1 CHAPTER I INTRODUCTION Glucagon is a 29-amino acid peptide hormone secreted by the alpha cells of the pancreas, which was originally identified as a hyperglycemic hormone in pancreatic extracts in 1923 by Kimball and Murlin. The regulation of glucagon secretion is complex; it involves the effects of several metabolic substrates, hormones and neurotransmitters. The main physiological role of glucagon is the maintenance of hepatic glucose production during fasting, hypoglycemia, exercise and infection/trauma. The goal of this dissertation is to describe the acute in vivo regulation of hepatic glucose production by glucagon during insulin-induced hypoglycemia in the overnight- fasted conscious dog. This chapter will provide an introduction to the following: 1) Counterregulatory response to hypoglycemia, 2) Glucagon action and signaling, 3) Insulin action and signaling, 4) Insulin and glucagon interaction. Counterregulatory response to hypoglycemia Under physiological conditions glucose is metabolized by all tissues throughout the body, but is a critical metabolic fuel for the nervous system. The reason for this is that the brain can not synthesize glucose or store more than a small amount of glycogen; it relies mainly on the continuous uptake of glucose from the circulation to supply its metabolic needs (1). As a result, hypoglycemia is a dangerous condition that can lead to brain damage, coma and even death. Therefore, maintenance of the plasma glucose concentration is critical for survival and it is normally tightly regulated by various control
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CHAPTER I
INTRODUCTION
Glucagon is a 29-amino acid peptide hormone secreted by the alpha cells of the
pancreas, which was originally identified as a hyperglycemic hormone in pancreatic
extracts in 1923 by Kimball and Murlin. The regulation of glucagon secretion is
complex; it involves the effects of several metabolic substrates, hormones and
neurotransmitters. The main physiological role of glucagon is the maintenance of hepatic
glucose production during fasting, hypoglycemia, exercise and infection/trauma.
The goal of this dissertation is to describe the acute in vivo regulation of hepatic
glucose production by glucagon during insulin-induced hypoglycemia in the overnight-
fasted conscious dog. This chapter will provide an introduction to the following: 1)
Counterregulatory response to hypoglycemia, 2) Glucagon action and signaling, 3)
Insulin action and signaling, 4) Insulin and glucagon interaction.
Counterregulatory response to hypoglycemia Under physiological conditions glucose is metabolized by all tissues throughout
the body, but is a critical metabolic fuel for the nervous system. The reason for this is
that the brain can not synthesize glucose or store more than a small amount of glycogen;
it relies mainly on the continuous uptake of glucose from the circulation to supply its
metabolic needs (1). As a result, hypoglycemia is a dangerous condition that can lead to
brain damage, coma and even death. Therefore, maintenance of the plasma glucose
concentration is critical for survival and it is normally tightly regulated by various control
2
mechanisms. These counterregulatory signals are so efficient that hypoglycemia is a rare
clinical condition in normal individuals. Clinical conditions most commonly associated
with hypoglycemia are: ethanol-consumption, certain drugs, insulin-secreting islet cell
tumors, pituitary or adrenal insufficiency, hepatic and renal failure, sepsis and ectopic
production of an insulin-like growth factor (2). However, hypoglycemia is the most
frequent complication experienced by insulin-requiring individuals with diabetes. It is
also the principal factor limiting the glycemic control in people with type 1 diabetes and
late stage type 2 diabetes (1).
For many years investigators performed studies to understand hypoglycemia by
using an acute intravenous bolus of insulin, which resulted in a rapid increase in insulin
concentration followed by a short term hypoglycemia. Garber et al. (3) conducted studies
in healthy humans using insulin injections (0.15 U/kg). The insulin injection resulted in a
rapid fall in glucose production (~30%) followed by a doubling of glucose production by
40 minutes due to an increase in glucagon secretion. The increase in glucose production
was attributable mainly to glucagon’s effects on glycogenolysis (3). This model doesn’t
represent a common clinical condition seen in patients with Type 1 Diabetes in which
hypoglycemia develops gradually and can be present for several hours (4). To
understand better the mechanisms involved in the increase in glucose production during
prolonged hypoglycemia, Lecavalier et al. (5) and Caprio et al. (6) in the human and
Frizzell et al. (7) in the dog, studied the contribution of glycogenolysis and
gluconeogenesis to the regulation of hepatic production during prolonged hypoglycemia.
Frizzell infused a high dose of insulin (5mU/kg/min) intraportally for 3 hours into
overnight fasted conscious dogs. Glucose production fell initially and then doubled by 60
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minutes. They found that glycogenolysis accounted for ~79% of glucose production
during the first hour of hypoglycemia and gluconeogenesis played a major role by the
third hour of hypoglycemia (~68%). Studies in humans have also concluded that
glycogenolysis accounted for the increase in glucose production in the first hour
following establishment of hypoglycemia while gluconeogenesis played a mayor role
during the subsequent hours of prolonged hypoglycemia (5; 6).
Defense against hypoglycemia
The fall in arterial plasma glucose is sensed in widespread regions of the brain,
portal vein, carotid body and pancreas. When arterial plasma glucose decreases (~80-85
mg/dl) in response to an increase in insulin, there is a reduction of insulin secretion and
enhancement of hepatic glucose production (8; 9). It has been suggested that
glucokinase-mediated sensing in the pancreatic beta cells is involved in this response
(10). As the arterial plasma glucose concentration decreases to ~65-70 mg/dl the
secretion of glucagon and epinephrine increases (8; 9). Under physiological conditions
this response can restore euglycemia without the development of hypoglycemic
symptoms. Glucagon secretion from α cells is regulated by many factors, including
plasma insulin levels, blood substrate concentrations and the autonomic nervous system
(10). Under the control of the CNS, epinephrine is secreted from the adrenal medullae
during hypoglycemia (1). In patients with type 1 diabetes the counterregulatory
mechanisms mentioned above are impaired (8). When plasma glucose decreases to ~60
mg/dl it results in the secretion of norepinephrine, cortisol and growth hormone and to
the development of symptoms (8; 9). Like the epinephrine response, increases in
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circulating levels of norepinephrine, cortisol and growth hormone are mediated through
the CNS (1).
The glycemic thresholds for the counterregulatory responses described above
apply to insulin induced hypoglycemia. Most studies in vivo used insulin as a
pharmacologic agent to induce hypoglycemia. A study conducted by Flattem et al. (11)
used a glycogen phosphorylase inhibitor to induce hypoglycemia in conscious dogs.
They found that during non insulin-induced hypoglycemia the glycemic threshold for the
increase in glucagon secretion was ~94 mg/dl, which is much higher than the threshold
during insulin-induced hypoglycemia. Therefore, there seems to be a difference in the
glycemic threshold required for the counterregulatory response of the α cell when
hypoglycemia is accompanied by hyperinsulinemia. The mechanism for this increase in
the sensitivity of the α cell to insulin remains unclear but recent studies have shown that
perhaps is attributable to a loss of the fall in intraislet insulin that normally triggers an
increase in glucagon secretion as glucose levels fall (12). For the purpose of our studies
we are going to focus on insulin-induced hypoglycemia.
Hormone Action
The counterregulatory response to hypoglycemia involves the release of glucagon,
epinephrine, norepinephrine, cortisol and growth hormone (1; 4; 13). Studies in humans
and dogs have demonstrated the primary role of glucagon during insulin induced
hypoglycemia (14-17). Studies performed by Gerich et al. (15) in normal and
adrenalectomized humans showed the primary role of glucagon and the secondary role of
epinephrine during insulin-induced hypoglycemia. Dobbins et al. (18) performed studies
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in overnight fasted conscious dogs to characterize the role of the hormone during insulin-
induced hypoglycemia. A 6 fold rise in glucagon (Δ140 pg/ml) significantly increased
glucose production (Δ 4.5 mg/kg/min) in the presence of hypoglycemia despite an arterial
insulin level that was increased 20 fold (Δ328 µU/ml). The effect of the increment of
glucagon on hepatic glucose production was primarily due to a rapid, time dependent
effect on glycogenolysis and a modest, prolonged effect on gluconeogenesis.
Epinephrine, like glucagon, has been shown to increase production in a rapid,
time-and dose-dependent manner in response to a fall in glucose (19; 20). Studies
performed by Cherrington et al. (21) in overnight fasted conscious dogs showed that an
acute physiological rise in plasma epinephrine was associated with a initial increase in
glucose production due to a glycogenolytic response followed later by a gluconeogenic
response. The effect of epinephrine on glycogenolysis wanes with time like glucagon
(21; 22). This similarity may be explained by the fact that epinephrine exerts its effect by
binding to the β-adrenergic receptors on the liver (23; 24). In addition, Chu et al. (25)
demonstrated that effects of epinephrine on glycogenolysis are the result of a direct effect
of the hormone on the liver. On the other hand, the effects of epinephrine on
gluconeogenesis are the result of its action on peripheral tissues (22; 26-28), specifically
an increase in muscle glycogenolysis and adipose tissue lipolysis.
Norepinephrine is also involved in the counterregulatory response. Circulating
norepinephrine reflects release of the catecholamine from the adrenal medullae but more
importantly its release from sympathetic postganglionic neurons (1; 29). The ability of
norepinephrine to restrain a fall in plasma glucose, while not as potent as epinephrine’s,
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involves a stimulatory effect on gluconeogenesis which results from a glycogenolytic
effect in muscle and a lipolytic effect in fat (30-32).
Cortisol and growth hormone are referred as “slow acting” hormones because
their effects are seen a few hours after their increase in plasma. Boyle et al. (33)
conducted studies in humans that provide evidence that cortisol and growth hormone are
involved in the defense against hypoglycemia but they are not critical for recovery from a
low blood sugar. Additionally, the authors suggested that the roles of these hormones in
the defense of hypoglycemia are permissive rather than direct. Further, De Feo et al.
