Somatostatin Receptor Type 2 Anatagonism Improves Glucagon Counter-regulation in … · 2013. 10....
Transcript of Somatostatin Receptor Type 2 Anatagonism Improves Glucagon Counter-regulation in … · 2013. 10....
Somatostatin Receptor Type 2 Antagonism Improves Glucagon Counter-Regulation in
Biobreeding Diabetes-Prone Rats
By
Negar Karimian
A thesis submitted in conformity with the requirements for the degree of Masters of Science
Institute of Medical Science
University of Toronto
© Copyright by Negar Karimian (2013)
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Somatostatin Receptor Type 2 Antagonism Improves Glucagon Counter-Regulation in
Biobreeding Diabetes-Prone Rats
Negar Karimian
Master of Science
Institute of Medical Science
University of Toronto
2013
Abstract
Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly
due to inadequate pancreatic islet alpha-cell glucagon secretion. We hypothesized that
hypoglycemia can be prevented in autoimmune T1D by selective somatostatin receptor
type 2 (SSTR2) antagonism of alpha cells to relieve SSTR2 inhibition, thereby increasing
glucagon secretion. Diabetic biobreeding diabetes prone (BBDP) rats (D) vs non-diabetic
BBDP (N) rats, underwent infusion of vehicle or SSTR2 antagonist (SSTR2a) during
insulin-induced hypoglycaemia. D rats, treated with SSTR2a, needed little or no glucose
to maintain hypoglycemia. To monitor real-time glucagon secretory response directly, we
developed the technique of thin slices of the pancreas from D and N rats as well as
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normal human pancreas, subjected to perifusion with vehicle vs SSTR2a. SSTR2a
treatment enhanced glucagon secretion in N and D rats and human pancreas. We
conclude that SSTR2 antagonism can enhance hypoglycemia-stimulated glucagon release
sufficient to achieve normoglycemic control.
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Acknowledgments I would like to thank my supervisor Dr. Herbert Y. Gaisano and my co-supervisor
Dr. Mladen Vranic for their endless support throughout my graduate training. Their
guidance at the scientific and personal levels has helped me greatly in completing this
thesis project.
I am very grateful to my M.Sc, supervisory committee, Dr. Minna Woo and Dr.
Adria Giacca. They have given me invaluable comments and guidance in the committee
meetings.
I would also like to express my sincere gratitude to Dr. Mary Seeman, Dr. Howard
Mount and Ms. Hazel Pollard for their endless support from the very beginning of my
studies as a M.Sci. candidate at the IMS.
I am also very grateful for the help and support I have received from my colleagues
in the lab, Dr. Youhou Kang and Huanli Xie and particularly from Tairan Qin and Ya-chi
Huang throughout this project.
Special thanks to Dr. Mark Cattral and Ms. Erin Winter for providing the human
pancreas specimens.
Finally, I would like to express my deepest thanks to my parents and sisters, Golnar
and Negin, whose unlimited support and love have made everything possible. To Saber
Ghadakzadeh, my dear husband, your love has been the source of courage for me to
withstand the tough times and the motivation for me to complete this study.
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Table of Contents Abstract………………………………………………………………………………………….……..ii
Acknowledgments……………………………………………………………………………….…….iv
Table of Contents………………………………………………………………………………….…...v
List of Figures………………………………………………………………………………….……..vii
Chapter 1 Introduction ........................................................................................................................... 1
1.1 Hypoglycemia ............................................................................................................................. 1
1.1.1 Normal counter-regulatory hormone responses to hypoglycemia ........................................ 2
1.1.1.1 Insulin ......................................................................................................................... 3
1.1.1.2 Glucagon ....................................................................................................................... 3
1.1.1.3 Epinephrine and Norepinephrine and Glucocorticoids ................................................. 5
1.2 Hypoglycemia in Type 1 and Type 2 Diabetes mellitus ............................................................. 6
1.3 Defective Counter-regulation in Diabetes ................................................................................... 7
1.3.1 Glucagon counter-regulation in diabetes .............................................................................. 7
1.3.2 Epinephrine and norepinephrine and glucocorticoids counter-regulation in diabetes ..... 8
1.4 Somatostatin .............................................................................................................................. 11
1.4.1 Somatostatin functions in pancreatic islets ......................................................................... 11
1.4.2 Somatostatin receptors ........................................................................................................ 12
1.4.3 Somatostatin in diabetes ..................................................................................................... 13
1.4.4 Somatostatin receptor type 2 antagonist PRL-2903 ........................................................... 14
1.5 Animal models of type 1diabetes:BBDP diabetic rats ss Streptozotocin induced diabetic rats14
1.5.1 BBDP diabetic rats .............................................................................................................. 15
1.5.2 STZ induced diabetic rats ................................................................................................... 16
1.6 Islet cell composition, normal vs. type 1 diabetes ..................................................................... 17
1.7 Rationales .................................................................................................................................. 19
1.8 Hypothesis ................................................................................................................................. 22
1.9 Objectives .................................................................................................................................. 24
Chapter 2 Research design and methods .............................................................................................. 25
2.1 In-vivo Studies .......................................................................................................................... 25
2.1.1 Animals .............................................................................................................................. 25
2.1.2 Somatostatin receptor type2 antagonist (PRL-2930) solution ........................................... 25
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2.1.3 Surgical procedures and hypoglycemic clamp experiments .............................................. 26
2.1.4 Plasma hormone measurements ......................................................................................... 27
2.1.5 Total pancreatic glucagon and somatostatin protein content ............................................. 28
2.2 In-vitro studies ...................................................................................................................... 29
2.2.1 Animals .............................................................................................................................. 29
2.2.2 Human pancreas specimens ............................................................................................... 29
2.2.3 Pancreatic slices preparation .............................................................................................. 29
2.2.4 Pancreas slices perifusion studies....................................................................................... 32
3.2.5 Pancreatic slices perifusion secretory glucagon and insulin measurements ...................... 33
2.3 Statistical analysis ..................................................................................................................... 34
Chapter 3 Results ................................................................................................................................. 35
3.1 In-vivo studies ........................................................................................................................... 35
3.1.1 Plasma glucose levels during the hyperinsulinemic hypoglycemic clamp experiments .... 35
3.1.2 Glucose infusion rates during the hyperinsulinemic hypoglycemic clamp experiments ... 35
3.1.3 Insulin infusion rates during hyperinsulinemic hypoglycemic clamp experiments ........... 35
3.1.4 Plasma hormones secretory levels during hyperinsulinemic hypoglycemic clamp experiments .................................................................................................................................. 36
3.1.5 The whole pancreatic glucagon and somatostatin protein levels immediately following the hyperinsulinemic hypoglycemic clamp experiments ................................................................... 37
3.1.6 The whole pancreatic glucagon and somatostatin protein levels in the diabetic and non diabetic bb rats not undergone the clamp ..................................................................................... 39
3.2 In-vitro studies .......................................................................................................................... 39
3.2.1 Diabetic BBDP and non diabetic BBDP rat pancreatic slices glucagon secretion in perifusion studies ......................................................................................................................... 39
3.2.2 Human Pancreatic Slices Glucagon and Insulin Secretion in Perifusion Studies .............. 40
Chapter 4 Discussion ............................................................................................................................ 65
Chapter 5 Conclusion and future directions ......................................................................................... 75
Chapter 7 References ........................................................................................................................... 80
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List of Figures
Figure 1-1 Physiology and pathophysiology of insulin and glucagon in health and diabetes ……………………………………………………………………………………………….11
Figure 1-4 Alpha, Beta and Delta cell interactions during hypoglycaemia………………...26
Figure 2-1 In Vivo studies and hyyperinsulinemic hypoglycaemic clamps………………...30
Figure 2-2 Agarose embedded pancreatic blocks…………………………………………...33
Figure 2-3 The vibrotome machine and pancreatic slicing procedure……………………....34
Figure 2-4 Perifusion pump and chambers.…………………………………………………34
Figure 2-5 Diabetic or non diabetic BBDP rats pancreatic slice perifusion studies………...36
Figure 2-6 Normal human pancreatic slice perifusion studies……………………………...36
Figure 3-1 Plasma glucose levels during hyperinsulinemic hypoglycemic clamps experiments…………………………………………………………………………………..45
Figure 3-2 Glucose infusion rates during hyperinsulinemic hypoglycemic clamp experiments…………………………………………………………………………………..46
Figure 3-3 Insulin infusion rates distribution between the four groups during hyperinsulinemic hypoglycemic clamp experiments………………………………………...46
Figure 3-4. A & B Plasma glucagon secretory levels during hyperinsulinemic hypoglycemic clamp experiments…………………………………………………………………………...48
Figure 3- 5. A & B Plasma corticosterone secretory levels during hyperinsulinemic hypoglycemic clamp experiments…………………………………………………………...50
Figure 3-6. A & B Plasma epinephrine secretory levels during hyperinsulinemic hypoglycemic clamp experiments…………………………………………………………...52
Figure 3-7. A & B Plasma norepinephrine secretory levels during hyperinsulinemic hypoglycemic clamp experiments…………………………………………………………...54
Figure 3-8. A & B Plasma somatostatin levels during hyperinsulinemic hypoglycemic clamp experiments…………………………………………………………………………...56
Figure 3-9 Whole pancreatic glucagon protein contents following hyperinsulinemic hypoglycemic clamp experiments…………………………………………………………...57
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Figure 3-10 Whole pancreatic somatostatin protein content following hyperinsulinemic hypoglycemic clamp experiments…………………………………………………………...58
Figure 3-11 The whole pancreatic glucagon protein contents of BBDP rats that have not undergone hypoglycemic clamp……………………………………………………………..59
Figure 3-12 The whole pancreatic somatostatin protein contents of BBDP rats that have not undergone hypoglycemic clamp……………………………………………………………..60
Figure 3-13. A & B Glucagon secretion from diabetic BBDP rat pancreatic slices……….61
Figure 3-14.A & B Glucagon secretion from Non diabetic BBDP rat pancreatic slices…..63
Figure 3-15.A & B Glucagon and insulin secretion from perifused human pancreatic slices…………………………………………………………………………………………65
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Chapter 1 Introduction
1.1 Hypoglycemia
The American Diabetes Association Workgroup on Hypoglycemia (Cryer, 2005)
defined hypoglycemia in diabetes as “all episodes of abnormally low plasma glucose
concentration that expose the individual to potential harm.” They recommended that at a
plasma glucose concentration of 3.9 mmol/l the patients should be concerned about the
possibility of developing hypoglycemia (Cryer, 2005).
People with type 1 diabetes can suffer at least two episodes of symptomatic
hypoglycemia and a number of asymptomatic hypoglycemia per week (Cryer, 2005;
Cryer, Davis, & Shamoon, 2003). However, hypoglycemia is less frequent in type 2
diabetes and becomes a major problem later in the course of the disease (Cryer, 2008a;
Cryer, et al., 2003). Hypoglycemia is a limiting factor in the glycemic management of
diabetes (Cryer, 2008a, 2012a). It causes recurrent physical and psychological morbidity
and even some mortality in most people with type 1 diabetes and advanced type 2
diabetes who are absolutely insulin-deficient (Cryer, 2012a).
Symptoms of hypoglycemia (Towler, Havlin, Craft, & Cryer, 1993) are categorized
as neuroglycopenic (those that are the direct result of brain glucose deprivation per se)
and neurogenic (or autonomic), those that are largely the result of the perception of
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physiological changes caused by the sympathoadrenal (largely the sympathetic neural)
(Towler, et al., 1993) discharge triggered by hypoglycemia. Neuroglycopenic
manifestations include cognitive impairments, behavioral changes and psychomotor
abnormalities, and, at lower plasma glucose concentrations, seizure and coma.
Adrenergic neurogenic symptoms include palpitations, tremor, and anxiety/arousal
(Towler, et al., 1993). Cholinergic neurogenic symptoms include sweating, hunger, and
paresthesias (Towler, et al., 1993) . Central, as well as peripheral, mechanisms may be
involved in the generation of some symptoms such as hunger (Cryer, et al., 2003).
