain Res Bull 1985;15:287-92.
70
139. Lam TK, Gutierrez-Juarez R, Pocai A et al. Regulation of blood glucose by hypothalamic pyruvate metabolism. Science 2005;309:943-7.
140. Yang CS, Lam CK, Chari M et al. Hypothalamic AMP-activated protein kinase regulates glucose production. Diabetes 2010;59:2435-43.
141. Lam CK, Chari M, Su BB et al. Activation of N-methyl-D-aspartate (NMDA) receptors in the dorsal vagal complex lowers glucose production. J Biol Chem 2010;285:21913-21.
142. Lam CK, Chari M, Rutter GA et al. Hypothalamic nutrient sensing activates a forebrain-hindbrain neuronal circuit to regulate glucose production in vivo. Diabetes 2011;60:107-13.
143. Dostmann WR. (RP)-cAMPS inhibits the cAMP-dependent protein kinase by blocking the cAMP-induced conformational transition. FEBS Lett 1995;375:231-4.
144. Murray AJ. Pharmacological PKA inhibition: all may not be what it seems. Sci Signal 2008;1:re4.
145. Young AA, Cooper GJ, Carlo P et al. Response to intravenous injections of amylin and glucagon in fasted, fed, and hypoglycemic rats. Am J Physiol 1993;264:E943-E950.
146. Inokuchi A, Oomura Y, Nishimura H. Effect of intracerebroventricularly infused glucagon on feeding behavior. Physiol Behav 1984;33:397-400.
147. Campos RV, Lee YC, Drucker DJ. Divergent tissue-specific and developmental expression of receptors for glucagon and glucagon-like peptide-1 in the mouse. Endocrinology 1994;134:2156-64.
148. Unson CG, Gurzenda EM, Merrifield RB. Biological activities of des-His1[Glu9]glucagon amide, a glucagon antagonist. Peptides 1989;10:1171-7.
149. Baron AD, Schaeffer L, Shragg P et al. Role of hyperglucagonemia in maintenance of increased rates of hepatic glucose output in type II diabetics. Diabetes 1987;36:274-83.
150. Morton GJ, Cummings DE, Baskin DG et al. Central nervous system control of food intake and body weight. Nature 2006;443:289-95.
151. Coll AP, Farooqi IS, O'Rahilly S. The hormonal control of food intake. Cell 2007;129:251-62.
152. Kleinridders A, Konner AC, Bruning JC. CNS-targets in control of energy and glucose homeostasis. Curr Opin Pharmacol 2009;9:794-804.
153. Norsted E, Gomuc B, Meister B. Protein components of the blood-brain barrier (BBB) in the mediobasal hypothalamus. J Chem Neuroanat 2008;36:107-21.
154. Rajala MW, Obici S, Scherer PE et al. Adipose-derived resistin and gut-derived resistin-like molecule-beta selectively impair insulin action on glucose production. J Clin Invest 2003;111:225-30.
71
155. Ruiter M, La Fleur SE, van HC et al. The daily rhythm in plasma glucagon concentrations in the rat is modulated by the biological clock and by feeding behavior. Diabetes 2003;52:1709-15.
156. Vranic M, Kawamori R, Pek S et al. The essentiality of insulin and the role of glucagon in regulating glucose utilization and production during strenuous exercise in dogs. J Clin Invest 1976;57:245-55.
157. Marubashi S, Tominaga M, Katagiri T et al. Hyperglycaemic effect of glucagon administered intracerebroventricularly in the rat. Acta Endocrinol (Copenh) 1985;108:6-10.
158. Atrens DM, Menendez JA. Glucagon and the paraventricular hypothalamus: modulation of energy balance. Brain Res 1993;630:245-51.
159. Menendez JA, Atrens DM. Insulin and the paraventricular hypothalamus: modulation of energy balance. Brain Res 1991;555:193-201.
160. Herman JP, Cullinan WE. Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis. Trends Neurosci 1997;20:78-84.
161. Peruzzo B, Pastor FE, Blazquez JL et al. A second look at the barriers of the medial basal hypothalamus. Exp Brain Res 2000;132:10-26.
162. Amir S. Central glucagon-induced hyperglycemia is mediated by combined activation of the adrenal medulla and sympathetic nerve endings. Physiol Behav 1986;37:563-6.