(34) have reported that growth hormone effects were evident after 3 hours of insulin-
induced hypoglycemia at which time it enhanced glucose production and decreased
glucose utilization. Goldstein et al. (35; 36) also showed that acute increases in cortisol
have minimal effects on hepatic glucose production whereas chronic infusion of cortisol
(5 days) increased glucose production by maintaining substrate availability to support
gluconeogenesis and by maintaining hepatic glycogen availability. It also had effects in
peripheral tissues where it decreased glucose utilization in muscle and enhanced lipolysis
in adipose tissue.
Autoregulation and other factors
It has been suggested that the liver is capable of adjusting its glucose output in
response to changes in the plasma glucose concentration per se, independent of changes
in the hormones that normally control glucose homeostasis (37; 38). In vitro studies in
perfused rat liver have reported that hepatic glucose production can vary inversely with
the perfusate glucose levels (39). In vivo studies have shown that the hormonal changes
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are not the only means by which counterrregulation is brought about. Frizzell et al. (40)
performed studies in overnight-fasted conscious dogs to assess the role of the
counterregulatory hormones per se in the response to insulin-induced hypoglycemia. In
one group the counterregulatory hormone response was simulated in the presence of
euglycemia to separate the effects of hypoglycemia per se from those associated with the
counterregulatory hormones. The other groups included a control for the previous group
(insulin + euglycemia) and a group in which insulin was infused alone. They concluded
that the counterregulatory hormones alone accounted for 50% of the response, while the
other 50% resulted from some aspect of hypoglycemia per se. In addition, Bolli et al.
(41) demonstrated the contribution of hepatic autoregulation to hypoglycemic
counterregulation in humans. They assessed the role of hepatic autoregulation during
moderate (~50mg/dl) and severe (~30 mg/dl) hypoglycemia by using somatostatin and
pharmacologic agents to inhibit the secretion of glucagon, growth hormone, cortisol and
to block the action of epinephrine and norepinephrine. Glucagon and growth hormone
were fixed at basal levels while insulin was infused. During moderate hypoglycemia
insulin infusion resulted in complete inhibition of glucose production whereas during
severe hypoglycemia there was an initial suppression of glucose production followed by
an increased in glucose production two times higher than the moderate hypoglycemic
group. Therefore, the authors concluded that hepatic autoregulation is a component of
the counterregulatory response during severe hypoglycemia. Further, Connolly et al. (42)
conducted studies in adrenalectomized overnight-fasted conscious dogs to control for
epinephrine and cortisol release and used somatostatin to clamp insulin and glucagon.
During the euglycemic-hyperinsulinemic control period the liver displayed net hepatic
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glucose uptake, but as the plasma glucose levels dropped there was a stepwise increase in
net hepatic glucose output despite the absence of counterregulatory hormones. The
authors, therefore, concluded that non-hormonal mechanisms including autoregulation
and direct neural input to the liver can stimulate glucose production in response to
insulin-induced hypoglycemia.
The brain is known to be responsible for most of the rise in the counterregulatory
hormones during hypoglycemia but it also affects glucose production directly (43). It
has been reported that stimulation of the VMH results in an increase in hepatic glucose
production (44) and that electrical stimulation of hepatic nerves results in hyperglycemia
(45). Furthermore, Borg et al. (46) have reported that the VMH stimulates the
counterregulatory response during hypoglycemia in rats. In addition, Connolly et al.
(47) conducted studies to determine if the increase in glucose production seen in the
absence of the counterregulatory hormones is either initiated by liver (autoregulation) or
the brain (neural input) in overnight-fasted conscious dogs. They observed that in the
absence of counterregulatory hormones, hypoglycemia sensed at the liver results in an
increase of hepatic glucose production whereas hypoglycemia sensed at the brain
stimulates the lipolytic and ketogenic responses. Taken together, these studies clearly
indicate that non hormonal mechanisms (autoregulation and neural input to the liver) also
play a role in the metabolic response to hypoglycemia.
Although much about the counterregulatory response during hypoglycemia is
known a controversy still remains regarding the site at which the change in the plasma
glucose level is sensed. The brain and the hepato-portal region have both been postulated
to contain glucose sensing neurons that are responsible for triggering the
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counterregulatory response. Biggers et al. (43) performed studies in which euglycemia
was maintained in the brain but hypoglycemia was allowed to occur elsewhere. Under
these circumstances, the plasma glucagon levels decreased, the sympathetic nervous
system response to hypoglycemia was blunted and a rise in glucose production was
attenuated by 75%. On the other hand, Donovan et al. (48) has shown that when
glucose was infused into the hepato-portal region during insulin-induced hypoglycemia
there was an inhibition of the sympathetic response to hypoglycemia. Therefore, the
authors suggested that glucose sensing neurons in the hepato-portal region are important
in the response of the sympathetic nervous system to hypoglycemia, supporting the view
that hypoglycemic sensing occurs at peripheral sites. On the other hand, Jackson et al.
(49; 50) have conducted vagal blockade and liver denervation studies resulting in no
prevention of the counterregulatory response to hypoglycemia. More recently, Saberi et
al. (51) conducted studies in chronically cannulated rats that underwent afferent ablation
of spinal afferent nerve endings in the portal vein (PV) or portal and superior mesenteric
veins (PMV) nerve endings to determine if the rate by which glucose falls determines the
primacy of the hypoglycemic sensing. Their data showed that when PV and PMV were
ablated using capsaicin, the sympathetic response was suppressed when hypoglycemia
developed slowly (~80 min). However, when hypoglycemia was reached quickly (~ 20
min) the responses were minimally decreased (15-30 %). Therefore, it seems that low
blood glucose levels are sensed by central and peripheral mechanisms and the
predominance between them is rate sensitive. It should be noted however that glucagon
secretion is solely under the control of central rather than peripheral glucose sensors.
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Glucagon action and signaling The main physiological role of glucagon is to stimulate hepatic glucose
production. Studies in humans and dogs have established the dose response relationship
between plasma glucagon levels and hepatic glucose production (52; 53). Stevenson at
al. (20) showed using overnight fasted conscious dogs that a selective rise in glucagon (2-
,4-,8-, and 12-fold) for 3 hours resulted in a sensitive dose-dependent increase in glucose
production. In addition, studies in our laboratory have demonstrated that in the presence
of basal insulin a fourfold rise of the hormone produces a half-maximal activation of
hepatic glucose production (~Δ 5.0 mg/kg/min) despite mild hyperglycemia (52).
Additionally, a small change (<10 pg/ml) in arterial plasma glucagon results in an
increase in glucose production of ~ 0.5 mg/kg/min (52; 53). Not only are glucagon’s
effects on hepatic glucose production potent, they also have been shown to be rapid since
it takes ~4.5 minutes for the hormone to half-maximally activate the liver (54). All
together glucagon is a potent and rapid stimulator of hepatic glucose production, and a
small change of the hormone can result in significant changes in hepatic glucose output.
Glucose production by the liver is the result of either glycogen breakdown
(glycogenolysis) or de novo synthesis of glucose from gluconeogenic precursors
(gluconeogenesis). In the dog and the human, the effect of an increment in glucagon on
hepatic glucose production has been shown to be primarily due to a rapid, time dependent
stimulation of glycogenolysis and a modest more prolonged effect on gluconeogenesis
(55-57). The time dependence of glucagon’s effect on glycogenolysis is in part related to
the progressive inhibitory effect of hyperglycemia that occurs in response to the hormone
and in part to factors endogenous to the liver that limit the action of the hormone (58; 59).
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Glucagon’s effects on gluconeogenesis are more modest. Studies have shown that the
hormone regulates amino acid transport into liver via the transcriptional expression of the
hepatic Na+-dependent amino acid transport system A (60). In addition the hormone is
known to stimulate transcription of gluconeogenic enzymes like PEPCK and G-6-Pase
(61-65). It also enhances the phosphorylation of pyruvate kinase, and
phosphofructokinase and decreases intracellular levels of fructose-2, 6-P2, resulting in
inhibition of glycolysis and stimulation of gluconeogenesis (61). The reason for the
limited effect of glucagon on gluconeogenesis, despite its hepatic effects, lies in its
inability to increase gluconeogenic substrate mobilization from the peripheral tissues
such as muscle and fat (66). In fact there are no glucagon receptors in muscle and there
are very few in adipose tissue (67). As one would predict from this observation
glucagon does not have effects on glucose utilization by adipose tissue or skeletal muscle
(20; 68; 69). Likewise it has minimal effects on lipolysis and protein metabolism.
Glucagon exerts its effects by binding to the glucagon receptor (Figure 1.1). The
glucagon receptor belongs to the superfamily of heptahelical transmembrane G protein-
coupled receptors, which is divided into subfamilies based on amino acid sequence. A
large number of G proteins have been identified: Gs, Gi and Gq and subsets of these
proteins. Each G protein consists of three subunits, α, β and γ (70-74). The binding of
glucagon to the receptor results in conformational changes of the latter, leading to
subsequent activation of the coupled G proteins. Upon G protein-coupled receptor
activation, guanosine diphosphate (GDP) is exchanged for guanosine triphosphate (GTP),
which dissociates the G protein complex into 2 units: the Gα and Gβγ subunits. These
subunits in turn activate or inhibit enzymes. Activation of Gq results in the activation of
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phospholipase C (PLC) which causes the production of inositol 1,4,5-triphosphate and
subsequent release of intracellular calcium (70; 75). The extent to which this pathway
contributes to glucose production remains unclear. There are in fact inconsistencies in
the data; some investigators have found that in the presence of a physiological increment
of glucagon there is an increase in intracellular calcium (76), while others have found that
calcium only increases in response to a supra-physiological increment in glucagon (77).