Awareness of hypoglycemia is largely the result of the perception of neurogenic
symptoms (Towler, et al., 1993). Pallor and diaphoresis (the result of adrenergic
cutaneous vasoconstriction and cholinergic stimulation of sweat glands, respectively) are
common signs of hypoglycemia (Cryer, 2008a). Rarely, hypoglycemia causes sudden,
presumably cardiac arrhythmic, death or, if it is prolonged and profound, brain death
(Adler et al., 2009; Cryer, 2007). Recent reports indicate that 6% to 10% of deaths of
people with type 1 diabetes mellitus are caused by hypoglycemia (Jacobson et al., 2007;
Skrivarhaug et al., 2006)
1.1.1 Normal counter-regulatory hormone responses to hypoglycemia
Multiple mechanisms are involved in the normal defense against falling plasma
glucose concentrations, but some are more important than others (Cryer, 2008a) . Falling
plasma glucose concentrations elicit a sequence of responses that normally prevent or
rapidly correct hypoglycemia. The physiological defenses against declining plasma
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glucose concentrations include 1) a decrease in insulin secretion, 2) an increase in
glucagon secretion, and, in absence or deficiency of the latter, an increase in epinephrine
secretion (Cryer, 2006).The behavioral defense is the ingestion of carbohydrates
prompted by the awareness of hypoglycemia (Cryer, 2008a; DeRosa & Cryer, 2004;
Towler, et al., 1993). I will discuss each of these below.
1.1.1.1 Insulin
The first physiological defense against hypoglycemia is a decrease in pancreatic
islet β-cell insulin secretion. That occurs as plasma glucose concentrations decline within
the physiological range (4.4-4.7 mmol/l) and increases hepatic (and renal) glucose
production with virtual cessation of glucose utilization by insulin-sensitive non-neural
tissues(Cryer, 2008a). Reduced insulin secretion is believed to be a result of direct
effect of hypoglycemia on pancreatic β-cells (Cryer, 1993). Additionally, norepinephrine
from pancreatic sympathetic nerves and epinephrine from adrenal medulla have been
suggested to play a role in inhibiting insulin secretion from β-cells (Sherck et al.,
2001)(Figure 1-1).
1.1.1.2 Glucagon
The second physiological defense is an increase in pancreatic islet α-cell glucagon
secretion. That occurs as plasma glucose concentrations fall just below the physiological
range (3.6-3.9 mmol/l)(Cryer, 2008a) . To increase blood glucose levels, glucagon
promotes hepatic glucose output by stimulating glycogenolysis and gluconeogenesis and
by decreasing glycogenesis and glycolysis in a concerted fashion via multiple mechanism
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(Gromada, Franklin, & Wollheim, 2007)(Figure1-1). Whether α-cells directly sense and
respond to fluctuations in plasma glucose or whether the response is mediated by the
autonomic nervous system and/or the paracrine/endocrine effects of secretory products
from other islet cell types have been very hotly debated and in fact remain unresolved
(Gromada, et al., 2007). While some studies suggest a direct effect of glucose on α-cell
secretory function (Ravier & Rutter, 2005; Vieira, Salehi, & Gylfe, 2007), other studies
have suggested that a direct stimulatory effect of low glucose on the α-cell seems to be
of less physiological importance, and in fact, others have postulated that high glucose
could paradoxically augment glucagon release (Franklin, Gromada, Gjinovci, Theander,
& Wollheim, 2005; Olsen et al., 2005; Salehi, Vieira, & Gylfe, 2006). More recently,
more dominant roles for glucagon secretion have been conferred to intra-islet paracrine
and autocrine interactions. For example, reduced intra-islet insulin and the co-released
zinc from the pancreatic β-cells, have been suggested to promote glucagon secretion
(Hope et al., 2004; Ishihara, Maechler, Gjinovci, Herrera, & Wollheim, 2003; Kawamori
et al., 2009; Robertson, Zhou, & Slucca, 2011; Slucca, Harmon, Oseid, Bryan, &
Robertson, 2010; Zhou et al., 2004; Zhou, Zhang, Harmon, Bryan, & Robertson, 2007;
Zhou et al., 2007). This is an underlying concept of the switch-off hypothesis in the
induction of glucagon secretion (Hope, et al., 2004). There are other regulators that the
fall in their concentrations as part of the switch off hypothesis have been indicated to
stimulate the glucagon secretory response from pancreatic α-cells during hypoglycemia
in normal physiological conditions, including γ-amino-butyric acid (GABA) (Rorsman et
al., 1989) and somatostatin (Gerich et al., 1974; Unger & Orci, 1977). In addition, central
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and autonomic nervous systems also play a role in regulating glucagon secretion (Ahren,
2000; Evans et al., 2004; Marty et al., 2005).
1.1.1.3 Epinephrine and Norepinephrine and Glucocorticoids
The third physiological defense, which becomes critical when glucagon secretion is
deficient, is an increase in drenomedullary epinephrine secretion. That, too, occurs as
plasma glucose concentrations fall just below the physiological range (3.6-3.9mmol/l)
and raises plasma glucose concentrations through increasing glucose production and
decreasing glucose utilization by the peripheral tissues (Clarke et al., 1979; Cryer, 2006,
2008a; Rizza, Cryer, & Gerich, 1979). Epinephrine response may not be critical in
correcting hypoglycemia unless glucagon secretion is deficient, which is the case in type
1 diabetes or advanced type 2 diabetes (Clarke, et al., 1979; S. N. Davis, Fowler, &
Costa, 2000; Rizza, et al., 1979).
It has been shown that norepinephrine contributes to increasing blood glucose
during hypoglycemia via the same mechanisms as epinephrine (Hansen, Firth, Haymond,
Cryer, & Rizza, 1986); however, its concentration in the peripheral blood is much less
than epinephrine (Shum et al., 2001)
Glucocorticoids, cortisol in human and corticosterone in rodents, are not part of the
primary defense against hypoglycemia (Hoffman, Sinkey, Dopp, & Phillips, 2002). They
support glucose production during prolonged hypoglycemia and are normally triggered at
the glycemic threshold of 3.2mmol/l (Mitrakou et al., 1991). They stimulate
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gluconeogenesis and reduce glucose utilization (De Feo et al., 1989).They also suppress
insulin’s inhibitory effect on glucose production (Dirlewanger et al., 2000).
1.2 Hypoglycemia in Type 1 and Type 2 Diabetes mellitus
Because of the effectiveness of physiologic defenses, and in particular in intact
glucagon secretory response, hypoglycemia is a distinctly uncommon clinical event
except in people with diabetes who use medications, such as a sulfonylurea, or
exogenous insulin, that raise circulating insulin concentrations to lower their plasma
glucose levels (Cryer, 2012a). In that setting where in there is a defective glucagon
response (see below), hypoglycemia is common. Indeed, hypoglycemia is the limiting
factor in the glycemic management of diabetes (Cryer, 2012a) .
Hypoglycemia in diabetes is fundamentally iatrogenic, the result of relative or
absolute therapeutic hyperinsulinemia that causes the plasma glucose concentration to
decline. However, that alone seldom results in hypoglycemia. Rather, hypoglycemia is
typically the result of the interplay of therapeutic hyperinsulinemia and compromised
counter-regulatory defenses against the falling plasma glucose concentrations (Cryer,
2012a; Dagogo-Jack, Craft, & Cryer, 1993; Segel, Paramore, & Cryer, 2002). With
different time courses, the pathophysiology of glucose counterregulation is the same in
type 1 diabetes and advanced (i.e., absolutely endogenous insulin deficient) type 2
diabetes (Cryer, 2008a, 2012a; Dagogo-Jack, et al., 1993; Segel, et al., 2002). Because
absolute β-cell failure occurs rapidly in type 1 diabetes but slowly in type 2 diabetes, the
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syndromes of defective glucose counterregulation and hypoglycemia unawareness
develop early in type 1 diabetes but later in type 2 diabetes (Cryer, 2012a).
1.3 Defective Counter-regulation in Diabetes
The compromised defenses include loss of the decrease in insulin and loss of the
increase in glucagon as plasma glucose concentrations fall (Cryer, 2012a; Dagogo-Jack,
et al., 1993; Segel, et al., 2002). Some studies also include attenuated adrenomedullary
and sympathetic neural responses to falling plasma glucose concentrations resulting in
the clinical syndromes of defective glucose counterregulation and hypoglycemia
unawareness, respectively, collectively termed hypoglycemia-associated autonomic
failure in diabetes (Cryer, 2012a).
1.3.1 Glucagon counter-regulation in diabetes
In type 1 diabetes and advanced type 2 diabetes, the absence of an increment in
glucagon secretion, in the setting of an absent decrement in insulin secretion and an
attenuated increment in sympathoadrenal activity, in response to falling plasma glucose
concentrations plays a key role in the pathogenesis of iatrogenic hypoglycemia (Bolli et
al., 1983; Cryer, 2008a, 2012a; Cryer, et al., 2003; Dagogo-Jack, et al., 1993; Rizza, et
al., 1979; Segel, et al., 2002). In type 1 diabetes, this defect usually occurs within the first
few years of the onset of the disease (Bolli, et al., 1983).This defect may be specific to
insulin induced hypoglycemia as the glucagon response to neurogenic stress (Miles,
Yamatani, Lickley, & Vranic, 1991; J. T. Y. Yue et al., 2006) and exercise (Purdon et al.,
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1993; Sigal et al., 1994) is normal. The exact mechanisms underlying the defect in
glucagon response are still unclear. It has been suggested that the glucagon secretion is
impaired as the result of loss of the decrement reduction in intra-islet insulin due to loss
of β-cell function, which has been postulated to be the underlying basis of loss of insulin
switch-off mechanisms in type1 diabetes. Others have suggested the increased α-cell
sensitivity to insulin due to absent β-cell function in type 1 diabetes (Taborsky, Ahren, &
Havel, 1998) and a defect in autonomic response to hypoglycemia as another
contributing underlying mechanisms of dysregulated glucagon secretion (Taborsky,
Ahren, Mundinger, Mei, & Havel, 2002). Also, it has been shown that infusion of
somatostatin evoked a switch off effect on α-cell during hypoglycemia, which when
blocked, can restore glucagon secretion (Farhy et al., 2008). Other studies have
suggested the increased pancreatic somatostatin in diabetes as the cause of impaired
glucagon secretion in type 1 diabetes (Rastogi, Lickley, Jokay, Efendic, & Vranic, 1990).
In addition to local intra-pancreatic networks, there are a number of studies that have
suggested that the central nervous system contribute to the perturbation of glucagon
secretion (Borg et al., 1999; Paranjape et al., 2010).
1.3.2 Epinephrine and norepinephrine and glucocorticoids counter-regulation in diabetes
The epinephrine response to hypoglycemia is critical in a setting of deficient
glucagon counterregulation to hypoglycemia (Cryer, 2008a). The individuals lacking
both glucagon and epinephrine counterregulation are at 25-fold or greater risk of severe
hypoglycemia compared to individuals lacking glucagon but have an intact epinephrine
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response (Cryer, 2008a, 2008b, 2012b). Epinephrine counterregulation to hypoglycemia
has been shown to be either impaired (Bolli, et al., 1983; Chan et al., 2002; Dagogo-Jack,
et al., 1993; K. Inouye et al., 2002) or not impaired (Kinsley & Simonson, 1996) in type-
1 diabetes. The epinephrine defect in diabetes could also be stressor specific as it is
reduced in response to hypoglycemia and cold stress (Kinsley, Widom, Utzschneider, &
Simonson, 1994) but not to exercise (Marliss & Vranic, 2002).
It has been suggested that the prior glycemic control profile of the diabetic
individuals contributes to the extent of impairment in the epinephrine response (M.
Davis, Mellman, Friedman, Chang, & Shamoon, 1994). In addition, since tight control of
blood glucose levels is associated with increased frequency of hypoglycemic episodes,
recurrent hypoglycemia may be the possible cause of impaired epinephrine response in
type 1 diabetes (K. E. Inouye, Chan, Yue, Matthews, & Vranic, 2005). Many studies
have suggested the same central nervous system regulatory mechanisms for epinephrine
and glucagon responses particularly in regard to the role of the ventromedial
hypothalamus (Chan, Zhu, Ding, McCrimmon, & Sherwin, 2006).
Lastly, in regard to norepinephrine and glucocorticoid responses to hypoglycemia,
there are various reports suggesting their decreased (K. Inouye, et al., 2002)or unchanged
(Dagogo-Jack, et al., 1993) responses in diabetes. Similar to epinephrine, prior strict
glycemic control and the consequent recurrent hypoglycemia may influence the
impairment of these responses (Kinsley & Simonson, 1996).
