Under certain conditions, α-cells can produce physiologically significant amounts of GLP-1 (rather than glucagon) by changes in its expression of prohormone converting enzymes. This locally produced GLP-1 can have important intra-islet effects including the stimulation of insulin secretion, inhibition of glucagon and increase in β-cell mass, mediated by enhancement of proliferation and protection from apoptosis. Similarly, CCK is produced by islets, this too may play an important role in mechanisms underlying islet cell compensation.
Diabetes mellitus is a disease that affects over 171 million people worldwide (Figures from WHO for 2000). Type 2 diabetes accounts for 95% of all cases of diabetes. The Predicted global prevalence by 2025 is 300million. It has recently been recognised by the United Nations as a major global health problem, second only to AIDS. Not only has diabetes got a severe impact on the life expectancy, and the quality of life of a sufferer, but also it places an enormous strain on healthcare systems. It is estimated that type 2 diabetes cost the UK NHS £1.3bn per year (Wild et al, 2004).
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Type 2 diabetes is a multifactorial disease that results from insulin resistance and culminating in total β-cell failure (Polonsky et al, 1996). Where insulin resistance is present, normal physiological concentrations of insulin aren't sufficient for it to wield its biological effect, and increasing levels of insulin are required to regulate glucose metabolism. Insulin resistance increases the demand on the pancreatic β-cell. In some individuals this increased demand for insulin eventually causes exhaustion of the β-cells and β-cell failure (Rhodes et al, 2005). This eventually leads to insulin deficiency, postprandial and fasting hyperglycaemia, and thus Type 2 diabetes (Lebovitz et al, 1999).
Type 2 Diabetes is the result of a combination of insulin resistance and defective β-cell function.
Physiological actions of Insulin:
-suppresses hepatic glucose production
-promotes glucose storage as glycogen
-stimulates glucose uptake by adipose tissue and muscle
-regulates protein turnover
-suppresses hepatic ketogenesis and lipolysis
-effects electrolyte balance
Other actions (longer-term):
-regulates growth and development (in utero and post-utero)
-regulates expression of certain genes
Insulin secretion is inhibited by adrenaline and somatostatin, secretion of insulin is enhanced by glucagon, GIP and GLP-1. The action of insulin at tissue level might be antagonised by: glucagon, glucocorticoids, catecholamines, and growth hormone. Hyperinsulinaemia is a result of resistance to the actions of insulin or a deficit in insulin secretion. Resulting Type 2 diabetes is related to progressive failure of β-cell compensation. GLP-1 augments the endogenous insulin response to meals. (Krentz et al.,2001).
Patients with Type 2 Diabetes have fasting hypergluconaemia with impaired postprandial glucagon suppression in the presence of defective insulin secretion and/or action. A reduction in postprandial glucagon secretion in diabetic subjects has been shown to considerably reduce blood glucose, signifying that glucagon contributes considerably to the hyperglycaemia seen in subjects with type 2 diabetes (Shah et al, 2000).
The major biological action of glucagon is to counteract the actions of insulin and preserve normoglycaemia during the fasting state by inducing hepatic glucose production. Glucagon exerts its effects on target tissues via activation of the GPCR, a G-protein-coupled receptor member of the class II G protein-coupled receptor superfamily (Jelinek et al., 1993).
The regulation of plasma glucose concentrations is under tight control. In normal subjects concentrations are usually kept between 3.9-5.6mM by a equilibrium between glucose uptake by the peripheral tissues, and by hepatic glucose production (Kruger et al, 2006). This is co-ordinated by insulin and glucagon secretion. The intricate control of the insulin: glucagon ratio is important in maintaining the balance of gluconeogenesis and glycogenolysis in the liver. During fasting, glucagon increases hepatic glucose production via glycogenolysis and gluconeogenesis. During the fed state, elevated plasma glucose concentrations promote insulin secretion from the pancreas which reduces plasma glucose. Insulin elicits its effects by stimulating glucose uptake from peripheral tissues such as fat and skeletal muscle; and it inhibits hepatic glucose output (Aronoff et al, 2004). Current therapies for type 2 diabetes increase insulin secretion by blocking ATP-sensitive K+ channels in B-cells (insulin secretagogues, e.g. sulfonylureas), decrease hepatic glucose production and increase glucose uptake striated muscle (biguanides, e.g. metformin), or, enhance the response of tissues to insulin (thiazolidinediones, e.g. troglitazone).