A study performed by Yamatani et al. (78) showed that glucagon increased glucose
production mainly through the cAMP pathway and that Ca2+ dependency was only
observed when the cAMP pathway was inhibited and when supra-physiological levels of
glucagon were present (78).
On the other hand, activation of Gs leads to the activation of adenyl cyclase, and
elevation of cAMP (61; 67; 70; 79). The rise in cAMP causes the activation of c-AMP-
dependent protein kinase or PKA (80), leading to the phosphorylation of a number of
cellular proteins involved in glycogenolysis, gluconeogenesis, glycolysis and glycogen
synthesis (67; 70; 79).
Glucagon stimulates glycogenolysis through the activation of PKA. PKA
catalyzes the phosphorylation of a single serine residue in each subunit of glycogen
phosphorylase. The phosphorylation of the serine-14 residue leads to major changes in
the catalytic and physical properties of the enzyme (81). This in turn increases glycogen
breakdown and net hepatic glucose output. Another effect of glucagon is inhibition of
glycogenesis. Glucagon controls glycogenesis by inducing the phosphorylation and
inactivation of glycogen synthase. Studies have shown that the enzyme is subject to
multi-site phosphorylation, some of which results in the inactivation of the enzyme.
13
Recent studies have suggested that PKA activation by cAMP leads to the
phosphorylation of cAMP response element-binding protein (CREB). PKA
phosphorylates CREB at serine 133 leading to its activation (82). CREB is a
transcription factor that induces the expression of key genes involved in the
gluconeogenic pathway such as PEPCK and G-6-Pase (83). PGC-1 (Peroxisome
proliferator-activated receptor- coactivator) is a transcriptional target of CREB and its
expression is triggered by elevated cAMP levels (84). Studies performed by Yoon et al.
(85) showed that overexpression of PGC-1 in liver increased glucose production and the
transcription of genes encoding gluconeogenic enzymes. In addition Heizig et al. (86)
provided evidence that the metabolic effects of cAMP in the liver may be mediated
through PGC-1. Furthermore studies have shown that the nuclear transcription factor
hepatocyte nuclear factor-4 (HNF-4) acts together with PGC-1 to increase the
transcription of PEPCK (85). Transcription factors function through the docking of
specific coactivitors or corepressors proteins. Recently Koo et al. (87) identified the
transcriptional regulator TORC2 (Transducer of regulated CREB activity 2) as an
important component of the gluconeogenic gene regulation (87; 88). Furthermore,
glucagon has been shown to activate glucose-6-phosphatase activity (89). Hornbuckle et
al. (90) have shown that glucagon increased the G-6-Pase activity by selectively
stimulating the transcription of the G-6-Pase catalytic subunit but not the G-6-Pase
transporter and they found that the effect is cAMP dependent (90).
In addition glucagon via cAMP and PKA enhances the phosphorylation of
pyruvate kinase and phosphofructokinase and decreases intracellular levels of fructose-2,
6-P2, resulting in the inhibition of glycolysis (61; 91). Fructose-1, 6-P2ase catalyzes the
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hydrolysis of the C-1 phosphate in fructose 1, 6-P2 into fructose 6-P. Fructose-1, 6-P2ase
is allosterically inhibited by fructose 2, 6-P2. The levels of fructose 2, 6-P2 are regulated
by the hepatic bifunctional enzyme, 6PF-2-K/Fru-2, 6-P2ase. Studies have shown that
upon glucagon stimulation, activated PKA phosphorylates, 6PF-2-K/Fru-2, 6-P2ase in the
liver at serine-32, leading to the inhibition of the kinase and activation of the
phosphatase. This in turn reduces the intracellular levels of the fructose-2, 6-P2, thereby
relieving the inhibition of fructose-1, 6-P2ase and stimulating gluconeogenesis (91-93).
Phosphofructokinase is allosterically activated by fru-2,6-P2ase therefore the
activated PKA by reducing the levels of the biphosphate also causes the inhibition of the
phosphofructokinase (92; 94). In addition, glucagon inhibits pyruvate kinase due to the
PKA phosphorylation and it also inhibits transcription of the pyruvate kinase gene and
increases the degradation of pyruvate kinase mRNA (92; 95).
Insulin has a wide variety of physiologic effects in different tissues. Insulin
stimulates cell growth and differentiation and promotes the storage of substrates in fat,
liver and muscle by stimulating lipogenesis, glycogen synthesis and protein synthesis and
by inhibiting lipolysis, glycogenolysis, gluconeogenesis and protein breakdown (96). It
has been known for many years that increasing plasma insulin levels results in an
inhibition of glucose production. In addition, there was a dose-dependent relationship
between hepatic sinusoidal insulin levels and glucose production (52; 53).
Insulin rapidly inhibits hepatic glucose production, but it requires several hours
(~3 hours) to reach its steady state effect (97). A number of investigators have studied
the ability of insulin to inhibit glycogenolysis and gluconeogenesis. In vitro studies have
shown that insulin represses gluconeogenesis by inhibiting PEPCK and G-6-Pase gene
transcription (61; 98) More recently Hall et al. (99) found that addition of insulin to
dexamethasone-treated cells results in a rapid dissociation of the glucocorticoid receptor,
polymerase II, and other transcriptional regulators from the PEPCK and G-6-Pase gene
promoter. They suggested that insulin caused the demethylation of arginine-17 on
histone H3 of both genes, leading to the reduction in gene transcription of both genes.
On the other hand in vivo studies performed in humans and dogs have shown that the
effect of insulin in gluconeogenesis is minimal and that its main effect comes about
through an inhibition of glycogenolysis (100; 101). Edgerton et al. (101) conducted
studies in overnight fasted conscious dogs in which they used three different methods to
determine gluconeogenesis and glycogenolysis. They found that the liver glycogenolysis
is markedly sensitive to small changes in insulin whereas the gluconeogenic flux is not.
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For many years insulin was thought to decrease hepatic glucose production by a
direct interaction with its hepatic receptor. That was until in 1987 when Prager et al.
(102) suggested that the hormone can suppress glucose production through indirect
actions. In these studies carried out using insulin-resistant obese subjects insulin was
infused peripherally in the presence of euglycemia and hepatic glucose production was
suppressed by 82%. There was a decrease in endogenous insulin secretion in response to
peripheral insulin infusion such that portal insulin levels were calculated to have changed
minimally. Thus the authors concluded that indirect effects of the hormone caused the
inhibition of glucose production since the insulin level at the liver did not change
appreciably (102). This concept has subsequently been supported by others (103; 104)
but the indirect mechanisms by which insulin suppresses hepatic glucose production was
probably best demonstrated in a study performed by Sindelar et al (105). The authors
used overnight fasted conscious dogs to investigate the mechanism of a selective increase
in either peripheral or portal insulin in changing hepatic glucose production. A selective
rise of 14µU/ml in either the arterial insulin or portal insulin was associated with a
decrease in NHGO of ~ 50%. Even though the extent to which insulin inhibited hepatic
glucose production was similar in both groups, the time required for the inhibition and
the mechanism for the inhibition was markedly different. The response of the liver to a
selective increase in portal insulin (direct action) was observed at 15 minutes and it was
attributable to an inhibition in glycogenolysis. On the other hand, the response of the
liver to a selective rise in arterial insulin (indirect action) occured slowly (~ 1 hour) and
resulted from the suppression of hepatic gluconeogenic precursor uptake secondary to a
reduction in gluconeogenic amino acid flux from muscle and glycerol from adipose tissue
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and from the redirection of glycogenolytic carbon to lactate due to a decrease in NEFA
levels (105; 106). Therefore, insulin inhibits hepatic glucose production by directly
inhibiting glycogenolysis and indirectly by inhibiting net gluconeogenic flux and
lipolysis.
In addition to insulin’s indirect effects in the muscle and fat, it has been reported
that insulin can inhibit the alpha cell leading to inhibition of glucagon secretion. In
unpublished data from our laboratory a rise of insulin of ~ 20µU/ml resulted in a decrease
in glucagon to ~15pg/ml. In addition, in perfused pancreas from rats a retrograde
infusion of ~0.3mU/ml of insulin significantly inhibit glucagon secretion (107). Recent
investigations have provided some insight into the possible mechanisms by which insulin
inhibits glucagon secretion. It appears that insulin increases α-cell KATP channel activity
in PI-3K dependent manner thus resulting in hyperpolarization of the membrane and
inhibition of α-cell electrical activity and glucagon secretion (108; 109). Another
mechanism proposed recently, involves the GABA-GABAA receptor system. Insulin has
been reported to activate GABAA receptors in the α-cells through receptor translocation
via an AKT kinase-dependent pathway, leading to hyperpolarization and ultimately
inhibition of glucagon secretion (110). In any event any insulin induced decrease in
glucagon levels would reduce glucose production by the liver.
Furthermore, it has been suggested that insulin’s action in the brain may explain
part of insulin’s indirect actions in the liver. Studies performed by Davis et al. have
shown that the brain can sense circulating insulin levels (111). It is also known that the
brain provides neural drive to the liver (112). Most recently, Obici et al. (113) showed
that infusion of insulin into the third ventricle in six hour fasted conscious rats resulted in
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suppression of glucose production. They also showed that blockade of the insulin
receptor using an antisense oligonucleotide injection into the hypothalamus impaired the
ability of a rise in plasma insulin to inhibit hepatic glucose production. Thus they
concluded that hypothalamic insulin signaling could be important to the action of insulin
on the liver. On the other hand, Edgerton et al. (114) carried out a study to determine the
effect of a 4-fold rise in the head insulin on hepatic glucose production during peripheral
hyperinsulinemia and hepatic insulin deficiency in overnight fasted conscious dogs.