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Figure 1-1. Physiology (normal) and pathophysiology (endogenous insulin-deficiency in
type 1 diabetes) in glucagon secretory responses to hypoglycemia. Normally, a decrease
in plasma glucose causes a decrease in β-cell insulin secretion that signals an increase in α-
cell glucagon secretion during hypoglycemia.. In the setting of β-cell failure in type 1
diabetes, a decrease in plasma glucose cannot cause a decrease in β-cell insulin secretion, and
the absence of that signal results in no increase in pancreatic α-cell glucagon secretion during
hypoglycaemia (adapted from (Cryer, 2012a)).
Figure 1-1
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1.4 Somatostatin
1.4.1 Somatostatin functions in pancreatic islets
Somatostatin tightly controls the secretion of glucagon and insulin, two major
hormones regulating the glucose homeostasis (Mathias Z. Strowski & Blake, 2008).
There are two major circulating SST-isoforms, which consist of 14 and 28 amino acids
respectively and are processed from a common precursor protein (Mathias Z. Strowski &
Blake, 2008). The primary secretory product of pancreatic D-cells is somatostatin-14
(Patel, Wheatley, & Ning, 1981),which contributes to less than 5% of circulating
somatostatin in adult human (Taborsky & Ensinck, 1984). However, the increase in
postprandial plasma concentration of somatostatin is mostly due to increased circulating
somatostatin-28,which is derived from the intestinal epithelium (Patel & O'Neil, 1988).
The main function of somatostatin in the pancreas is to inhibit the insulin and
glucagon secretion (D. J. Koerker et al., 1974). In addition to its potent antisecretory
effect, somatostatin also inhibits the gene expression of glucagon and insulin (Moller,
Stidsen, Hartmann, & Holst, 2003).
There is much evidence in support of paracrine mechanism of somatostatin-14
action on pancreatic α and β-cells. Firstly, D-cells are in close proximity to α- and β-cells
shown in rat and human islets (Orci & Unger, 1975). Secondly, administration of anti-
somatostatin antibody to bind to endogenous somatostatin in isolated islets resulted in
increased secretion of both insulin and glucagon (Itoh, Mandarino, & Gerich, 1980).
Thirdly, immune-neutralization of pancreatic somatostatin in the perfused isolated
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pancreata showed similar effects (Brunicardi et al., 1994; Kleinman et al., 1994).
Fourthly, the oscillatory pulses of somatostatin and glucagon release are temporally
anti-synchronous (Salehi, Qader, Grapengiesser, & Hellman, 2007). Lastly, the use of
specific somatostatin receptor agonists (Singh et al., 2007), antagonists (Cejvan, Coy, &
Efendic, 2003), somatostatin (Hauge-Evans et al., 2009), and somatostatin receptor
knock-out mice (M. Z. Strowski, Parmar, Blake, & Schaeffer, 2000) have collectively
and uniformly confirmed the paracrine inhibitory action of pancreatic somatostatin on
glucagon and insulin secretion from α- and β-cells.
1.4.2 Somatostatin receptors
Somatostatin induces its effects on the target cells trough five different somatostatin
receptor subtypes (SSTR1-5).
Rat and human pancreatic islets express all five SSTR subtypes, with each SSTR
subtype displaying a distinct distribution on specific islet cells (Mathias Z. Strowski &
Blake, 2008). Human pancreatic α and β-cells show a high expression of SSTR2 (Singh,
et al., 2007; Mathias Z. Strowski & Blake, 2008). The use of specific SSTR agonists
confirmed SSTR2 activity on both α- and β cells in isolated human islets (Singh, et al.,
2007). In isolated human pancreatic islets, SSTR2 is the predominant subtype that
mediates the suppression of glucagon release by somatostatin (Singh, et al., 2007).
Somatostatin nhibition on insulin secretion from β-cell was also shown to be mediated
bySSTR2 in isolated perfused human pancreas (Brunicardi et al., 2003; Moldovan et al.,
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1995) as well as in isolated pancreatic islets of hamsters (Yao, Gill, Martens, Coy, &
Hsu, 2005).
In rodent islets, SSTR2 is the predominant subtype on α-cells while SSTR5 is the
predominant SSTR subtype on β-cells (Mitra et al., 1999). This is consistent with
subsequent studies showing that SSTR2 mediated suppression of glucagon secretion
from α-cells (Cejvan, et al., 2003; M. Z. Strowski, et al., 2000), and that SSTR5 mediated
inhibition of insulin release from pancreatic β-cells (Kailey et al., 2012; Mitra, et al.,
1999; Rossowski & Coy, 1994; M. Z. Strowski et al., 2003; M. Z. Strowski, et al., 2000).
1.4.3 Somatostatin in diabetes
The number of D-cells has been reported to be increased in patients with type 1
diabetes (Orci et al., 1976; Rahier, Goebbels, & Henquin, 1983). These patients also have
an elevated level of basal plasma somatostatin (Segers, De Vroede, Michotte, & Somers,
1989) and pancreatic somatostatin content (Orci, et al., 1976). There are numerous
studies demonstrating increased pancreatic and plasma somatostatin levels as well as the
number of pancreatic D-cells in models of type-1 diabetes, including streptozotocin
(STZ) diabetic rats, Non obese diabetic (NOD) mice and alloxan diabetic dogs (Mancera,
Gomez, & Pisanty, 1995; Orci, et al., 1976; Papachristou, Pham, Zingg, & Patel, 1989;
Patel & Weir, 1976; Rastogi, et al., 1990; Shi et al., 1996). In diabetic bio-breeding
diabetic prone (BBDP) rats, the model we have used in this thesis, the plasma level of
somatostatin was reported to be increased; however the pancreatic content of
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somatostatin was reported to be reduced (Patel, Ruggere, Malaisse-Lagae, & Orci,
1983).
There is evidence that insulin treatment can reduce and/or partially normalize the
elevated plasma somatostatin in diabetic animal models (Papachristou, et al., 1989; Patel,
et al., 1983; Rastogi, et al., 1990) and in type 1 diabetic patients (Segers, et al., 1989).
1.4.4 Somatostatin receptor type 2 antagonist PRL-2903
Various antagonists with different pharmacological selectivity for particular SSTRs
have been synthesized. Of these, the SSTR2 antagonist peptide used in the present thesis
is PRL-2903, also known as DC-41-33 and BIM-234548 (Hocart, Jain, Murphy, Taylor,
& Coy, 1999).This octapeptide, synthesized by Dr. David Coy at Tulane University
(New Orleans, LA, USA) (Hocart, et al., 1999), has a molecular weight of 1160 da, PRL-
2903 is selective for SSTR2 over SSTR3 and SSTR5 by 10 and 40 fold, respectively, and
have negligible binding affinity to SSTR1 and SSTR4 (Hocart, et al., 1999).
1.5 Animal models of type 1diabetes:BBDP diabetic rats Vs Streptozotocin induced diabetic rats
Type 1 diabetes comprises ∼10% of all patients with diabetes mellitus, and its
prevalence is increasing. Affected individuals require lifelong injections of insulin for
survival (Green & Patterson, 2001). The disease results from inflammatory infiltration of
15
the islets of Langerhans (insulitis) and selective destruction of insulin-producing beta
cells (Atkinson & Eisenbarth, 2001) .
There are different rodent models to study type 1 diabetes. Two major models are
streptozotocin (STZ) diabetic rats and Biobreeding (BB) diabetic rats. In the STZ model,
STZ, an antibiotic, induces diabetes by chemical destruction of β-cells. The BB model is
considered to be an autoimmune type 1 diabetic model, thus more closely mimic the
human type-1 diabetes. I will discuss each of these two models.
1.5.1 BBDP diabetic rats
BB rats are the most extensively studied rat model of type-1 diabetes. They are
derived from a Canadian colony of outbred Wistar rats developed in the 1970s, which
exhibited spontaneous hyperglycemia and ketoacidosis (Nakhooda, Like, Chappel,
Murray, & Marliss, 1977). These original colonies were the founders for two subsequent
colonies that were later used to establish all other BB rat colonies. One colony was
established in Worcester, Massachusetts (BB/Wor), and were inbred, and spontaneously
diabetic, and this colonywas formally designated as Worcester diabetes-prone BB rats
(BBDP/Wor) (BIOMERE LLC, Worcester, MA,USA) (Mordes, Bortell, Blankenhorn,
Rossini, & Greiner, 2004). This colony became the commercial source used by many
investigators, including ourselves in this thesis.
BBDP/Wor rats of both sexes develop pancreatic insulitis that is rapidly followed
by selective destruction of beta cells and frank diabetes between 50 and 90 days of age
(Stubbs, Guberski, & Like, 1994). It is noteworthy that the natural course of insulitis in
16
the spontaneously diabetic BB rat is different from other models of autoimmune diabetes
such as the non-obese diabetic (NOD ) mouse, the most commonly used mouse model of
type-1 diabetes. In the BBDP rat, there is no significant or persistent infiltration in the
regions adjacent to the islet (“peri-insulitis”) before progression to frank insulitis and
overt diabetes, which is observed in NOD mouse islets. Insulitis in BB rats is
morphologically similar to that observed in human type 1 diabetes and features a
predominance of Th1-type lymphocytes (Kolb et al., 1996). After the onset of
hyperglycemia, residual “end-stage” islets are small, distorted, and composed
predominantly of non β-cells. Unless treated with exogenous insulin, hyperglycemic BB
rats quickly progress to diabetic ketoacidosis that is fatal (Mordes, et al., 2004).
In the current study, we have employed the diabetic BBDP/Wor rats as the model
of autoimmune diabetes because it etiologically more closely mimics the human
autoimmune diabetes, and because we can track the course of the disease in established
type 1 diabetes. Towards the latter, we wanted the type-1 diabetic model to also mimic
the clinical treatment course of the human disease, that is to be dependent and to receive
exogenous insulin as treatment. This also mimics type 1 diabetic patients who have to
insulin treatments. The STZ-induced diabetic rat model, develop diabetes as the result of
a chemical destruction of the insulin secreting β-cells, which is completely different from
the disease process in human type 1 diabetes.
1.5.2 STZ induced diabetic rats
17
Streptozotocin, an antibiotic derived from Streptomyces achromogenes, is
structurally a glucosamine derivative of nitrosourea. The types of diabetes that is induced
and other characteristics of beta cell damage differ depending on the dose and frequency
of the STZ administered and also the different animal species used. Single diabetogenic
dose of STZ (70- 250mg/kg, body weight) has been demonstrated to induce complete
destruction of beta cells in most species within 24 hour. STZ induces diabetes in almost
all species (Junod et al., 1967). Diabetes can be induced by STZ either by single injection
of STZ or by multiple low dose injections of STZ. STZ is the most commonly used drug
for induction of diabetes in rats. Initial hyperglycemia is observed at one hour after the
STZ injection followed by hypoglycemia; and thena hyperglycemic state observed at 48
hours which peaks at 48- 72 hours; this hyperglycemic state is maintained thereafter
(Bonner-Weir, Trent, Honey, & Weir, 1981).
1.6 Islet cell composition, normal vs. type 1 diabetes
The islets of Langerhans are located in the pancreas. The primary cell types within
the rodent islet are alpha-, beta-, and delta-cells which contribute approximately 15-20%,
60-80%,and 5-10%, respectively (Unger & Orci, 1981). In human, the relative
percentages are 40%, 50% and 10%, respectively (Cabrera et al., 2006).
In a normal rodent islet, alpha-cells form a thin layer of cells on the islet surface
that enwraps the large population of beta-cells which are clustered within the islet core.
In between alpha- and beta-cells interspersed the delta-cells (Unger & Orci, 1976). This
fine anatomical subdivision of rodent islets allows immediate contact of alpha-cells with
18
other islet cell types, and is particularly crucial for the secretion, regulation, and inter-
cellular communication of alpha-cells with other islet cell types. At present, the
underlying mechanisms of these intraislet paracrine interactions remain largely unclear
(Unger & Orci, 1976).
This cellular arrangement within rodent islets does not exist within the human
islets. Alpha-, beta-, and delta-cells are randomly scattered throughout the human islets
and are frequently found aligning along the blood vessels (Cabrera, et al., 2006).