163. Langhans W, Duss M, Scharrer E. Decreased feeding and supraphysiological plasma levels of glucagon after glucagon injection in rats. Physiol Behav 1987;41:31-5.
164. Geary N, Smith GP. Pancreatic glucagon and postprandial satiety in the rat. Physiol Behav 1982;28:313-22.
165. Honda K, Kamisoyama H, Saito N et al. Central administration of glucagon suppresses food intake in chicks. Neurosci Lett 2007;416:198-201.
166. Kurose Y, Kamisoyama H, Honda K et al. Effects of central administration of glucagon on feed intake and endocrine responses in sheep. Anim Sci J 2009;80:686-90.
167. Geary N, Kissileff HR, Pi-Sunyer FX et al. Individual, but not simultaneous, glucagon and cholecystokinin infusions inhibit feeding in men. Am J Physiol 1992;262:R975-R980.
168. Sheriff S, Chance WT, Iqbal S et al. Hypothalamic administration of cAMP agonist/PKA activator inhibits both schedule feeding and NPY-induced feeding in rats. Peptides 2003;24:245-54.
169. Schwartz MW, Sipols AJ, Marks JL et al. Inhibition of hypothalamic neuropeptide Y gene expression by insulin. Endocrinology 1992;130:3608-16.
170. Day JW, Ottaway N, Patterson JT et al. A new glucagon and GLP-1 co-agonist eliminates obesity in rodents. Nat Chem Biol 2009;5:749-57.
72
171. Pocai A, Carrington PE, Adams JR et al. Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009;58:2258-66.
172. Woods SC, D'Alessio DA. Central control of body weight and appetite. J Clin Endocrinol Metab 2008;93:S37-S50.
173. Cartwright H. Boehringer Ingelheim licenses Zealand Pharma's drug candidates for type 2 diabetes and obesity. PharmaDeals Review 2011[6], 62. 2012.
174. Van Tine BA, Azizeh BY, Trivedi D et al. Low level cyclic adenosine 3',5'-monophosphate
accumulation analysis of [des-His1, des- Phe6, Glu9] glucagon-NH2 identifies glucagon antagonists from weak partial agonists/antagonists. Endocrinology 1996;137:3316-22.
175. Sacca L. Role of counterregulatory hormones in the regulation of hepatic glucose metabolism. Diabetes Metab Rev 1987;3:207-29.
176. Vranic M, Gauthier C, Bilinski D et al. Catecholamine responses and their interactions with other glucoregulatory hormones. Am J Physiol 1984;247:E145-E156.
177. Beguin P, Nagashima K, Nishimura M et al. PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation. EMBO J 1999;18:4722-32.
178. van den Hoek AM, Voshol PJ, Karnekamp BN et al. Intracerebroventricular neuropeptide Y infusion precludes inhibition of glucose and VLDL production by insulin. Diabetes 2004;53:2529-34.
179. Cabou C, Vachoux C, Campistron G et al. Brain GLP-1 signaling regulates femoral artery blood flow and insulin sensitivity through hypothalamic PKC-delta. Diabetes 2011;60:2245-56.
180. Holst JJ. Glucagon, glucagon-like peptide-1 and their receptors: an introduction. Acta Physiol Scand 1996;157:309-15.
181. Moens K, Flamez D, Van SC et al. Dual glucagon recognition by pancreatic beta-cells via glucagon and glucagon-like peptide 1 receptors. Diabetes 1998;47:66-72.
182. Scrocchi LA, Brown TJ, MaClusky N et al. Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene. Nat Med 1996;2:1254-8.
183. Milanski M, Arruda AP, Coope A et al. Inhibition of hypothalamic inflammation reverses diet-induced insulin resistance in the liver. Diabetes 2012;61:1455-62.
184. Mighiu PI, Filippi,BM, Lam,TK. Linking inflammation to the brain-liver axis. Diabetes 2012;61:1350-2.
185. Christ B. Inhibition of glucagon-signaling and downstream actions by interleukin 1beta and tumor necrosis factor alpha in cultured primary rat hepatocytes. Horm Metab Res 2008;40:18-23.
186. Belgardt BF, Okamura T, Bruning JC. Hormone and glucose signalling in POMC and AgRP neurons. J Physiol 2009;587:5305-14.