They found that an acute 4-fold rise of insulin in the head did not reduce hepatic glucose
production. Furthermore, they demonstrated that the direct effects of insulin on hepatic
glucose production are dominant. The different results obtained in these studies might be
explained by the differences between the animal model used (rodents and dogs) and acute
effects vs. chronic effects of insulin. The glucose production rate is much greater in the
rodents compared to the dog or human. The hepatic glucose production rate of a rat or
mouse is ~12 and 20 mg/kg/min, respectively, whereas in the dog or human it is ~2-
3mg/kg/min. . It is conceivable that this might result in the existence of higher neural
drive to the liver in the rodent than in the dog or human (115; 116). Furthermore, it is
possible that an acute increment in insulin is not able to acutely regulate hepatic glucose
production via an action on the brain whereas a chronic rise in insulin might be able to
(115; 116).
Insulin exerts its effect by binding to the insulin receptor. The insulin receptor
(IR) is a tetrameric protein that consists of two extracellular α-subunits and two
intracellular β-subunits linked together by disulfide bonds. It belongs to a subfamily of
receptor tyrosine kinases which also includes the insulin growth factor-1 receptor
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(IGF1R) and IR-related receptor (IRR) (117). Binding of insulin to the α-subunit induces
a conformational change resulting in the autophosphorylation of several tyrosine residues
present in the β-subunit (96; 118). These residues are recognized by phosphotyrosine-
binding (PTB) domains of adaptor proteins such as members of the insulin receptor
substrate family (IRS), Gab-1, Shc and Cbl. (96; 118; 119). Upon tyrosine
phosphorylation, these proteins interact with signaling molecules through their SH2
domains. This results in the activation of PI 3-Kinase and downstream PtdIns(3,4,5)P3,
ras, MAP kinase cascade, Cbl/CAP and TC10 (96). Cbl/CAP and TC 10 are involved in
stimulation of glucose uptake and GLUT4 translocation. The MAPK pathway regulates
the expression of some genes and cooperates with the PI3K pathway to control cell
growth and differentiation. The PI 3-kinase pathway is responsible for the metabolic
aspects of insulin action. For the purpose of this thesis we will focus on the PI-3K
pathway.
The metabolic effects of insulin are mediated through downstream effectors of
Phosphoinositide 3-kinase (PI3K), atypical protein kinase (aPKC) and Protein Kinase B
(PKB) or Akt. Previous studies have reported that the increase in plasma insulin that
follows a carbohydrate meal results in a decreased transcription and translation of
PEPCK in vitro (61; 120). In addition, studies have shown that insulin represses G-6-
Pase gene expression in vitro and in vivo (121; 122). Furthermore, overexpression of the
catalytic subunit of PI 3-kinase is sufficient to markedly inhibit PEPCK and G-6-Pase
gene expression (123). In addition more recently studies conducted by Dentin et al. (124)
have reported that insulin inhibits the gluconeogenic gene expression during re-feeding
by promoting the phosphorylation and degradation of TORC2, a cAMP-responsive
21
CREB coactivator. All together these effects of insulin on gluconeogenic gene
expression contribute to the reduction in gluconeogenesis seen after the ingestion of a
carbohydrate meal.
After a carbohydrate meal, insulin stimulates glycogen synthesis and inhibits
glycogen breakdown. Glycogen synthase, an enzyme that catalyzes the rate-determining
step in glycogen synthase, is regulated by insulin through changes in phosphorylation.
Insulin activates glycogen synthase by promoting its dephosphorylation via the inhibition
of GSK-3 (96). This results in the inactivation of GSK-3 and in the disinhibition of
glycogen synthase, leading to an increase in glycogen synthesis. In addition, PP1 also
reduces GSK-3 activity and inhibits glycogen phosphorylase, a key enzyme in glycogen
breakdown (96). In addition, insulin stimulates PDE3B which promotes the degradation
of cAMP in the liver. The reduction in cAMP results in decreased activation of PKA and
a subsequent decrease in glycogenolysis in the liver (91; 125-127).
In addition, PI3K is an upstream regulator of mTOR (mammalian target of
rapamysin) which is a central regulator of ribosome biogenesis, protein synthesis, cell
growth. mTOR controls the translation machinery, in response to aminos acids and
growth factors via activation of p70 ribosomal S6 Kinase and inhibition of eIF-4E
binding protein (128). Therefore, insulin effects on GSK-3, PP1, mTOR and PDE inhibit
glucose production, promote glycogen, FFA, protein and triglycerides synthesis, all
together, opposing glucagon’s action.
22
Figure 1.2. Insulin-receptor signaling pathway Insulin receptor substrate family (IRS), Gab-1, Shc and Cbl. Phosphoinositide 3-kinase (PI3K); atypical protein kinase (aPKC); Protein Kinase B (PKB) or Akt; mammalian target of rapamysin (mTOR); Protein Phosphatase 1 (PP1); Glycogen Synthase Kinase 3 (GSK-3). From reference (96).
cAMP5’-AMP PDE3
mTORcAMP5’-AMP PDE3
mTOR
23
Insulin and glucagon interaction
Insulin and glucagon are potent regulators of carbohydrate metabolism and their
interaction is usually the main determinant of gluconeogenic and glycogenolytic flux in
the liver. After an overnight fast, glucagon plays a major role in stimulating hepatic
glucose production while insulin acts as a potent inhibitor of the process. Glucagon can
be considered to provide the positive drive to the liver which allows insulin to exert its
controlling effects on glucose production.
In response to carbohydrate ingestion, insulin secretion increases whereas
glucagon secretion decreases (52; 53). These changes in hormone secretion, along with
the hyperglycemia that results from the glucose load and the portal glucose signal, inhibit
hepatic glucose production and convert the liver to net glucose consumtion (53; 129).
Insulin is an anabolic hormone that promotes storage of substrates in fat, liver and
skeletal muscle by stimulating triglyceride, glycogen and protein synthesis, and inhibiting
lipolysis, and glycogen and protein breakdown (130)
Furthermore, Steiner at al. (131) has previously examined the interaction between
insulin and glucagon in controlling glucose production using a pancreatic clamp in the
conscious dog. A constant replacement of basal amounts of insulin and glucagon did not
change glucose production. A selective four-fold rise in glucagon resulted in an
increment in glucose production of ~4.5 mg/kg/min at 30 minutes. In contrast, a
selective four-fold rise in insulin resulted in a decrement in glucose production of ~1.3
mg/kg/min at 30 minutes. When both hormones were simultaneously increased fourfold,
the decrement in glucose production at 30 minutes was only ~0.6 mg/kg/min. Therefore,
glucagon’s effect was 4.5 mg/kg/min in the presence of basal insulin despite a developing
24
hyperglycemia and only 0.7 mg/kg/min in the presence of high insulin, a reduction of
almost 85%. Consequently, insulin dominates glucagon’s action on the liver even if the
increments are equimolar (131). This was not the case in the presence of hypoglycemia
as seen in another previous study (18). A 6 fold rise in glucagon (Δ140 pg/ml)
significantly increased glucose production (Δ 4.5 mg/kg/min) in the presence of
hypoglycemia despite an arterial insulin level that was increased 20 fold (Δ328 µU/ml).
Therefore, glucagon appears to be more effective during hypoglycemia than during
Despite the fact that previous studies have suggested that the liver is more
sensitive to glucagon during hypoglycemia, a direct comparison of the effects of a
controlled rise in glucagon on glucose production in the presence of euglycemia versus
hypoglycemia has never been carried out. Therefore, the aim of this work was to
examine the interaction of a selective rise in insulin and glucagon in controlling hepatic
glucose production under euglycemic and hypoglycemic conditions.
25
CHAPTER II
MATERIALS AND METHODS
Animal Care
Studies were conducted on twenty-four 18 h fasted conscious mongrel dogs (18-
25 kg) of either sex that had been fed a standard diet of meat (Kal Kan, Vernon, CA) and
chow (Purina Lab Canine Diet No. 5006; Purina Mills, St. Louis, MO) composed of 34%
protein, 14.5% fat, 46% carbohydrate, and 5.5% fiber based on dry weight (1500
kilocalories). Water was available at all times. Only dogs that had good appetite, a
leukocyte count < 18,000/mm3, a hematocrit >35%, and normal stools were used for
studies. The animals were housed in a facility which met the American Association for
Accreditation of Laboratory Animal Care guidelines, and the protocol was approved by
the Vanderbilt University Medical Center Animal Care Committee.
Surgical Procedures
Approximately 16 days prior to the metabolic study, surgery was performed on
each dog while it was under general anesthesia. Anesthesia was induced with propofol
(given until induction) preceded by buprenorphine HCl (0.02 mg/kg, presurgery) 30 min
earlier. Anesthesia was maintained by isoflurane (1.5-2.0% with oxygen) inhalation. The
dog was placed in a supine position on a surgical table with an 8.5 mm inner diameter
(ID) endotracheal tube (Concord/Protex, Kenee, NH), and ventilated with a tidal volume
of 400 ml at 14 breaths per minute.
26
A laparotomy was performed by making a midline incision 1.5 cm caudal to the
xyphoid process through the skin, subcutaneous layers and linea alba, and extending
caudally 15-20 cm. Silastic catheters (0.03 in ID; HelixMedical, Carpintera, CA) were
placed in the following manner: A portion of the jejunum was exposed and a branch of a
jejunal vein was selected for cannulation. A small section of the vessel was exposed by
blunt dissection and ligated with 4-0 silk (Ethicon, Inc, Sommerville, NJ). A silastic
infusion catheter was inserted into the vessel through a small incision and passed
antegrade until the tip of the catheter lay approximately 1 cm proximal to the coalescence
of two jejunal veins. Another silastic catheter was inserted into a distal branch of the
splenic vein and advanced until the tip of the catheter lay 1 cm beyond the bifurcation of
the main splenic vein. The catheters were secured in place with 4-0 silk.