However, despite this difference, alpha-cells remain largely in contact with adjacent
delta- and beta-cells, which, similar to rodent alpha-cells, would have similarly important
functional implications on paracrine regulation.
Studies on STZ diabetic rats showed a highly significant decrease in the number
and volume of insulin immunofluorescent cells per pancreas indicating beta cell
destruction; and an increase in the total number and volume of somatostatin
immunofluorescent delta cells per pancreas. But the number and volume of glucagon
immunofluorescent alpha cells per pancreas did not differ significantly from control rats
despite the apparent increases per diabetic islet. They interpreted these findings as
evidence of D-cell hypertrophy and hyperplasia in diabetes (Orci, et al., 1976).
Studies on BB diabetic rats showed the virtual elimination of betacells and severe
reduction of deltacells early in diabetes. The alphacells were preserved initially, but
eventually they, too, underwent attrition. Thus, the "terminal" islets from chronic diabetic
insulin-treated rats, exhibit 85 to 100% loss of all islet cells. Such a generalized
involvement of all islet cells is probably caused by the islet inflammatory process and is a
19
feature of other forms of diabetes also characterized by insulitis, for example virus-
induced diabetes in mice (Patel, et al., 1983).
In a study of two diabetic human pancreata, islets were clearly less numerous than
in the normal pancreases. No insulin immunofluorescent cells could be identified in the
diabetic islets, which consisted largely of glucagon-immunofluorescent cells;
somatostatin-immunofluorescent cells were also abundant. Morphometric analyses
revealed both cell types to be significantly increased above the control values (Orci, et
al., 1976).
1.7 Rationale
Hypoglycemia has been well recognized to be one of the most serious acute
complications of type-1 diabetes; thus a major limiting factor in intensive insulin
treatment targeted at tight control of blood glucose (Cryer, 2008a).The main objective of
this thesis is to determine a means to improve or prevent hypoglycemia in type-1
diabetes.
In type-1 diabetes, hypoglycemia counter-regulation is impaired, the major
component being the a greatly diminished glucagon secretory response to hypoglycemia
in type =1- diabetes (Cryer, 2008a, 2008b).The precise mechanisms underlying the
impaired glucagon secretory response in type-1 diabetes during hypoglycemia are yet to
be elucidated (Cryer, 2012a). There is considerable evidence that accentuated inhibitory
effect of excessive pancreatic somatostatin on glucagon release in the absence of the
tonic inhibitory effect of insulin compromise the impairment of glucagon counter-
20
regulatory response to hypoglycemia in type-1 diabetes(Gaisano, Macdonald, & Vranic,
2012; Rastogi, et al., 1990; J. T. Yue et al., 2012).
First, it has been suggested that somatostatin released from islet D-cells tightly
controls glucagon release through a local paracrine action (Hauge-Evans, et al., 2009;
Mathias Z. Strowski & Blake, 2008; Unger & Orci, 1977). Second, the number of D-cells
is increased in type-1 diabetic humans and rodents (Mathias Z. Strowski & Blake, 2008).
Third, it has been shown that plasma somatostatin and the pancreatic prosomatostatin
mRNA and somatostatin protein levels are increased in diabetic humans (Orci, et al.,
1976), dogs (Rastogi, et al., 1990), and rodents (Patel, et al., 1983; J. T. Yue, et al.,
2012). Fourth, exogenously administered somatostatin (SST) inhibits the release of
glucagon (Donna J. Koerker et al., 1974)from the endocrine pancreas. Fifth,
administration of a somatostatin receptor antagonist increases the glucagon secretory
response to hypoglycemia in STZ-diabetic rats (J. T. Yue, et al., 2012); and lastly, the
stimulated glucagon secretion was increased in SSTR2 knockout mice (M. Z. Strowski,
et al., 2000).
As mentioned above, it is well established that somatostatin induces its inhibitory
effect on glucagon secretion from α-cells through SSTR2 and this finding has been
supported by studies using the specific SSTR2 agonist (Singh, et al., 2007) and
antagonist (Cejvan, et al., 2003) as well as SSTR2 knockout mice (M. Z. Strowski, et al.,
2000). The use of specific SSTR2 antagonist, PRL-2903 (Hocart, et al., 1999) in isolated
perifused pancreatic islets and isolated perfused pancreata of non-diabetic rats has
confirmed that SST can act as an intra-islet regulator of glucagon secretion in rats,
21
thereby antagonizing the SSTR2, which would explain the enhancement of e arginine -
stimulated glucagon secretion (Cejvan, et al., 2003).
We had previously hypothesized that antagonising the somatostatin action by a
specific SSTR2 antagonist can restore the impaired hypoglycemia countergulation in
type-1 diabetes (J. T. Yue, et al., 2012). However, in that previous study employing the
STZ-induced diabetic rat model, insulin treatment was not required, thus not truly
mimicking the human disease. Nonetheless, antagonising the SSTR2 by PRL-2903 could
normalize the glucagon and corticosterone counter-regulatory responses to insulin-
induced hypoglycemia in the STZ-treated rat model (J. T. Yue, et al., 2012).
The effect of SSTR2 antagonism has been never tested in the autoimmune type-1
BB rat modelwhich resembles human type 1 diabetes in many aspects. Furthermore, it
would be of particular interest to explore the effect of SSTR2 antagonism on restoring the
counter-regulatory hormones secretory responses to hypoglycemia in diabetic BB rats
that receive exogenous insulin treatment using slow releasing subcutaneous insulin
pellets which clinically mimics the insulin therapy in diabetic patients.
0To investigate the specific and direct effects of SSTR2 blockade on the pancreatic
alpha-cells to cause increasein glucagon secretion independent of any confounding
systemic factors, we have employed the thin pancreatic slices perifusion method.
Previously, we had used the thin pancreatic slices to study the in-situ electrophysiology
of the particular ion channels in mouse pancreatic α- and β-cells (Huang, Rupnik, &
Gaisano, 2011; Huang et al., 2012). This is the first time that the thin fresh pancreatic
slices have been used in a perifusion setting to explore the real-time hormonal secretion
22
from diabetic rat and normal human pancreata. By using this approach we have
surmounted the technical limitations of the conventional methods being used to study the
pancreatic alpha cells (Huang, et al., 2011; Speier & Rupnik, 2003), particularly in
diabetic pancreatic islets. Since the islets are extremely damaged in diabetes,
conventioanl methods of islet isolation and dispersion into single cells are not reliable;
and this in part explains why previous reports have been inconsistent with regards to the
functions and cellular interactions of islet cells (Speier & Rupnik, 2003; Speier, Yang,
Sroka, Rose, & Rupnik, 2005). The focus of my thesis study is to investigate the
paracrine interaction of D-cellsomatostatin and alpha-cell glucagon secretory response in
a type-1 BB rat model. This assay of pancreas slice perifuion would be a more ideal
method to study the pancreas without interference of other factors like the central and
autonomic nervous systems present in the in-vivo settings or in the ex-vivo isolated
whole-pancreas perfusion assay. Furthermore, by deploying this technique to human
pancreas, we have been able to study the normal human pancreatic islets also in situ in
their normal architecture by using a very small fresh pancreatic specimens obtained from
surgical resection of pancreatic cancer. The latter would provide initial insight as to
whether these findings of SSTR2 blockage in enhancing glucagon secretion during
hypoglycemia can be translatable to humans.
1.8 Hypothesis
We hypothesise that the impaired glucagon counter-regulation in type 1
diabetes is due to the accentuated somatostatin inhibitory effect on α-cells in the
23
absence of endogenous insulin; thus antagonising the somatostatin receptor type 2,
in order to block the somatostatin inhibitory effect on α-cells would improve the
compromised glucagon counter-regulatory response to hypoglycemia in
autoimmune type 1 diabetic BBDP rats. (Figure 1-4)
Figure 1-4. Alpha, beta and delta cell interactions during hypoglycemia. In absence of
the tonic effect of insulin, somatostatin is the only endogenous inhibitor of glucagon release
that exerts a strong inhibitory effect on α-cell. Therefore, when an antagonist blocks the
somatostatin receptors on α-cell, despite the inhibitory effect of the injected insulin, α-cell
can release normal amount of glucagon during hypoglycemia (Gaisano, et al., 2012; Vranic,
2010).
24
1.9 Objectives
1. To investigate in the autoimmune type-1diabetic model - diabetic BB rats the in
vivo effects of SSTR2 antagonism on restoring counter-regulatory glucagon
secretory responses by employing hyper-insulinemic hypoglycemic clamps.
2. To explore the catecholamines (epinephrine and noreoinephrin) and corticosterone
responses to hypoglycemia in type 1 diabetic BBDP rats and whether these
responses will be improved by antagonising the SSTR2 during the hyper-
insulinemic hypoglycemic clamp.
3. To unequivocally demonstrate and confirm the specific action of SSTR2 blockade
on increasing glucagon secretion by employing the pancreatic slices perifusion
method in diabetic, non-diabetic BB rats.
4. To deploy the pancreatic slices perifusion technique to normal human pancreas in
order to demonstrate the specific action of SSTR2 blockade on increasing
glucagon secretion.
25
Chapter 2 Research design and methods
2.1 In-vivo Studies
2.1.1 Animals
All procedures were in accordance with Canadian Council on Animal Care
Standards and were approved by the University of Toronto Animal Care Committee.
Male diabetic prone BB/Wor rats (BIOMERE LLC, Worcester, MA,USA) were
housed in a sterile animal facility, fed a standard pellet diet, and maintained on a 12-h/12-
h day/night cycle. The age-matched BBDP rats which did not become diabetic at age
range of 60-150 days, were used as controls. Diabetic rats were treated by subcutaneous
implantation of 1.5-2 insulin pellets (Linplants; LinShin Canada, Scarborough, Toronto,
ON, Canada) once they become diabetic (random BS> 22mmol/l on two sequential
measurements). The implants released insulin at ∼2 units/24 h.
2.1.2 Somatostatin receptor type2 antagonist (PRL-2930) solution
SSTR2a (PRL-2930) was generously provided by Dr. David H. Coy (Tulane
University, New Orleans, LA)(Hocart, et al., 1999). The SSTR2a solution was prepared
by dissolving a calculated dose of the antagonist in 1% acetic acid and 0.9% saline.
26
2.1.3 Surgical procedures and hypoglycemic clamp experiments
Animals were studied in 4 different groups: Diabetic vehicle (n=8),
Diabeitic+SSTR2a (n=8), Non-Diabetic vehicle (n=5) and Non-Diabetic+SSTR2a (n=6).
The Surgery and the clamp procedures were performed in accordance with our
previous study in STZ-treated diabetic rats (J. T. Yue, et al., 2012). Surgery was
performed 7 days prior to the hypoglycaemic clamp experiments. In case of BBDP
diabetic rats, this was 14 days after the insulin pellet implantation. Under general
anaesthesia, by isoflurane inhalation, the left carotid artery and right jugular vein were
catheterized for blood sampling and infusion of test substances, respectively.
Immediately after the clamp experiments, the rats were sacrificed and the whole
pancreata were dissected, frozen on dry ice and transferred to -80°C for latter
determinations of total pancreatic content of somatostatin and glucagon. The whole
pancreata of the age-matched BBDP Diabetic (± insulin pellet implantation) and non-
diabetic rats which had not undergone hypoglycemic clamp study were also dissected and
preserved.
On the day of the clamp experiment, the rats were weighed, connected to infusion
catheters and acclimatized for 1 hour. Any stress trigger was strictly avoided during this
period and throughout the whole hypoglycemic clamp procedure. After obtaining a
baseline blood sample at t = -60 min, the rats underwent a 3-hour infusion of vehicle (1%
acetic acid in 0.9% saline) or SSTR2a (1500 nmol/kg/h) at an infusion rate of 1ml/h with
a digital syringe infusion pump (Harvard Apparatus, Holliston, MA, USA). In order to
induce hypoglycaemia to a target level of 3±0.5mmol/l, insulin infusion at the constant
27
rate of 20-50 mU/kg/min together with variable rates of glucose infusion were started at t
= 0 min.
During the clamp, blood samples were obtained from the carotid catheter every 10
minutes in capillary tubes coated with Kalium-EDTA (Microvette CB 3000, Sarstedt
Inc., Montreal, QC Canada) and immediately centrifuged in room temperature in order to
measure the plasma glucose levels using a glucose analyzer (Analox Glucose analyzer,
Analox instrument, London, UK).
Figure 2-1
28
2.1.4 Plasma hormone measurements
Blood samples were obtained at t = -60 min, then every 30 minutes from t=0 min in
ice-chilled tubes containing 5µl of 100mM EDTA (Ethylenediaramine tetraacetic acid)
solution and 30 KIU aprotinin (APR600, BioShop Canada Inc., Burlington, ON, Canada
); followed by centrifugation at 12000 rpm at 4°C to separate the plasma. The plasma
was aliquoted in multiple tubes and immediately frozen on dry ice and transferred to -
80ºC awaiting measurements of glucagon by radioimmunoassay (rat glucagon RIA kit,
EMD Millipore, Darmstadt, Germany), and corticosterone, nor/epinephrine by enzyme-
linked immunosorbent assays (corticosterone ELISA kits by ALPCO Diagnostics, Salem,
NH, USA; nor/epinephrine by 2-Cat Plasma ELISA kit, Labor Diagnostica Nord GmbH
& Co. KG, Nordhorn, Germany), and somatostatin by enzyme immunoassay (Extraction
free, rat somatostatin-14 EIA kit, Bachem Group, Bubendorf, Switzerland).
2.1.5 Total pancreatic glucagon and somatostatin protein content
The frozen harvested pancreata were placed in acid-ethanol mixture (1.5%HCl in
70% EtOH), incubated twice overnight at -20ºC. Between the two incubation periods, the
pancreata were homogenized; following centrifugation, and neutralization by 1M pH 7.5
Tris buffer, somatostatin and glucagon levels were determined by RIA and EIA,
respectively. The derived hormone concentrations were then normalized to the total
29
pancreatic protein content determined by modified Lowry method (Markwell, Haas,
Bieber, & Tolbert, 1978).
2.2 In-vitro studies
2.2.1 Animals
Male diabetic prone BB/Wor rats (BIOMERE LLC, Worcester, MA, USA) (N=6)
were used for the in-vitro studies. The age-matched BBDP rats which did not become
diabetic at age range of 60 to 150 days, were used as controls (N=6). The insulin
treatment was as previously mentioned in the in-vivo section.
2.2.2 Human pancreas specimens
All procedures which involved human pancreas tissue were approved by the
University Health Network Research Ethics Board (Protocol #10-0393-T) and written
consents were obtained from the source patients.
The human pancreas specimens were obtained from the normal margins of the
resected pancreata of the pancreatic cancer patients who had been operated at Toronto
General Hospital. Immediately after the resection, the specimens were sent to the surgical
pathology laboratory to be evaluated, and a normal portion not required for pathology
review, kept in extra cellular solution prior to the experiments.
2.2.3 Pancreatic slices preparation
30
Pancreatic slices were prepared as previously described (Huang, et al., 2011;
Huang, et al., 2012). After sacrificing the rat, the rat abdominal cavity was immediately
opened, and the common bile duct clamped. 3 ml of 37◦C low melting 1.9% agarose gel
(15517-022, Invitrogen, Camarillo, CA) was injected into the pancreas via the proximal
end of the common bile duct using a PE-50 tube. The injected pancreas was resected and
cut into smaller pieces which were then embedded in 1.9% agarose gel and cooled down
to become solidified (Figure 2-1). The tissue blocks were placed in Carbogen (5% CO2,
95% O2) bubbled ice-cold extracellular solution and were sliced into 4×4 mm, 140μm -
thick slices at a blade frequency of 70 Hz by a vibrating blade microtome (Vibratome,
Leica Microsystems, Mannheim, Germany)(Figure 2-2 A&B). The extracellular solution
bathing the pancreas slices was composed of (in mmol/l) 125 NaCl, 2.5 KCl, 1 MgCl2, 2
CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 2 Na pyruvate, 0.25 ascorbic acid, 3 myo-inositol,
6 lactic acid, 7 glucose.
The human pancreas slices were cut out of the agarose gel embedded tissue blocks
using the same procedures as mentioned above.
Figure 2-2
Figure 2-2. Pancreatic tissue portions embedded in 1.9% agarose gel and cooled down to become solidified. Courtesy of Dr.Ya-chi Huang 2012
31
Figure 2-3. The tissue blocks were placed in Carbogen (5% CO2, 95% O2) bubbled ice-cold extracellular solution and were sliced into 4×4 mm, 140μm -thick slices at a blade frequency of 70 Hz by a vibrating blade microtome (Vibratome, Leica Microsystems, Mannheim, Germany) .Courtesy of Dr. Ya-chi Huang 2012.
Figure 2-3.
Figure 2-4
Figure 2-4. Perifusion pump and chambers. The pancreatic slices were loaded into the perifusion chambers at 37°C and were perifused by different solutions.The perfusate was collected in ice chilled tubes containing aprotinin.
32
2.2.4 Pancreas slices perifusion studies
HEPES balanced KRBB solution (KRBH) containing in mmol/l: 135 Nacl, 3.6
KCl, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2, 2 NaHCO3, 10 HEPES, 1 or 7 glucose (as
indicated in the perifusion protocol) and 1 g/L BSA was used as the perifusion solution
with the flow rate of 1ml/min. Arginine and SSTR2a (PRL-2903) at concentrations of 20
mmol/l and 30 µmol/ml, respectively, were added to the perifusion solution as indicated
in the study protocols.
Rat pancreas slice perifusion studies were carried out in the following 4 groups:
Diabetic, Diabetic+SSTR2a, Non-Diabetic, Non-Diabetic+SSTR2a. 10 pancreatic slices
where loaded into each perifusion chamber (Figure 2-3); the perifusion protocol was
initiated with a 20-min equilibration period with KRBH containing 7mmol/l glucose
which was followed by KRBH containing 1mmol/l glucose (30 minutes), 7mmol/l
glucose (10 minutes) and 1mmol/l glucose plus 20mmol/l arginine (30 minutes),
respectively. For the Non-diabetic and Diabetic groups treated with SSTR2a, the slices
were perifused with SSTR2a at a concentration of 30µmol/ml for the whole duration of
the perifusion procedure (including the equilibration period).
The human pancreas slices were perifused by loading 15-20 slices per chamber.
The perifusion protocol was as follows: perifusion of KRBH containing 7mmol/l glucose
(15 minutes as the equilibration period) followed by 1mmol/l glucose (30 minutes),
1mmol/l glucose + 30 µmol/ml SSTR2a (30 minutes) and finally a KRBH containing
1mmol/l glucose and 20mmol/l arginine (20 minutes).
33
The samples were collected at 1-min intervals in ice chilled tubes containing
1000KIU aprotinin; the tubes were frozen and stored at -80°C awaiting determination of
glucagon levels by RIA. At the end of the experiments, the slices were collected from
each chamber and were preserved in acid-ethanol mixture (1.5% HCl in 70% ethanol)
and kept at -80°C for the purpose of determining the total glucagon content of the slices.
Figure 2-5
Figure 2-6
34
3.2.5 Pancreatic slices perifusion secretory glucagon and insulin measurements
The secretory glucagon and insulin levels from the perifusion samples were
measured using radioimmunoassay (rat glucagon and insulin RIA kit, EMD Millipore,
Darmstadt, Germany). The total glucagon and insulin content of the slices were measured
by Acid ethanol (1.5%HCl in 70%EtOH) extraction method as previously mentioned in
the in-vivo section of the methods.
2.3 Statistical analysis
Data are presented as mean ± SEM for a given number of observations. Groups of
data were compared using ANOVA with Tukey post-hoc test or two tailed
paired/unpaired t test (Graphpad, PRISM software, USA), with significance being
accepted if P < 0.05.
35
Chapter 3 Results
3.1 In-vivo studies
3.1.1 Plasma glucose levels during the hyperinsulinemic hypoglycemic clamp experiments
The animals received 1500 nmol/kg/hr SSTR2a or Vehicle solution IV from the
beginning of the clamp experiments. Insulin infusion at the rate of 20-50 mU/kg/min
along with 50% glucose solution infusion at variable rates were started at t=0. All of the
groups reached the target hypoglycemic level (3±0.5 mmol/l) 70 minutes after the start of
the insulin infusion and remained at the same level until the end of the experiment.
(Figure 3-1)
3.1.2 Glucose infusion rates during the hyperinsulinemic hypoglycemic clamp experiments
The Non-Diabetic SSTR2a group, Non Diabetic Vehicle group and the Diabetic
SSTR2a group required very low to no glucose to become and remain hypoglycemic at
the target level of 3±0.5 mmol/l. In contrast, the Diabetic Vehicle group had very high
glucose requirements in order to maintain the hypoglycemia at the target level of 3±0.5
mmol/l. (Figure 3-2)
3.1.3 Insulin infusion rates during hyperinsulinemic hypoglycemic clamp experiments
36
The Diabetic Vehicle, Diabetic SSTR2a and the Non Diabetic Vehicle groups
received 20 or 30 mU/Kg/min insulin infusion via their jugular vein in order to induce
and maintain the target hypoglycemic level during the clamp experiments. The mean ±
SEM infusion rates were not significantly different between the Diabetic Vehicle
(25±1.89), Diabetic SSTR2a (25.56±1.75) and Non Diabetic Vehicle (26±2.44) groups;
however we had to infuse 50 mU/Kg/min for the animals in the Non Diabetic SSTR2a
group in order to induce the target hypoglycemic level. These results would suggest that
the non-diabetic BB rats receiving SSTR2a treatment became insulin resistant. (Figure 3-
3)
3.1.4 Plasma hormones levels during hyperinsulinemic hypoglycemic clamp experiments
Glucagon secretion during hypoglycemia was much blunted in Diabetic Vehicle
group compared to the Non Diabetic Vehicle group (p<0.0001). Glucagon secretion
during hypoglycemia was increased in Diabetic SSTR2a group by almost 2.3 fold
compared to Diabetic Vehicle group (p=0.005), and this increase showed full restoration
to the same level as the Non Diabetic Vehicle group (p=0.94). SSTR2a treatment also
enhanced the glucagon secretion in Non Diabetic SSTR2a group by nearly 3.3 fold
compared to Non Diabetic Vehicle group, Diabetic SSTR2a and Diabetic Vehicle
groups(p<0.0001). (Figure 3-4 A & B)
Corticosterone secretion in Diabetic Vehicle was not blunted and in fact increased
during hypoglycemia, compared to Non Diabetic Vehicle group (p=0.009). SSTR2a
treatment in Diabetic SSTR2a group resulted in enhancement of corticosterone secretion
37
compared to Diabetic Vehicle (p=0.019), Non Diabetic Vehicle (p=0.0041) and Non
Diabetic SSTR2a (p=0.006). Corticosterone secretion remained unchanged in Non
Diabetic SSTR2a group compared to Non-Diabetic Vehicle group (p=0.412). (Figure 3-5.
A & B)
Epinephrine secretion was unchanged by the SSTR2a treatment in Diabetic
(p=0.975) and Non Diabetic (p=0.249) groups. Also, the epinephrine response was not
compromised in Diabetic vehicle compared to Non Diabetic Vehicle (p=0.412). (Figure
3-6 A & B)
Norepinephrine secretion remained unchanged by the SSTR2a treatment in Diabetic
(p=0.626) and Non Diabetic (p=0.059) groups. Diabetes did not affect the norepinephrine
response to hypoglycemia. (Figure 3-7 A & B)
Plasma Somatostatin AUC calculations demonstrated that SSTR2a treatment
increased the plasma somatostatin during hypoglycemia in Non Diabetic SSTR2a group
by almost 3 fold compared to Non Diabetic Vehicle, Diabetic Vehicle and Diabetic
SSTR2a groups (p<0.0001). Plasma somatostatin remained unchanged during
hypoglycemia in Diabetic SSTR2a group compared to Diabetic Vehicle (p=0.735) and
also in Diabetic Vehicle group compared to Non Diabetic Vehicle (p=0.173). (Figure 3-8
A &B)
3.1.5 The whole pancreatic glucagon and somatostatin protein levels immediately following the hyperinsulinemic hypoglycemic clamp experiments
38
The whole pancreatic glucagon protein content was significantly lower in the
Diabetic SSTR2a group immediately after the hypoglycemic clamp compared to Diabetic
Vehicle group (p=0.0009), suggesting that the α-cells could secrete more glucagon in
response to hypoglycemia in SSTR2a-treated diabetic rats compared to the Diabetic
Vehicle group. There was no significant difference between the glucagon protein content
of the pancreata between the Diabetic Vehicle, Non-Diabetic Vehicle and Non-Diabetic
SSTR2a groups following hypoglycemia, confirming that while there might be enough
synthesis of glucagon in α-cells in diabetic rats treated by insulin (Diabetic Vehicle
group), the glucagon response to hypoglycemia still remains blunted. (Figure 3-9)
The pancreatic somatostatin content following hypoglycemia was slightly lower in
Diabetic SSTR2a group compared to Diabetic Vehicle; however, the difference was not
significant (p=0.057). SSTR2a treatment elevated the total pancreatic somatostatin
content in the Non Diabetic SSTR2a group compared to Non Diabetic Vehicle; however,
the difference was not significant (p=0.055). Diabetic Vehicle and Diabetic SSTR2a had
lower pancreatic somatostatin contents compared to Non Diabetic SSTR2a group
(p=0.002 and 0.0003 respectively). Also, while the pancreatic somatostatin content was
not different between the Diabetic Vehicle and the Non Diabetic Vehicle (p=0.102),
SSTR2a treatment significantly decreased the pancreatic somatostatin content following
hypoglycemia in Diabetic SSTR2a group compared to Non Diabetic Vehicle (p=0.004).