For blood sampling, silastic catheters (0.04 in ID) were placed into the left hepatic
vein, the hepatic portal vein and left femoral artery. The central and left lateral lobes of
the liver were retracted cephalically and caudally, respectively. The left common hepatic
vein and the left branch of the portal vein were exposed. A 14-gauge angiocath (Benton
Dickinson Vascular Access, Sandy, UT) was inserted in the left branch of the portal vein
2 cm from the central liver lobe. A silastic catheter (0.04 in ID) was inserted into the hole
created by the angiocath, advanced retrograde about 4 cm into the portal vein so that the
tip of the catheter lay 1 cm beyond the bifurcation of the main portal vein. It was then
secured with three ties of 4-0 silk through the adventitia of the vessel and around the
catheter. An angiocath was inserted into the left common hepatic vein 2 cm from its exit
from the left lateral lobe. A silastic sampling catheter was inserted into the hole and
passed antegrade 2 cm and secured into place with three ties of 4-0 silk suture.
27
For sampling of arterial blood, a catheter was inserted into the left femoral artery
following a cut-down in the left inguinal region. A 2 cm incision was made parallel to
the vessel. The femoral artery was isolated and ligated distally. A silastic catheter (0.04
in ID) was inserted and advanced 16 cm in order to place the tip of the catheter in the
abdominal aorta. It was then secured into place with 4-0 silk suture.
All catheters were filled with normal saline (Baxter Healthcare Corp, Deerfield,
IL) containing 200 U/ml heparin (Abbott Laboratories, North Chicago, IL) and knotted.
Abdominal catheters were secured to the abdominal wall and placed in a subcutaneous
pocket prior to closure of the skin. The arterial sampling catheter was also placed in a
subcutaneous pocket prior to closure of the skin.
Ultrasonic flow probes (Transonic System Inc, Ithaca, NY) were positioned
around the hepatic artery and portal vein, to determine liver blood flow during
experiments. The duodenum was retracted laterally to expose a section of the hepatic
artery and portal vein. A small section of the portal vein was exposed by blunt dissection,
taking care not to disturb the nerve bundle located on the vessel. A 6 or 8 mm ID
ultrasonic flow probe (Transonic Systems Inc, Ithaca, NY) was placed around the vessel.
A small portion of the common hepatic artery was also carefully exposed and a 3 mm ID
ultrasonic flow probe was secured around the vessel. To prevent blood from entering the
portal vein beyond the site of the flow probe, the gastroduodenal vein was isolated and
ligated. Blood that would normally flow through the gastroduodenal vein was shunted
through the caudal pancreatoduodenal vein draining the tail of the pancreas. The
ultrasonic flow probe leads were positioned in the abdominal cavity and secured with the
ends of the catheters to the abdominal wall.
28
After all abdominal surgeries, the subcutaneous layer was closed with a
continuous suture of 2-0 chromic gut (Ethicon, Inc.). The skin was closed with
horizontal mattress sutures of 3-0 Dermalon (Ethicon, Inc.). Immediately following
surgery, the dogs received an intramuscular injection of penicillin G (106 U, Procaine;
Anthony Products, Irwindale, CA) to minimize the possibility of infection. In addition,
Flunixin (Meglumine 50mg/ml; Phoenix Scientific, Inc., St. Joseph, MO) was injected
intramuscularly (1 mg/kg body weight) after wound closure for acute pain relief.
Animals awoke from surgery within 2 h, were active, and ate normally approximately 8 h
after surgery. Post-operatively, each dog also received 500 mg ampicillin (Principen;
Bristol-Myers Squibb, Princeton, NJ) orally twice a day for 3 days.
Experimental Procedure
On the day of the experiment following an 18h fast, the free ends of the catheters
and ultrasonic leads were removed from their subcutaneous pockets under local
anesthesia (2% lidocaine; Abbott Laboratories, North Chicago, IL). The contents of each
catheter were aspirated, and they were flushed with saline. Blunt needles (18 gauge;
Monoject, St. Louis, MO) were inserted into the catheter ends and stopcocks (Medex,
Inc, Hilliard, OH) were attached to prevent the backflow of blood between sampling
times.
Twenty gauge Angiocaths (Beckton Dickson) were inserted percutaneously into
the left and right cephalic veins and into a saphenous vein for the infusion of
somatostatin, tracers, dye and glucose. A continuous infusion of heparinized (1U/ml;
Abbott Laboratories, North Chicago,IL) normal saline was started via the femoral artery
29
at a rate to prevent any clotting in the line. Animals were allowed to rest quietly in a
Pavlov harness for at least 100 min before the start of the experiment.
Experimental Design
The study included four groups of animals: saline-euglycemia (SE), saline-
hypoglycemia (SH), glucagon-euglycemia (GE) and glucagon-hypoglycemia (GH). Each
experiment consisted of equilibration (-140 to -40 min), basal (-40 to 0 min) and
experimental (0 to 180 min) periods (Figure 2.1). At -140 min a priming dose of [3-3H]
glucose (33 µCi) was given, followed by a constant infusion of [3-3H] glucose
(0.35µCi/min) and indocyanine green (0.08 mg/min). The equilibration period was
followed by a control period and an experimental period which was divided into period 1
(0-60 min) and period 2 (60-180 min). In period 1, somatostatin (0.8µg/kg/min) and
intraportal insulin (5.0 mU/kg/min) were infused and glucose was monitored every five
minutes in order to maintain euglycemia using glucose infusion through the saphenous
vein as required (20% Dextrose). In period 2, the somatostatin and insulin infusions were
continued and in addition either glucagon (2.3ng/kg/min) or saline were infused
intraportally. Glucose was infused as required to bring about euglycemia (~100 mg/dl) or
hypoglycemia (~50 mg/dl).
30
Figure 2.1: Experimental Design
Po Glucagon (2.3 ng/kg/min) or Saline
Euglycemia
Hypoglycemia
Po Glucagon (2.3 ng/kg/min) or Saline
180-40 min 0 60CP P2P1
Pe Somatostatin (0.8 g/kg/min) + Po Insulin (5.0 mU/kg/min)
Pe Glucose(Euglycemia)
[3-3H]-Glucose (0.35µCi/min) + Indocyanine Green (0.08 mg/min)
SAL + EU (n=6)
GGN + EU (n=6)
SAL + HYPO (n=6)
GGN + HYPO (n=6)
Pe - Peripheral; Po - PortalCP - Control Period P1 - Period 1; P2 - Period 2
Po Glucagon (2.3 ng/kg/min) or Saline
Euglycemia
Hypoglycemia
Po Glucagon (2.3 ng/kg/min) or Saline
180-40 min 0 60CP P2P1
Pe Somatostatin (0.8 g/kg/min) + Po Insulin (5.0 mU/kg/min)
Pe Glucose(Euglycemia)
[3-3H]-Glucose (0.35µCi/min) + Indocyanine Green (0.08 mg/min)
SAL + EU (n=6)
GGN + EU (n=6)
SAL + HYPO (n=6)
GGN + HYPO (n=6)
Pe - Peripheral; Po - PortalCP - Control Period P1 - Period 1; P2 - Period 2 Pe - Peripheral; Po - PortalCP - Control Period P1 - Period 1; P2 - Period 2
31
Collection and Processing of Samples
Blood samples were drawn from the femoral artery and portal and hepatic veins at
the predetermined time points. Additionally, whenever the experimental design required
a glucose clamp, small (~0.5 ml) arterial samples were drawn every 5 min to facilitate
maintenance of the plasma glucose concentration. Before samples were taken, the
sampling catheter was cleared by withdrawing 5 ml of blood into a syringe. After
sampling, this blood was re-infused and the catheter was flushed with heparinized saline
(1 U/ml; Abbott Laboratories, North Chicago, IL). The total volume of blood withdrawn
did not exceed 20% of the animal’s blood volume, and two volumes of normal saline
(0.9% sodium chloride; Baxter Healthcare Co., Deerfield, Il) were given for each volume
of blood withdrawn. No significant decrease in hematocrit occurred throughout duration
of study.
Before the experiment started, an arterial blood sample was drawn and
centrifuged (3000 rpm for 7 min). The plasma from this blood sample was used to
prepare hormone infusates and the indocyanine green standard curve. When samples
were taken from all vessels, the arterial and portal blood samples were collected
simultaneously ~30 s before the collection of the hepatic vein samples in an attempt to
compensate for the transit time through the liver, and thus allow for the most accurate
estimates of net hepatic substrate balance (132).
Immediately following each sample collection, the blood was processed. A 20 l
aliquot of arterial whole blood was used for the immediate duplicate measurement of
hematocrit using capillary tubes (0.4 mm ID; Drummond Scientific Co., Broomall, PA).
One ml of the collected blood was placed in a tube containing 20µl of 0.2M glutathione
32
(Sigma Chemical Co.) and 1.8mg EGTA (Sigma Chemical Co.) for catecholamine
measurements. This tube was vortexed, centrifuged at 3000 rpm for 7 minutes, and the
supernatant was stored in a separate tube for later analysis. The remaining blood was
placed into tubes containing potassium ethylenediaminetetraacetate (EDTA, 1.6 mg/ml;
Sarsdedt, Newton, NC), inverted and gently mixed. One ml aliquot of whole blood was
lysed with 3 ml of 4% perchloric acid (PCA; Fisher Scientific, Fair Lawn, New Jersey),
centrifuged and the supernatant was stored for later analysis of metabolites levels (lactate,
alanine, -hydroxybutyrate and glycerol). The remainder of the whole blood was
centrifuged at 3000 rpm at 4º C to obtain plasma.