Overall it can be postulated that hypoglycemia results in more somatostatin-14 synthesis
in D-cells of the non-diabetic BB rats. (Figure 3-10)
39
3.1.6 The whole pancreatic glucagon and somatostatin protein levels in the diabetic and non diabetic BB rats not undergone the clamp
The insulin-treated diabetic BBDP rats had a significantly lower pancreatic
glucagon protein content compared to untreated diabetic and non diabetic BB rats
(p=0.005 and 0.003 respectively), suggesting that the α-cells become more primed to
secrete glucagon in insulin-treated diabetic rats. There was no significant difference
between the pancreatic glucagon content of untreated diabetic rats and the non diabetic
rats (p=0.827). (Figure 3-11)
The insulin-treated diabetic BBDP rats had also a significantly lower pancreatic
somatostatin protein content compared to untreated diabetic and non diabetic BB rats
(p=0.0006 and 0.004 respectively), suggesting that insulin treatment could correct the
high levels of pancreatic somatostatin observed in the untreated diabetic BB rats to some
extent. Although the pancreatic somatostatin content was slightly higher in untreated
diabetic rats compared to the non diabetic rats, the difference was not statistically
significant (p=0.277). (Figure 3-12)
3.2 In-vitro studies
3.2.1 Diabetic BBDP and non diabetic BBDP rat pancreatic slices glucagon secretion in perifusion studies
We next assessed the direct effects of SSTR2a on a-cell glucagon secretion without
any confounding systemic factors. We thus employed the pancreatic slice technique
40
(Huang, et al., 2011; Huang, et al., 2012; Speier & Rupnik, 2003). SSTR2a treatment
significantly increased glucagon secretion from the diabetic BBDP pancreatic slices in
hypoglycemic and basal conditions by approximately 2 fold compared to the control
(p=0.015 and 0.047 respectively). In addition, SSTR2a treatment significantly enhanced
the arginine-stimulated glucagon secretion from the diabetic BBDP pancreatic slices
compared to control diabetic BBDP rats (p=0.003). (Figure 3-13 A & B)
SSTR2a treatment also significantly increased the glucagon secretion from the non-
diabetic BBDP pancreatic slices in hypoglycemic and basal conditions by approximately
2.8 fold compared to the control (P=0.001 and 0.009 respectively). In addition, SSTR2a
treatment significantly enhanced the arginine stimulated glucagon secretion from the
non-diabetic BBDP pancreatic slices compared to the control non-diabetic BBDP rats
(p=0.015)(Figure 3-14 A & B.)
3.2.2 Human Pancreatic Slices Glucagon and Insulin Secretion in Perifusion Studies
We next examined whether these observations made on the rat model are
translatable to the human clinical situation, employing human pancreas slices which were
prepared from normal portions of pancreatic cancer resections. The glucagon secretion
from the human pancreatic slices after a short transition showed an obvious enhancement
in response to low glucose when SSTR2a was added to the perifusion solution. SSTR2a
also enhanced arginine-stimulated release in the low glucose (1mmol/l) condition,
which was observed as a prominent peak. AUC calculations of these data from perifusion
41
assay on human pancreas slices confirmed the enhanced glucagon secretory responses to
hypoglycemia by the SSTR2a treatment (Figure 3-15 A & B).
We also measured the simultaneous insulin secretion from the human pancreatic
slices during hypoglycemia, as well as when treated with SSTR2a or when arginine.
Shortly after adding SSTR2a to the solution, insulin secretion from the slices was
increased. Adding arginine resulted in a sharp increase in the insulin secretion from the
slices (Figure 3-15 C). This served as a positive control. However, since we had only
two replicates of these experiments, we were not able to perform any statistical analysis.
The human samples were scarce, and I intend to obtain at least two more human samples.
42
Figure 3-1. Plasma glucose levels during hyperinsulinemic hypoglycemic clamps experiments. The animals have received 1500 nmol/kg/hr SSTR2a or Vehicle solution IV from the beginning of the clamp experiments. Insulin infusion at the rate of 20-50 mU/kg/min along with 50% glucose solution infusion at variable rates were started at t=0. Plasma glucose levels are shown as mean ± SEM for each group at different time points. All of the groups have reached the target hypoglycemic level (3±0.5 mmol/l blood glucose) from t=70min.
43
Figure 3-2.Glucose infusion rates during hyperinsulinemic hypoglycemic clamp experiments. The Non Diabetic SSTR2a, Non Diabetic Vehicle and the Diabetic SSTR2a required very low to no glucose to become and remain hypoglycemic at the target level of 3±0.5 mmol/l blood glucose. In contrast, the Diabetic Vehicle group had very high glucose requirements in order to maintain the hypoglycemia at the target level of 3±0.5 mmol/l blood glucose.
44
Figure 3-3. Insulin infusion rates distribution between the four groups during hyperinsulinemic hypoglycemic clamp experiments. The Diabetic Vehicle, Diabetic SSTR2a and the Non Diabetic Vehicle groups received 20 or 30 mU/Kg/min Insulin infusion via the jugular vein in order to induce and maintain the target hypoglycemic level during the clamp experiments. The mean ±SEM insulin infusion rates were not significantly different between the Diabetic Vehicle (25±1.89), Diabetic SSTR2a (25.56±1.75) and Non Diabetic Vehicle (26±2.44) groups; however we had to infuse more insulin at 50 mU/Kg/min for the animals in the Non Diabetic SSTR2a group in order to induce the target hypoglycemic. Data are shown as the individual insulin infusion rates for each animal in each group along with the mean ± SEM for each group.
45
Figure 3-4. A. Plasma glucagon levels during hyperinsulinemic hypoglycemic clamp experiments. Data are shown as mean ± SEM of glucagon secretion for each time point for different groups. B. Area under curve analysis of glucagon secretion in plasma during the hypoglycemic period from t=60 to t=120 min. Data are shown as mean ± SEM of AUCs for each group. Glucagon secretion during hypoglycemia is increased in Diabetic SSTR2a group by almost 2.3 folds compared to Diabetic Vehicle group (p=0.005), and this increase shows full restoration to the same level as the Non Diabetic Vehicle group (p=0.94). Otherwise, glucagon secretion during hypoglycemia is much blunted in Diabetic Vehicle group compared to the Non Diabetic Vehicle group (p<0.0001). SSTR2a treatment also enhanced the glucagon secretion in Non Diabetic SSTR2a group by nearly 3.3 folds compared to Non Diabetic Vehicle group, Diabetic SSTR2a and Diabetic Vehicle groups(p<0.0001).
(***: p<0.001 and **: p<0.01)
Figure 3- 4. A.
46
Figure 3- 4. B.
47
Figure 3-5. A. Plasma corticosterone levels during hyperinsulinemic hypoglycemic clamp experiments. Data are shown as mean ± SEM of corticosterone secretion for each time point for different groups. B. Area under curve analysis of corticostrone secretion in plasma during the hypoglycemic period from t=60 to t=120 min. Data are shown as mean ± SEM of AUCs for each group. Corticosterone secretion in Diabetic Vehicle is not blunted and in fact increased during hypoglycemia, compared to Non Diabetic Vehicle group (p=0.009). SSTR2a treatment in Diabetic SSTR2a group resulted in enhancement of corticosterone secretion compared to Diabetic Vehicle (p=0.019), Non Diabetic Vehicle (p=0.0041) and Non Diabetic SSTR2a (p=0.006). Corticosterone secretion remained unchanged in Non Diabetic SSTR2a group compared to Non-Diabetic Vehicle group (p=0.412). (**: p<0.01 and *: p<0.05)
Figure 3- 5. A.
48
Figure 3- 5. B.
AUC
49
Figure 3-6. A. Plasma epinephrine levels during hyperinsulinemic hypoglycemic clamp experiments. Data are shown as mean ± SEM of epinephrine secretion for each time point for different groups. B. Area under curve analysis of epinephrine secretion in plasma during the hypoglycemic period from t=60 to t=120 min. Data are shown as mean ± SEM of AUCs for each group. Epinephrine secretion remained unchanged by the SSTR2a treatment in Diabetic (p=0.975) and Non Diabetic (p=0.249) groups. Also, the epinephrine response is not blunted in Diabetic vehicle compared to Non Diabetic Vehicle (p=0.412)
Figure 3- 6. A.
50
Figure 3-6. B.
51
Figure 3-7. A. Plasma norepinephrine secretory levels during hyperinsulinemic hypoglycemic clamp experiments. Data are shown as mean ± SEM of norepinephrine secretion for each time point for different groups. B. Area under curve analysis of norepinephrine secretion in plasma during the hypoglycemic period from t=60 to t=120 min. Data are shown as mean ± SEM of AUCs for each group. Norepinephrine secretion remained unchanged by the SSTR2a treatment in Diabetic (p=0.626) and Non Diabetic (p=0.059) groups. Also, the norepinephrine response is not blunted in Diabetic vehicle compared to Non Diabetic Vehicle (p=0.126)
Figure 3-7. A.
52
Figure 3- 7. B.
53
Figure 3-8. A. Plasma somatostatin levels during hyperinsulinemic hypoglycemic clamp experiments. Data are shown as mean ± SEM of somatostatin secreted in plasma for each time point for different groups. B. Area under curve analysis of somatostatin secretion in plasma during the hypoglycemic period from t=60 to t=120 min. Data are shown as mean ± SEM of AUCs for each group. Plasma somatostatin remained unchanged during hypoglycemia in Diabetic SSTR2a group compared to Diabetic Vehicle (p=0.735) and also in Diabetic Vehicle group compared to Non Diabetic Vehicle (p=0.173). SSTR2a treatment increased the plasma somatostatin during hypoglycemia in Non Diabetic SSTR2a group compared to Non Diabetic Vehicle, Diabetic Vehicle and Diabetic SSTR2a groups(p<0.0001). (***: p<0.001)
Figure 3- 8. A.
54
Figure 3- 8. B.