The plasma samples were used for all other measurements. Glucose
concentrations were immediately determined from four 10 l aliquots of plasma using the
glucose oxidase method with a glucose analyzer (Beckman Instruments, Fullerton, CA).
A 1 ml aliquot of plasma received 50 l of 10,000 KIU/ml Trasylol (FBA
Pharmaceuticals, New York, NY) and was stored for analysis of glucagon. Insulin, [3H]-
glucose, free fatty acids and cortisol were measured from aliquots of plasma (1.0, 1.0, 0.5
and 0.5 ml respectively) The arterial and hepatic insulin samples were used for
measurement of indocyanine green, as will be described later, and then frozen at -70ºC
until insulin was measured. After each sample was processed, it remained on wet ice for
the remainder of the experiment and was then stored at -70º C until analysis was
performed.
Following the study, the plasma samples for [3H]-glucose measurement were
deproteinized by stepwise addition of 5 ml of 0.067 N Ba(OH)2 and 5 ml 0.067 N ZnSO4
33
(Sigma Chemical Co.). These samples were then stored at 4ºC for 1-3 days and then
processed.
Sample Analysis
Plasma Glucose
Plasma glucose concentrations were determined during the experiment using the
glucose oxidase method (133) with a Beckman glucose analyzer (Beckman Instruments,
Fullerton, CA). The reaction sequence was as follows:
which is mainly oxidized or released as lactate. Therefore, in a net sense it is possible
that hepatic gluconeogenic and glycolytic flux occur simultaneously, with lactate output
and uptake occurring in different cells. To the extent that flux occurs in both directions
simultaneously the net hepatic balance method will result in an underestimation of the
absolute rate of gluconeogenic flux to G-6-P. Of note, net hepatic gluconeogenic and net
hepatic glycogenolytic fluxes can be calculated accurately without concern for the
assumptions related to whether or not simultaneous gluconeogenic and glycolytic
substrate flux occur. Ideally the gluconeogenic flux rate would be calculated using
unidirectional hepatic uptake and output rates for each substrate, but this would be
difficult, as it would require the simultaneous use of multiple stable isotopes which could
themselves induce a mild perturbation of the metabolic state.
Statistical Analysis
Data are expressed as means ± standard error (SE). The data were analyzed for
differences between saline-euglycemic vs. glucagon-euglycemic and saline-
hypoglycemic vs. glucagon-hypoglycemic. Statistical comparisons were carried out using
60
two-way repeated measures ANOVA and two-way ANOVA with post hoc data analysis
determined by Student-Newman-Kuels Method (Sigma Stat, SPSS Inc.). Significance
was established when P < 0.05.
61
CHAPTER III
THE SENSITIVITY OF THE LIVER TO GLUCAGON IS INCREASED DURING INSULIN-INDUCED HYPOGLYCEMIA
Aim
In the presence of insulin-induced hypoglycemia glucagon is the most important
stimulator of glucose production. In contrast under euglycemic conditions insulin is a
potent inhibitor of glucagon’s effect on the liver. The results of previous studies suggest
that the liver is more sensitive to glucagon during hypoglycemia. A comparison of the
effects of a controlled rise in glucagon on hepatic glucose production in the presence of
euglycemia or hypoglycemia has never been made. For this reason, the aim of the
present study was to examine the ability of a physiologic increase in glucagon to
overcome the inhibitory effect of insulin on glucose production under euglycemic or
hypoglycemic conditions.
Results
Hormone Concentrations
Arterial plasma insulin rose from baseline to between 230 and 300 U/ml in
response to insulin infusion (Figure 3.1A). Arterial plasma glucagon levels were similar
in all groups during the control period (39±1 pg/ml) and they fell during the first
experimental period to between 25±2 and 30±2 pg/ml. They remained low in
experimental period two in the SE and SH groups (26±5 and 23±6 pg/ml respectively)
but rose to ~ 100 pg/ml in response to intraportal glucagon infusion in the GE and GH
groups (Figure 3.1B). Arterial plasma cortisol was ~3.5±0.3 µg/dl in all groups
62
respectively during the control period and experimental period one (Figure 3.2A). It
remained unchanged in the SE and GE groups (3.7±0.8 and 4.8±2.0 µg/dl respectively)
during experimental period two. On the other hand, in response to insulin-induced
hypoglycemia it increased markedly (15±1 and 17±3 µg/dl, in SH and GH respectively;
P<0.05 vs. euglycemic groups). Arterial plasma epinephrine was basal (~130±2 pg/ml)
during the control period and experimental period one in all groups (Figure 3.2B).
During experimental period two, it was ~137±62 and 126±53 pg/ml in the SE and GE
groups respectively, but rose to 1917±376 and 1755±326 pg/ml, in the SH and GH
groups respectively (P<0.05 vs. euglycemic groups). Arterial plasma norepinephrine
levels remained basal and similar between the groups (161±15 pg/ml) during the control
period and experimental period one (Figure 3.2C). During experimental period two it
remained unchanged in the SE and GE groups (209±33 and 162±30 pg/ml), but increased
to 403±83 and 350±86 pg/ml (P<0.05) in the SH and GH groups. The arterial plasma
polypeptide level averaged ~ 181±37 pg/ml in all groups during the control period. It fell
during experimental period one to 101±19, 82±18, 149±44 and 173±59 pg/ml in the SE,
GE, SH and GH, respectively, in response to the intraportal somatostatin infusion and
remained low in period two in all groups (98±21, 129±65, 162±40 and 174±52 pg/ml in
the SE, GE, SH and GH groups, respectively) (Table 3.1)
Blood glucose levels and hepatic glucose balance
Euglycemia was maintained during experimental period one in each group and
during experimental period two in the SE and GE groups (Figure 3.3A). In the latter
groups glucose infusion rates were 14.7±2.6 and 13.1±2.4 mg/kg/min, respectively
63
(Figure 3.3B). On the other hand, hypoglycemia was allowed to occur in the SH and GH
groups (49±1 mg/dl). The glucose infusion rates required to maintain hypoglycemia of
50 mg/dl were 5.9±1.4 and 4.6±1.7 mg/kg/min respectively. NHGO was ~1.6±0.1
mg/kg/min in all groups in the control period. In response to intraportal insulin infusion
in the presence of euglycemia the liver switched to slight net hepatic glucose uptake in all
groups (~ 0.4±1.2 mg/kg/min; Figure 3.4A). During the second experimental period,
NHGU increased slightly over time (to ~1.5±0.4 mg/kg/min) in the SE group while in the
GE group the liver temporarily switched to net output (~0.4±0.6 mg/kg/min) after which
it switched back to net uptake (~0.9±0.7 mg/kg/min). In the SH and GH groups the liver
quickly switched to net glucose output and remained in a production mode until the end
of the study (1.3±0.2 and 3.1±0.5 mg/kg/min, respectively; P<0.05). The increase in net
hepatic glucose balance (60 to 180 minutes) caused by glucagon was significantly greater
in the presence of hypoglycemia (239 mg/kg/120 min; difference in NHGB between SH-
GH) than in the presence of euglycemia (106 mg/kg/120 min; difference in NHGB
between SE-GE) (Figure 3.4B). Changes in tracer-determined endogenous glucose
production (Ra) paralleled the changes in NHGO (Table 3.2)
Tracer determined glucose utilization (Rd) was between 2.2 and 2.8 mg/kg/min
during the control period. Intraportal infusion of insulin increased Rd in all groups (to
11.9±1.8, 10.6±1.3, 11.6±1.7 and 9.2±1.7 mg/kg/min; Table 3.2) during experimental
period one. Rd continued to increase over time in the SE and GE groups (reaching
19.6±2.2 and 18.6±1.3 mg/kg/min, respectively by the end of the study) but it decreased
in the SH and GH groups (6.2±0.9 and 5.3±0.7 mg/kg/min, respectively; P<0.05).
Glucose clearance increased in all groups during experimental period one (Table 3.2) and
64
it rose to a greater extent during experimental period two in the euglycemic groups than
in the hypoglycemic groups.
Metabolites
Arterial blood alanine had decreased in response to the rise in insulin in all groups
by the end of the study. There was no significant change in net hepatic alanine balance
over time and no difference between groups (Table 3.3). On the other hand, the
fractional extraction of alanine by the liver doubled in all groups although this change
was not significant in any individual group. Arterial blood lactate levels were basal
during control period and rose minimally in experimental period one in all groups (Table
3.3). During experimental period two they remained unchanged in both euglycemic
groups but increased markedly in the hypoglycemic groups (Table 3.3). The liver was
producing lactate in all groups during period one. By the end of the study all groups had
switched to net hepatic lactate uptake but the hypoglycemic groups were taking up almost
6 times as much lactate as the euglycemic groups. The presence of glucagon had no
effect in lactate metabolism in the euglycemic or hypoglycemic settings.
Arterial plasma glycerol levels fell in all groups when insulin rose (Table 3.4).
They remained suppressed during experimental period two in both euglycemic groups but
increased markedly in response to hypoglycemia. Net hepatic glycerol uptake followed
the changes in glycerol levels and was more than 10 fold greater in the presence of
hypoglycemia than in the presence of euglycemia. The addition of glucagon in the
presence of euglycemia or hypoglycemia had no effect in glycerol metabolism.
65
Arterial Plasma Free Fatty Acids and BOHB
Arterial plasma free fatty acid levels were basal in all groups during the control
period and fell markedly in response to insulin in experimental period one (Table 3.5).
They fell to less than 50 µmol/L during experimental period two in both euglycemic
groups whereas they increased markedly in the SH and GH groups (to 547±76 and
376±115 µmol/L, respectively). Net hepatic free fatty acid uptake paralleled the changes
in plasma free fatty acid levels (Table 3.5). Arterial blood BOHB levels and net hepatic
BOHB output tended to fall in response to elevated insulin. The decline was slightly less
in the presence of hypoglycemia. The presence of glucagon had no discernable effect in
FFA or BOHB metabolism (Table 3.4).
Net hepatic glycogenolytic and gluconeogenic flux
Net hepatic gluconeogenic(NHGNG) flux was ~ 0.1±0.1 mg/kg/min during the
control period (Figure 3.5A) and decreased to ~ -0.5±0.1 mg/kg/min in all groups during
experimental period one. It remained close to zero in the SE and GE groups during
experimental period two but increased significantly (~1.8 mg/kg/min) in the SH and GH
groups in response to hypoglycemia. Since the increase was virtually identical (1.7±0.4
and 1.8±0.4 mg/kg/min, respectively (P<0.05). it is clear that the drive for
gluconeogenesis was not attributable to glucagon. Net hepatic glycogenolytic (NHGLY)
flux was ~1.5±0.1 mg/kg/min in all groups at the end of the control period (Figure 3.5B)
and it decreased during experimental period one (to -0.5±0.6, -0.5±0.2, 0.0±0.4 and -
0.6±0.3 mg/kg/min in the SE, SH,GE and GH groups, respectively). During
experimental period two the SE group continued to exhibit net glycogen synthesis (-
66
1.3±0.7 mg/kg/min). Addition of glucagon (GE) caused a small glycogenolytic response
followed by a return to net glycogen synthesis. In the SH group net glycogen synthesis
remained near zero during experimental period two. On the other hand, in the GH group
NHGLY flux increased to ~2.9±1 mg/kg/min at 75 minutes and remained elevated
throughout the study. The increase in NHGLY flux between 60 to 180 minutes caused
by glucagon was much greater in the presence of hypoglycemia (279 mg/kg/120 min)
than in the presence of euglycemia (106 mg/kg/120 min) (P<0.05). Hypoglycemia thus
caused a 2.7 fold increase in the glycogenolytic response to glucagon (Figure 3.5C).
Molecular changes
Molecular indices from the last two dogs studied in each group were analyzed and
compared to control values in liver taken from 18 h, fasted dogs in which basal levels of
insulin, glucagon and glucose were maintained. These animals were part of another
study and were included for references purposes. Levels of phosphorylated (Ser473) Akt
were assayed as an index of activation of the insulin signaling pathway, as were the levels
of phosphorylated (Ser256) FOXO1 and (Ser9) GSK3-β, two downstream targets of Akt
that are relevant to hepatic glucose metabolism. Total protein levels of Akt, FOXO1, and
GSK3-β did not change with treatment, and were used to normalize quantification of the
respective phospho-proteins. (Figure 3.6A). Animals in the euglycemic (SE and GE)
groups featured similar 4.8-fold increases in P-Ser473 Akt relative to the control animals.
However, animals in both hypoglycemic groups had partially blunted Akt activation, with
only 2.1- and 1.5-fold increases in P-Ser473 Akt being observed in the SH and GH
groups, respectively. Relative to control animals, P-Ser9 GSK3-β was increased
67
substantially (7.0-fold increase) in SE animals, while progressively smaller increases
(5.7-, 4.4-, and 1.4-fold) were observed in the GE, SH, and GH groups, respectively.
FOXO1 (Ser256) phosphorylation was markedly increased (5.5-fold) in the SE group
relative to control animals, but only a 1.9-fold rise was observed in the GE group and
there was no increase apparent in either hypoglycemic test condition.
To assess glucagon signaling, we assayed levels of phosphorylated (Ser133)
cAMP-response element-binding protein (CREB), PPAR gamma coactivator-1α (PGC1α)
and PEPCK protein levels. Total levels of CREB protein did not vary between groups,
while P-Ser133 CREB was strongly suppressed in the SE group relative to control
animals, but this suppression was equivalently blocked by the presence of hypoglycemia
and/or glucagon in the other groups. Levels of PGC1α were depressed in the SE group to
~60% of that observed in control animals, but the presence of glucagon and/or
hypoglycemia (GE, SH, GH) led to 2.5-fold increases in PGC1α in these groups.
Likewise, the PEPCK protein level in the SE animals was reduced to ~60% of that in
control animals, but were unchanged from basal in the other three groups. Analysis of
gene transcription revealed that PEPCK mRNA levels were decreased by 89% in the SE
group relative to that in the control animals and this strong repression was decreased by
hypoglycemia and/or glucagon, leading to a doubling of PEPCK mRNA relative to that
evident in the SE animals (Figure 3.6B). Similarly, G6Pase mRNA expression was
reduced in the SE group by 87%. Both GE and SH groups exhibited a doubling in
G6Pase mRNA relative to the SE group, and the combination of glucagon and
hypoglycemia (GH) led to an even more substantial (3-fold) increase.
68
Art
eria
lPl
asm
a In
sulin
( U
/ml)
0
150
300
450
SAL + EUGGN + EUSAL + HYPOGGN + HYPO
Time (min)
-40 0 60 120 180
Art
eria
lPl
asm
a G
luca
gon
(pg/
ml)
0
50
100
150
A
B
Figure 3.1 - (A) Arterial plasma insulin (U/ml) and (B) glucagon (pg/ml) during basal(-40 to 0 min) and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence of euglycemia and hypoglycemia. Values are means ± SEM; n=6 groups. *P<0.05 vs. euglycemic group; †P<0.05 vs. saline group.
Pe SRIF + Po INS (5.0 mU/kg/min)EU EU or HYPO
Po GGN (2.3 ng/kg/min)
69
A
B
C
Art
eria
l Pl
asm
aEp
inep
hrin
e(p
g/m
l)
0
1000
2000
3000
4000 SAL + EUGGN + EUSAL + HYPOGGN + HYPO
Time (min)
-40 0 60 120 180
Art
eria
l Pl
asm
aN
orep
inep
hrin
e(p
g/m
l)
0
200
400
600
Figure 3.2 - (A) Arterial plasma cortisol, (B) epinephrine and (C) norepinephrine (pg/ml) during basal (-40 to 0 min) and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence of euglycemia and hypoglycemia. Values are means ± SEM; n=6 groups. *P<0.05 vs. euglycemic group; †P<0.05 vs. saline group.
**
** *
**
** *
*
**
**
*
**
**
*
** *
***
*
*
Pe SRIF + Po INS (5.0 mU/kg/min)EU EU or HYPO
Po GGN (2.3 ng/kg/min)
Art
eria
lPl
asm
a C
ortis
ol(
g/dl
)
0
5
10
15
20
25
70
Art
eria
lPl
asm
a G
luco
se(m
g/dl
)
0
406080
100120A
B
Time (min)
-40 0 60 120 180
Glu
cose
Infu
sion
Rat
e(m
g/kg
/min
)
0
5
10
15
20
SAL + EUGGN + EUSAL + HYPOGGN + HYPO
Pe SRIF + Po INS (5.0 mU/kg/min)EU EU or HYPO
Po GGN (2.3 ng/kg/min)
Figure 3.3 - (A) Arterial plasma glucose (mg/kg/min) and glucose infusion rate (mg/kg/min) between 60 to 180 caused by glucagon (mg/kg/min2) during basal (-40 to 0 min) and experimental periods (0 to 180 min) in 18h fastedconscious dogs exposed to a controlled rise of glucagon in the presence of euglycemia and hypoglycemia. Values are means ± SEM; n=6 groups. *P<0.05 vs. euglycemic group; †P<0.05 vs. saline group.
71
GGN-SALHYPO
GGN-SALEU
Time (min)-40 0 60 120 180
-2
0
2
4
SAL+EUGGN+EU SAL+HYPO GGN+HYPO
Net
Hep
atic
G
luco
se B
alan
ce(m
g/kg
/min
)A
B
NHGU
NHGO
** *
*†*† *†*†
*† *†
Figure 3.4 - (A) Net Hepatic Glucose Balance (mg/kg/min) and the Delta AUC: for the increase in NHGO between 60 to 180 caused by glucagon (mg/kg/min2) during basal (-40 to 0 min) and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence of euglycemia and hypoglycemia. Values are means ± SEM; n=6 groups. *P<0.05 vs. euglycemic group; †P<0.05 vs. saline group.
***
Pe SRIF + Po INS (5.0 mU/kg/min)EU EU or HYPO
Po GGN (2.3 ng/kg/min)
Del
ta A
UC
: Fo
r the
incr
ease
in N
HG
O
from
the
last
2 h
ours
ca
used
by
gluc
agon
(mg/
kg/1
20m
in)
0
50
100
150
200
250
300
72
Del
ta A
UC
: Fo
r the
incr
ease
in G
LY fr
om th
e la
st 2
hou
rs
caus
ed b
y gl
ucag
on(m
g/kg
bw
/120
min
)
0
100
200
300
Pe SRIF + Po INS (5.0 mU/kg/min)EU EU or HYPO
Po GGN (2.3 ng/kg/min)
Figure 3.5 - (A) Net hepatic gluconeogenic and (B) glycogenolytic flux (mg/kg/min) during basal (-40 to 0 min)and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence of euglycemia and hypoglycemia. Values are means ± SEM; n=6 groups. *P<0.05 vs. euglycemic group; †P<0.05 vs. saline group.