55
Figure 3-9. Whole pancreatic glucagon protein contents following hyperinsulinemic hypoglycemic clamp experiments. Data are shown as mean ± SEM of the whole pancreatic glucagon normalized to the total protein content of the pancreata. Diabetic SSTR2a group had a significantly lower pancreatic glucagon protein content compared to Diabetic Vehicle (p=0.0009), Non diabetic Vehicle (p=0.009) and Non Diabetic SSTR2a (p=0.002) groups. There was no significant difference between the pancreatic glucagon content of Diabetic Vehicle, Non Diabetic SSTR2a and Non Diabetic Vehicle groups. (***: p<0.001 and **: p<0.01)
56
Figure 3-10. Whole pancreatic somatostatin protein content following hyperinsulinemic hypoglycemic clamp experiments. Data are shown as mean ± SEM of the whole pancreatic somatostatin normalized to the total protein content of the pancreata. The pancreatic somatostatin content following hypoglycemia was slightly lower in Diabetic SSTR2a group compared to Diabetic Vehicle; however, the difference was not significant (p=0.057). SSTR2a treatment elevated the total pancreatic somatostatin content in the Non Diabetic SSTR2a group compared to Non Diabetic Vehicle; however, the difference was not significant (p=0.055). Diabetic Vehicle and Diabetic SSTR2a had lower pancreatic somatostatin contents compared to Non Diabetic SSTR2a group (p=0.002 and 0.0003 respectively). Also, while the pancreatic somatostatin content was not different between the Diabetic Vehicle and the Non Diabetic Vehicle (p=0.102), SSTR2a treatment significantly decreased the pancreatic somatostatin content following hypoglycemia in Diabetic SSTR2a group compared to Non Diabetic Vehicle (p=0.004). (***: p<0.001 and **:p<0.01)
57
Figure 3-11. The whole pancreatic glucagon protein contents of BBDP rats in absence of hypoglycemia. Data are shown as mean ± SEM of the whole pancreatic glucagon normalized to the total protein content of the pancreata. The insulin-treated diabetic BBDP rats had a significantly lower pancreatic glucagon protein content compared to untreated diabetic and non diabetic BB rats(p=0.005 and 0.003 respectively).There was no significant difference between the pancreatic glucagon content of untreated diabetic rats and the non diabetic rats(p=0.827)(**: p<0.01)
58
Figure 3-12. The whole pancreatic somatostatin protein contents of BBDP rats in absence of hypoglycemia. Data are shown as mean ± SEM of the whole pancreatic somatostatin normalized to the total protein content of the pancreata. The insulin-treated diabetic BBDP rats had a significantly lower pancreatic glucagon protein content compared to untreated diabetic and non diabetic BB rats (p=0.0006 and 0.004 respectively). Although the pancreatic somatostatin content was slightly higher in untreated diabetic rats compared to the non diabetic rats, the difference was not statistically significant (p=0.277)
(***: p<0.001 and **: p<0.01)
59
Figure 3-13.A. Glucagon secretion from diabetic BBDP rat pancreatic slices. Data are shown as the mean ± SEM of glucagon secreted from the slices normalized to the total glucagon content of the slices for each group during the perifusion. B. Area under curve analysis of the glucagon secretion. Data are shown as mean ± SEM of AUCs for each group. SSTR2a treatment has significantly increased the glucagon secretion by approximately 2 folds at 1mmol/l glucose (p=0.015), 7mmol/l glucose (p=0.047) and 7mmol/l glucose+ 20 mmol/l arginine (p=0.003) conditions compared to control. (**: p<0.01 and *: p<0.05)
Figure 3-13. A.
60
Figure 3-13. B.
61
Figure 3-14.A. Glucagon secretion from Non diabetic BBDP rat pancreatic slices. Data are shown as the mean ± SEM of glucagon secreted from the slices normalized to the total glucagon content of the slices for each group during the perifusion. B. Area under curve analysis of the glucagon secretion. Data are shown as mean ± SEM of AUCs for each group. SSTR2a treatment increased the glucagon secretion by approximately 2.8 folds in 1mmol/l glucose (p=0.001), 7mmol/l glucose (p=0.009) and 7mmol/l glucose+ 20 mmol/l arginine (p=0.015) conditions compared to control. (***: p<0.001, **: p<0.01 and *: p<0.05)
Figure 3-14. A.
62
Figure 3-14. B.
63
Figure 3-15. Glucagon and insulin secretion from perifused human pancreatic slices. A. Data shows the mean ± SEM of glucagon secreted from the pancreatic slices in different conditions normalized to the total glucagon content of the slices. B. Data shows the mean ± SEM of insulin secreted from the pancreatic slices in the different conditions normalized to the total insulin content of the slices. Since the N for experiments was 2, statistical analysis was not performed.
Figure 3-15. A.
64
Figure 3-15. B.
65
Chapter 4 Discussion
It is well established that the glucagon counterregulatory response to hypoglycemia
is compromised in type 1 diabetes (Cryer, 2008a, 2008b). There is a wealth of evidence
regarding the inhibitory effect of somatostatin on glucagon secretion from pancreatic α-
cells; there are also numerous studies suggesting a local paracrine inhibitory effect by
pancreatic somatostatin (Brunicardi, et al., 1994; Cejvan, et al., 2003; Itoh, et al., 1980;
Kleinman, et al., 1994; Salehi, et al., 2007; M. Z. Strowski, et al., 2000). It is also well
established that somatostatin induces its inhibitory effects on α-cells via SSTR2 in
humans (Singh, et al., 2007; Mathias Z. Strowski & Blake, 2008) and rodents (Cejvan, et
al., 2003; M. Z. Strowski, et al., 2000).
It had been proposed that the excessive somatostatin levels observed in type 1
diabetic human (Segers, et al., 1989), rodents and dog plays a role in the impairment of
glucagon counterregulation response to hypoglycemia (Rastogi, et al., 1990; J. T. Yue, et
al., 2012). Considering the exclusive role of SSTR2 on pancreatic α-cells in mediating
the inhibitory effects of somatostatin on glucagon secretion, it was hypothesised that
antagonising SSTR2 would improve glucagon counterregulation in response to
hypoglycemia (J. T. Yue, et al., 2012). This hypothesis was first tested in STZ diabetic
rats and it was observed that antagonising SSTR2 fully restores the glucagon and
corticosterone counterregulation to insulin-induced hypoglycemia in STZ diabetic rats (J.
T. Yue, et al., 2012). The STZ model is however not a genuine disease model of humans
66
in which T1D is an autoimmune disorder. Thus in the current study, I had employed a
well accepted autoimmune T1D model that resembled human T1D, and that is the
diabetic BBDP rat (Bortell & Yang, 2012). In the present study for the first time we
demonstrate that antagonising the SSTR2 in insulin treated autoimmune type 1 diabetic
BBDP rats, fully restores the glucagon counterregulation to insulin induced
hypoglycemia and indeed, increasing the glucagon secretion during hypoglycemia by 2.3
folds.
The autoimmune type 1 diabetes which occurs in the BBDP rats closely parallels
human type 1 diabetes in the most aspects compared to the other animal models of type1
diabetes and it is often used to investigate the potential intervention therapies for clinical
trials (Bortell & Yang, 2012). A most important feature of this model mimicking human
T1D, in addition to the autoimmune origin of diabetes, is the requirement of continuous
exogenous insulin therapy in these animals in order to prevent the lethal complications of
type1 diabetes. Thus, we can assume that the hyperinsulinemic hypoglycemic clamp in
this model closely mimicked the iatrogenic hypoglycemia in type 1 diabetes, simulating
the common situation in the diabetic patients who receive medications that raise the
circulating plasma insulin (Cryer, 2012a) as discussed previously in the introduction.
We also observed that SSTR2a treatment enhances the glucagon secretion during
insulin induced hypoglycemia in non diabetic BBDP rats, however the previous study on
normal Sprague Dawley did not showed any enhancement of glucagon secretion by
SSTR2a treatment, this could be probably because the non diabetic BBDP is not really
normal, but at a subclinical stage that could proceed to diabetes. Histologically, the non
67
diabetic BBDP pancreatic islets were observed also to be inflamed, and possibly some
inflammatory mediators might have undefined effects on alpha cells to have sensitized to
somatostatin inhibition.
To be consistent with the previous studies in the field, we also measured the
epinephrine, norepinephrine and corticosterone responses to hypoglycemia after SSTR2a
treatment. We did not observe any change in epinephrine and norepinephrine responses
by SSTR2a treatment; however, we did observe enhanced corticosterone response to
hypoglycemia. This finding is of particular importance; as in the clinical settings during
the prolonged hypoglycemia especially during sleep in children with type 1 diabetes,
cortisol response is significant and the current results suggest that SSTR2a treatment
may enhance the cortisol response during hypoglycemia in type 1 diabetes . The
epinephrine, norepinephrine and corticosterone responses were not blunted in the
Diabetic Vehicle group, suggesting that these responses are not affected in insulin treated
type 1 diabetic BBDP rats at least early in the time course of the disease, as has been
previously proposed (Jacob, Dziura, Morgen, Shulman, & Sherwin, 1996). The other
possibility may be that as we have studied the insulin treated diabetic rats in just 2 weeks
from the onset of diabetes; the time course of diabetes in these animals were likely too
short to compromise these counterregulatory responses to hypoglycemia observed in
latter stages of the disease. As previously discussed in the Introduction, the prior
glycemic control and the time course of the diabetes with regard to recurrent
hypoglycemic episodes in type 1 diabetic models are important factors affecting
68
nor/epinephrine and corticosterone counterregulation (Chan, et al., 2006; M. Davis, et al.,
1994; Kinsley & Simonson, 1996), which were not tested in my study.
We demonstrated that the pancreatic glucagon following hypoglycemia is not
decreased in the Diabetic Vehicle rats when compared to the Non-Diabetic Vehicle rats,
thus the defect in glucagon counterregulation may not be due to a decrement in glucagon
synthesis in autoimmune type 1 diabetes; this finding is well in line with the previous
study on the STZ diabetic rats that also showed unaffected pancreatic glucagon content in
diabetes following hypoglycemia (J. T. Yue, et al., 2012). SSTR2a administration
decreased the pancreatic glucagon protein content, while the secretory glucagon levels in
the plasma in response to hypoglycemia were fully restored. This finding suggests that
antagonizing the SSTR2 may increase α-cells potency to release glucagon in response to
hypoglycemia.
We observed that the pancreatic somatostatin protein content in the untreated
diabetic rats is increased; however, it did not reached to a statistical significant higher
level compared to the non-diabetic rats. Furthermore, our results indicate that insulin
treatment significantly decreases the pancreatic somatostatin level in the diabetic BBDP
rats, which is consistent with the previous studies suggesting that insulin treatment
corrects the elevated pancreatic somatostatin levels in type1 diabetes (Papachristou, et al.,
1989; Patel, et al., 1983; Rastogi, et al., 1990). The fact that the insulin treated diabetic
BB rats don’t have elevated pancreatic somatostatin and yet SSTR2a can normalize
hypoglycemia, demonstrates that the problem occurs not only when somatostatin is
increased, but also when it is normal. We would assume that in humans, where
69
somatostatin is increased, the effect would be even more marked. We also observed that
while the pancreatic somatostatin protein content remained unchanged following the
insulin induced hypoglycemia in Diabetic SSTR2a group compared to the Diabetic
Vehicle group, the plasma levels of somatostatin were slightly higher in the Diabetic
SSTR2a compared to the Diabetic Vehicle group. This finding suggests that the increased
plasma somatostatin during hypoglycemia may be originated from the extra pancreatic
sources particularly the gastrointestinal system (Patel, et al., 1983; Patel, Wheatley,
Malaisse-Lagae, & Orci, 1980; Ruggere & Patel, 1984). The same results for the Non
Diabetic SSTR2a group, further confirms the extra pancreatic origin of the increased
plasma somatostatin during hypoglycemia in this group. Nevertheless, the increased
pancreatic somatostatin content in Non Diabetic SSTR2a group, immediately after
hypoglycemia, may indicate the compensatory increase in somatostatin synthesis resulted
from blocking its action by antagonising the SSTR2.
The compensatory increase in soamtostatin synthesis and secretion in Non-diabetic
SSTR2a group may be one reason behind the requirement of more exogenous insulin
needed in this group in order to attain hypoglycemic during the clamp experiments. The
increased levels of somtostatin in Non-Diabetic SSTR2a group, further inhibits the
endogenous insulin secretion and increases the exogenous insulin requirements to induce
the target hypoglycemia (3±0.5 mmol/l). Indeed, we postulate that the main reason for
the high exogenous insulin requirement for Non-Diabetic SSTR2a group is the enhanced
plasma glucagon levels in this group as the result of SSTR2 blockade.
70
It has been postulated that the pancreatic somatostatin regulates glucagon secretion
by a local paracrine action (Unger & Orci, 1977). This notion has been supported by the
studies demonstrating the enhanced glucagon and insulin secretion by neutralizing the
intra-islet somatostatin by monoclonal antibodies in perfused human pancreas
(Brunicardi et al., 2001; Kleinman et al., 1995). Developing the specific SSTR2
antagonist (Hocart, et al., 1999)opened the way to investigate the paracrine somatostatin
action on glucagon and insulin action more accurately. It was shown that administration
of PRL-2903, the same SSTR2a that has been used in the current study, enhances the
arginine-stimulated glucagon secretion from the perifused rat isolated pancreatic islets
and perfused isolated pancreata (Cejvan, et al., 2003). Also it has been shown that
SSTR2a reverses the effects of SSTR2 specific agonist on insulin and glucagon secretion
in a dose response manner in perifused isolated human pancreatic islets (Singh, et al.,
2007). Developing somatostatin and SSTR2 knockout mice (Hauge-Evans, et al., 2009;
M. Z. Strowski, et al., 2000) also further confirmed the paracrine tonic inhibitory effect
of pancreatic somatostatin on insulin and glucagon secretion from β and α-cells.