A
Net GLY syn
Net GLY prod
Time (min)-40 0 60 120 180
Net
Hep
atic
G
lyco
geno
lytic
Flu
x(m
g/kg
/min
)
-2
0
2
4
SAL + EUGGN + EUSAL + HYPOGGN + HYPO
B*†
*†
*†*†
*†*†
GGN-SALHYPO
Net GNG
Net Gycolysis-1
0
1
2N
et H
epat
icG
NG
Flu
x(m
g/kg
/min
)
* * * *
* **
C
GGN-SALEU
73
A
B
Figure 3.6 - (A) Phosphorylation of Akt (Ser 473), GSK3- (Ser 9), FOXO1 (Ser 256), CREB(Ser 133) and PGC-1 and (B) relative gene expression of PEPCK and G-6-Pase of liver samples taken from 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence of euglycemia and hypoglycemia.
74
TABLE 3.1Pancreatic Polypeptide (pg/ml) during control (-40 to 0 min) and experimental periods (0-180 min) of studies conductedon 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia.
Mean ± SEM; n=6; *P < 0.05 vs. euglycemic group; †P<0.05 vs saline group.
P1 P2Experimental Period
180ControlPeriod 30-60 75 90 120 150
75
TABLE 3.2Tracer determined endogenous glucose production, utilization (mg/kg/min) and glucose clearance (ml/kg/min) during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia.
Tracer Determined Glucose Production Ra, mg/kg/min
Mean ± SEM; n=6; *P < 0.05 vs. euglycemic group; †P<0.05 vs saline group.
Experimental Period
30-60 75 90 120 150PeriodP1 P2Control
180
76
TABLE 3.3Lactate and alanine arterial blood levels and net hepatic balance during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia.
Mean ± SEM; n=6; *P < 0.05 vs. euglycemic group; †P<0.05 vs saline group.
PeriodP1 P2Control
180
Experimental Period
30-60 75 90 120 150
77
TABLE 3.4Glycerol and BOHB arterial blood levels and net hepatic balance during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia.
Mean ± SEM; n=6; *P < 0.05 vs. euglycemic group; †P<0.05 vs saline group.
Experimental PeriodP1 P2
180ControlPeriod 30-60 75 90 120 150
78
TABLE 3.5Arterial plasma free fatty acids levels and net hepatic FFA balance during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia.
mRNA still being reduced by almost 80% and PEPCK protein being unchanged from
control values. The question thus arises as to how the increase in gluconeogenesis comes
about. The answer lies in the large increases in the delivery of gluconeogenic precursors
to the liver during hypoglycemia.
In muscle, in the presence of hyperinsulinemic hypoglycemia (SH, GH) there
was a marked increase in the arterial blood lactate level and as a resulting in net hepatic
lactate uptake. This indicates that production of lactate by muscle increased dramatically
as a result of the rise in catecholamines (31; 32), neural input to muscle and/or
hypoglycemia per se. The increase in glucagon had no impact on the rise in blood lactate
or net hepatic lactate uptake.
Lipolysis is best estimated from the glycerol data since glycerol must be released
from the fat cell and can not be used for re-esterification. In the presence of
hyperinsulinemic euglycemia (SE) there was a marked inhibition of lipolysis as indicated
by a fall in blood glycerol levels. In the presence of the same conditions a physiologic
rise in glucagon had no effect on lipolysis. During insulin-induced hypoglycemia (SH,
GH), there was a marked increase in arterial blood glycerol and net hepatic glycerol
uptake indicating a dramatic rise in lipolysis. This was the result of the lipolytic effect of
86
catecholamines, hypoglycemia per se and/or neural input to fat (31; 32) Once again the
rise in glucagon had no effect on the response. Thus the marked increase in the net
hepatic gluconeogenesis in response to hypoglycemia was a function of increased
substrate delivery to the liver rather than a stimulation of the hepatic gluconeogenic
pathway per se. Addition of a physiologic rise in glucagon to hypoglycemia and its
various effects doubled the magnitude of the increase in glucose production which
occurred. This underscores the importance of glucagon to the increase in hepatic glucose
production seen during insulin-induced hypoglycemia. For this to be relevant to the
normal response to insulin-induced hypoglycemia it is important to point out that the
magnitude of the increment in glucagon which we used in the study represents the normal
physiologic response of glucagon to hypoglycemia of 50 mg/dl caused by insulin infusion
at 5.0 mU/kg/min and plasma glucose decrease to 50 mg/dl. Frizzell et al. (7) showed
that when insulin was infused at 5mU/kg/min, the increment in glucagon levels over 2
hours was Δ 9510 pg/kg bw/120 min. The increment in glucagon in our studies was Δ
8276 pg/kg bw/120 min. Therefore, the rise in glucagon which we used in our studies
represents a normal response of the hormone to hypoglycemia. It should be noted,
however, that we used a square wave elevation of glucagon to simplify the experimental
design whereas under normal circumstances the response would have had a spike decline
pattern.
In summary, hypoglycemia increased glucagon’s ability to overcome insulin’s
inhibitory effect on hepatic glucose production. This effect was attributable to a marked
(almost 3-fold) enhancement of net glycogen breakdown. It paralleled an increase in the
ability of glucagon to reduce the phosphorylation of GSK-3β in the presence of
87
hypoglycemia as opposed to euglycemia. At the same time hypoglycemia decreased
insulin’s activation of its signaling cascade. Proof that it is hypoglycemia per se, rather
than an increase neural input to the liver, or increases in some other component of the
counterregulatory response remains to be obtained.
Summary and Conclusions
In the United States, approximately 23.2 million people have diabetes (~8% of
population). Of those 18.6 million have been diagnosed and 4.6 million do not yet know
they have the disease. There are three types of diabetes: type1 diabetes, type 2 diabetes
and gestational diabetes. Type 1 diabetes is an autoimmune disease in which the immune
system destroys the insulin-producing β cells in the pancreas. Type 1 diabetes accounts
for about 5 to 10 percent of diagnosed diabetes. On the other hand, Type 2 diabetes
which is the most common form of diabetes accounts for ~ 90-95% of people with the
disease. Type 2 diabetes is characterized by insulin resistance and hyperglycemia.
Gestational diabetes is a type of diabetes that occurs only during pregnancy. Although
this form of diabetes usually disappears after birth of the baby, women who have had
gestational diabetes have a 20% to 50% chance of developing Type 2 diabetes within 5-
10 years.
Glycemic control is fundamental for the management of the disease. Reduction
of glucose levels prevents macrovascular and microvascular complications such as heart
disease, retinopathy, nephropathy and neuropathy in both type 1 and type 2 diabetes.
Another complication that is a limiting factor in the management of diabetes is
hypoglycemia. Hypoglycemia is the most frequent complication of insulin-requiring
diabetes and the principal factor limiting optimization of glycemic control. Is typically
88
the result of the interplay of insulin excess and compromised glucose counterregulation in
individuals with type 1 diabetes. In addition, it may contribute to recurrent morbidity in
patients with type 1 diabetes and may sometimes be fatal in patients with advanced,
insulin-requiring type 2 diabetes.
Hypoglycemia triggers the activation of a counterregulatory response to increase
glucose production. The counterregulatory response to hypoglycemia involves the
release of glucagon, epinephrine, norepinephrine and cortisol. Glucagon increases
glucose production by activating glycogenolysis and gluconeogenesis; however, its effect
on gluconeogenesis is limited by its inability to increase gluconeogenic substrate delivery
to the liver. Epinephrine stimulates hepatic glucose production through activation of
gluconeogenesis and glycogenolysis. Norepinephrine increases hepatic glucose
production by increasing gluconeogenesis which results from a glycogenolytic effect in
muscle and a lipolytic effect in fat. Cortisol stimulates hepatic glucose production by
maintaining substrate availability to support gluconeogenesis and limits glucose
utilization.
Glucagon is the primary hormone involved in the regulation of glucose
production. In addition, previous studies have shown glucagon remains the most
important regulator of glucose production even in the presence of very high insulin levels
(18). In contrast, under euglycemic conditions Steiner et al. (131) have shown that
insulin is a potent inhibitor of glucagon’s effect on the liver. Therefore, our aim was to
determine the extent to which hypoglycemia augments glucagon’s ability to increase
glucose production and at a molecular level, how it does so.
89
In the studies described in this dissertation, hypoglycemia increased glucagon’s
ability to overcome insulin’s inhibitory effect on hepatic glucose production 2.3 fold.
This effect was attributable to a marked (almost 3-fold) enhancement of net glycogen
breakdown which was associated with a 2.3 fold increase in the ability of glucagon to
reduce the phosphorylation of GSK3β caused by insulin.
It remains unclear which physiologic signal (increased cortisol, epinephrine,
norepinephrine, neural input to the liver or hypoglycemia per se) explains this adaptive
response. Therefore future studies should be conducted to determine which of these
physiological signals is responsible for the enhancement of glucagon action. It would be
of interest therefore to determine the effects of these other counterregulatory hormones
on glucagon’s action. One could conduct studies to assess the physiologic effects of
elevated cortisol, epinephrine and norepinephrine with or without a rise in glucagon in
the presence of euglycemia and hyperinsulinemia. The counterregulatory hormones
would be selectively increased to the levels seen during hypoglycemia and euglycemia
would be maintained in order discriminate between the effects of the counterregulatory
hormones and hypoglycemia per se. To assess the role of hypoglycemia per se in
overriding insulin’s inhibitory effect on glucagon action one could conduct studies in
adrenalectomized dogs. Using this approach we could eliminate the effects of
epinephrine and cortisol by removing the adrenal glands and because most of the
norepinephrine involve in the counterregulation process is released from sympathetic
postganglionic neurons one could use a α1-adrenergic blocker to inhibit norepinephrine
effects on the liver (24). Glucagon would be selectively increased and its physiologic
effect in overriding insulin’s inhibitory effect would be determined in the presence of
90
hyperinsulinemic-euglycemic or hyperinsulinemic-hypoglycemic conditions. These
studies would therefore determine if hypoglycemia itself is making glucagon more
effective during insulin-induced hypoglycemia.
91
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