In the present study to further investigate the local effect of the pancreatic
somatostatin on glucagon secretion and also to further explore the SSTR2a effect on
enhancing and restoring the perturbed glucagon counterregulatory response to
hypoglycemia in type 1 diabetes, we developed a new method. We deployed the
previously introduced fresh thin pancreatic slices (Huang, et al., 2011; Speier & Rupnik,
2003) in the perifusion setting. Employing this strategy, we could address the real-time
glucagon secretory responses to hypoglycemia in both healthy and diabetic conditions
71
devoid of the other intervening factors present in-vivo like the effects of the central and
autonomic nervous systems. This would not have been possible by employing the
conventional methods, as the pancreatic islets after T1D autoimmune injury and scarring
are considerably smaller in size, and much distorted shape and architecture, and any
attempt to isolate them by exposing them to enzymatic digestion will further damage
them (Huang, et al., 2012). Furthermore, since α-cells are located mostly in the islet
periphery in rodents (Cabrera, et al., 2006), they would be even more prone to injury
from the isolation procedures. In case of whole pancreatic perfusion, the interference of
the nervous system cannot be ruled out.
In the pancreatic slices perifusion studies, we observed that antagonizing the
SSTR2 significantly enhances the glucagon response during hypoglycemia both in
diabetic and non- diabetic BBDP rats. This finding further confirms the paracrine
inhibitory effect of pancreatic somatostatin on glucagon secretion which is mediated via
the SSTR2 on α-cells. In addition, we also observed that the basal glucagon secretion is
enhanced in both diabetic and non-diabetic animals.
There is abundance of studies suggesting that the clinical manifestations of type 1
diabetes are the result of the hyperglucagonemia which contributes to the hyperglycemia
(Lee et al., 2012; Lee, Wang, Du, Charron, & Unger, 2011; Unger & Cherrington, 2012).
Considering SSTR2a as a potential potent therapeutic for preventing hypoglycemia in
type 1 diabetes, by augmenting the glucagon secretion through antagonising the SSTR2
on α-cells, it is very important to investigate its effect on basal glucagon secretion. The
previous study on STZ diabetic rats well indicated that SSTR2a infused for 4 hours
72
during basal conditions in-vivo only resulted in a transient glucagon increase which
neither induced hyperglycemia nor altered corticosterone and catecholamine levels in the
plasma (J. T. Yue, et al., 2012).
The observed initial increase in basal glucagon secretion in the perifused pancreatic
slices by SSTR2a treatment may be because of the inherent limitation of our study.
Nonetheless, our in-vitro studies indicate that blockade of the actions of endogenous
pancreatic somatostatin on alpha cells particularly on its SSTR2, devoid of the other
confounding factors possibly present in-vivo which could prevent the hyperglucagonemia
induced hyperglycemia under basal conditions.
Previously it was shown that SST2a reversed the inhibitory effects of specific
SSTR2 agonist on insulin and glucagon secretion in a dose response manner in isolated
human islets (Singh, et al., 2007); however the effects of SSTR2a on enhancing the
glucagon secretion in hypoglycemia was never tested in human pancreas. Employing the
thin pancreatic slices perifusion technique, for the first time we demonstrate that SSTR2a
enhances the glucagon sceretory response to hypoglycemia stimulus in healthy human
pancreatic slices. Our results further demonstrate that the simultaneous insulin secretion
from the human pancreatic slices during hypoglycemia was also enhanced by the SSTR2
blockade. This could be explained by the observations of other groups who have
suggested that SSTR2 is the functionally predominant somatostatin receptor on both α
and β-cells in human pancreas which mediates the somatostain actions on these cells
(Brunicardi, et al., 2003; Kailey, et al., 2012; Moldovan, et al., 1995).
We are confident that our technical approach yielded an accurate estimate of
glucagon secretion from the pancreatic slices in the perifusion protocol which we
73
observed to be enhanced when the slices were treated either with SSTR2a or arginine;
and as well, also showed a downward trend in transition from 1 mmol/l to 7mmol/l
glucose concentration in the Non-Diabetic and Diabetic BBDP pancreatic slices. Thus,
our results are well in line with the previous reports, suggesting that glucose has the
highest inhibitory effect on glucagon release from α-cells at 6-7 mmol/l glucose
concentrations (Salehi, et al., 2006; Vieira, et al., 2007; Walker et al., 2011).
Overall, the response differences between pancreatic slices perifusion in-vitro and
the in-vivo experiments are due to the fact that the slices are not innervated, but also
because there is no blood flowing through the slices. For example, blood contains
arginine, and as indicated through the results, arginine by itself stimulates glucagon
release. Thus, we further hypothesize that since hypoglycemia by itself does not stimulate
glucagon release, it is possible that glucagon release will be stimulated when
hypoglycemia is accompanied by arginine and other amino acids. It’s also possible that
during hypoglycemia, blood flow through the islets changes and also hypoglycemia can
stimulate release of many amino acids.
The potent effects of the SSTR2a on normal human pancreas maybe because the
population of D-cells in human islets are probably twice that of rodent islets, whereas β-
cells populations at 55% are less than rodents at 75% (Cabrera, et al., 2006). This would
suggest that the proportionate inhibitory effects of insulin and somatostatin on human α-
cells may be different than rodents, with somatostatin probably having greater effect on
human alpha cells. Hence, SSTR2a actions were more evident on the normal human
alpha cells and not on normal Sprague Dawley rats. This has a strong clinical implication
in that human type 1diabetic patient with destroyed beta cells, leaving a potentially larger
74
D-cell population would predictably respond better to SSTR2a blockade than rodent type
1 diabetic models.
75
Chapter 5 Conclusion and future directions
We had hypothesised that the impaired glucagon counter-regulation in type 1
diabetes is due to the accentuated somatostatin inhibitory effect on α-cells in the absence
of endogenous insulin; thus antagonising the somatostatin receptor type 2, in order to
block the somatostatin inhibitory effect on α-cells would improve the compromised
glucagon counter-regulatory response to hypoglycemia in autoimmune type 1 diabetic
rats. In the present study we demonstrate for the first time that antagonising the SSTR2 in
insulin treated autoimmune type 1 diabetic BBDP rats, fully restores the glucagon
counterregulation to insulin induced hypoglycemia and indeed, increasing the glucagon
secretion during hypoglycemia by 2.3 folds. This proves our hypothesis that somatostatin
suppression of alpha cells in type 1 diabetes contributes to the impaired glucagon
secretory response to hypoglycemia.
The present findings further suggest that SSTR2 antagonism can enhance
hypoglycemia-stimulated glucagon and corticosterone release sufficient to restore
normoglycemic control in type 1 diabetic BB rats.
In the present study we further investigated the local effect of the pancreatic
somatostatin on glucagon secretion and also explored the SSTR2a effect on enhancing
and restoring the perturbed glucagon counterregulatory response to hypoglycemia in type
1 diabetes in BB diabetic rats by employing the pancreatic slices perifusion technique.
76
This is the first time that the thin pancreatic slices (Huang, et al., 2011; Huang, et al.,
2012; Speier & Rupnik, 2003) are employed perifusion studies. This study would not
have been possible employing the conventional methods, as the pancreatic islets after
T1D autoimmune injury and scarring are considerably smaller in size, and much
distorted shape and architecture, and any attempt to isolate them by exposing them to
enzymatic digestion will further damage them (Huang, et al., 2012).
In the pancreatic slices perifusion studies, for the first time we demonstrate that
antagonizing the SSTR2 significantly enhances the glucagon response during
hypoglycemia both in diabetic and non- diabetic BBDP rats. This finding further
confirms the paracrine inhibitory effect of pancreatic somatostatin on glucagon secretion
which is mediated via the SSTR2 on α-cells.
Benefiting from this novel technical approach, the present work is the first study to
show the improvement in glucagon secretion from the normal human pancreatic slices by
antagonizing the somatostatin receptor type 2 on α-cells by a specific SSTR2a (PRL-
2903).
These results suggest that SSTR2 blockade may be a novel treatment strategy to
improve the counter-regulatory responses, particularly of glucagon in type 1 diabetes.
SSTR2 antagonism should be explored to be ascertained that in patients with type 1
diabetes, the impaired glucagon and cortisol secretion during hypoglycemia can be
restored; this could be an effective treatment to prevent the acute and chronic
complications of hypoglycemia. Also by minimizing the risk of hypoglycemia, type 1
77
diabetic patients could benefit from intensive insulin treatment which ultimately would
lead to a lower incidence of diabetic complications.
In the future, we are also aiming to explore factors in the circulation that are
necessary to sensitize the response of α-cell to hypoglycemia and the mechanism of the
potential sensitization of α-cells to insulin in diabetes(Gaisano, et al., 2012).
Since the slice preparation has enabled us to begin to assess α-cell dysfunction in
type 1diabetes where in the very small islet mass and inflammation would have rendered
it impossible to reliably isolate and examine the α-cell (Gaisano, et al., 2012; Huang, et
al., 2012), in future we are planning to examine the precise action of SSTR2a on α-cell,
employing the pancreatic slices in electrophysiologic studies to see which ion channel or
exocytotic pathways might be affected (Huang, et al., 2011; Huang, et al., 2012). In our
recent study (Huang, et al., 2012) in fact, we found that α-cell of STZ-treated mice,
rendered T1D, there was perturbation of two ion channels, an increase in Na channel
density and reduction of voltage-gated K+ channel density, which contributed to
the increased action potential firing frequency and amplitude, coupled with larger
glucagon granules (by electron microscopy), together rendering the α-cell primed to
secrete more glucagon; hence revealing the elusive basis of hyperglucagonemia. It would
be of interest to see if similar ion channels in BB rat α-cells are perturbed, and if SSTR2
blockade then acts on the primed α-cell to stimulate more glucagon secretion. The major
distinction between the STZ models and the BB rat model is that the latter is
an autoimmune model, and hence involved a myriad of inflammatory processes. It would
78
be very exciting to test if any one of the inflammatory mediators might play a role in
modulating α-cell sensitivity for glucagon release.
Another strategy to explore this nature of glucagon secretion restored by the SSTR2
blockade is to directly examine glucagon exocytosis employing multi-photon imaging,
used extensively to examine beta-cell exocytosis (Takahashi, Ohno, & Kasai, 2012), in
order to precisely investigate the real time glucagon secretion from α-cells induced by
SSTR2a blockade. This is actually unprecedented and the precise exocytotic events in α-
cells are unknown. The major advantage of this approach is that we would be able to
subject the pancreatic slice to physiologic stimulus i.e. different glucose concentrations
as I have done to examine glucagon secretion on the slices; and then assess the precise
exocytotic event(s) that is modulated by the SSTR2 blockade. The electrophysiologic
study, which will reveal the precise ion channel events specifically altered by
somatostatin in disease, is nonetheless not a physiologic assay. Thus the combination of
both the exocytotic imaging and electrophysiologic assays will determine the underlying
mechanism of the effects of somatostatin and SSTR2a blockade on modulating glucagon
secretion in T1D that I have observed in this in vivo study.
Since we have demonstrated the enhancement of glucagon secretion
by antagonizing SSTR2 in the perifused thin human pancreatic slices, it would be of
particular interest to perform some of the above exocytotic imaging and
electrophysiologic studies on α-cells within human pancreas slices.
Finally, we need to put all these observations into the clinical perspective, which is
how these insights would better the lives of patients with type1diabetes. It would
79
therefore be of great interest to proceed to a small clinical study to see if SSTR2
blockade might work on T1D patients in preventing exogenous insulin
induced hypoglycemia by restoring low-glucose stimulated glucagon secretory response.
In fact, this ‘discovery’ has been patented by Professor Mladen Vranic, my co-supervisor
to precisely and eventually carry out such studies on diabetic patients.
80
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