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Glucagon is secreted into the blood stream from the pancreatic α-cells, through calcium dependant exocytosis, in reaction to hypoglycaemia. Glucagon acts through a GPCR expressed in many tissues but found predominantly in the liver. Glucagon increases intracellular cAMP levels which activate a number of enzymes responsible for glycogenolysis and gluconeogenesis including glycogen phosphorylase and PEPCK (Jiang et al, 2003). However, insulin counteracts this action by activating phosphodiesterases that decrease cAMP levels (Sloop et al, 2005).
The Islets of Langerhans:
Islets contain four main types of cells, beta cells that secrete insulin, the alpha cells that secrete glucagon, delta cells that secrete somatostatin, and pancreatic polypeptide (PP) producing cells. In recent times, an additional cell type has been identified, the epsilon cells. Epsilon cells express ghrelin, but are not immunopositive for any other islet hormones (Walia et al, 2008). The most prevalent cell type is the β-cell, making up around 55-70% of islet cells. In rodents there is a total separation of the cell types to well-defined regions of the islet. A large number of β-cells are grouped in the middle of the islet, and are surrounded by the α, δ and PP cells. Human islets, on the other hand, are much more interspersed, with no separation of the cell types as is seen in the rodent islet. Human islets contain a lesser percentage of β cells than the mouse islets (55% vs.77% respectively) (Cabrera et al, 2006). Additionally, the percentage of alpha cells in the human islet is much greater compared with the mouse islet (38% vs. 18%). There is also a random scattering of the various cell types around the blood vessels in the human islets signifying that there is no order in paracrine signalling.
The pancreas has an extraordinary compensatory mechanism in place to cope with changes in metabolic load (Rhodes et al, 2005). When required, the pancreas can expand its β-cell mass to increase insulin secretion. The best illustration of this is observed during pregnancy (Parsons et al, 1992). The increase in metabolic load during pregnancy can cause a significant change in islet function and β-cell mass. Beta cells have the ability to compensate for the increased necessity for increased β-cell proliferation and insulin biosynthesis (Parsons et al 1992). This is also the case in individuals with insulin resistance, where decreased insulin sensitivity leads to increased hepatic glucose output, and impaired uptake of glucose by peripheral tissues, and an eventual increase in circulating glucose. The increased requirement for insulin is compensated for by an expansion of the beta cells (Baggio et al, 2006a). Though, in many cases this modification in β-cell numbers ultimately fails giving rise to overt diabetes and the necessity for exogenous insulin.
Glucagon-like peptide-1 (GLP-1) is a 30-amino acid peptide hormone expressed in gut endocrine cells in response to nutrient ingestion that promotes nutrient absorption through regulation of gastrointestinal motility and islet hormone secretion (Drucker et al, 2003). Infusion of GLP-1 into normal or diabetic human subjects inhibits glucagon secretion and stimulates insulin, thus indirectly controlling peripheral glucose uptake and control of hepatic glucose production (Vella et al, 2000).
A proglucagon precursor contains amino acid sequences for GLP-1, GLP-2, oxyntomodulin, and glicentin. PC1/3 cleaves proglucagon in intestinal L cells and brain to liberate GLP-1, GLP-2, oxyntomodulin, and glicentin (Baggio et al, 2007). While proglucagon is expressed in pancreatic islet cells, PC2 is the principal processing enzyme in these cells, cleaving proglucagon to produce glucagon instead of GLP-1. However, under certain conditions, islet alpha cells do express PC1/3 and release GLP-1 from proglucagon instead, possibly to support the function and/or survival of adjacent beta cells. L cells are typically flask shaped and extend between adjacent epithelial cells to make direct contact with the lumen of the intestine. GLP-1 is produced in the L cells, which are located in high concentration in the distal intestine (Wideman et al, 2009).
Figure 1: GLP-1 actions in peripheral tissues. The majority of the effects of GLP-1 are mediated by direct interaction with GLP-1Rs on specific tissues. However, the actions of GLP-1 in liver, fat, and muscle most likely occur through indirect mechanisms (Baggio et al, 2007).
GLP-1 has a short half life (1-2 minutes) because of its fast degradation by DPPIV, a post-proline and post-alanine cleaving serine protease (Demuth et al, 2005). DPPIV is ubiquitously expressed as a transmembrane enzyme (present at the highest level in kidney, liver, brain and intestine, especially in the enterocyte brush border) but can additionally be found as a soluble form in the circulation. Because of degradation, only 10-15% of newly secreted GLP-1 enters the systemic circulation. Hence, it is not surprising that, in addition to endocrine pathways, a vagal reflex is involved in GLP-1 physiological actions such as the stimulation of insulin secretion (Ionut et al, 2005). Interestingly, sensory nerve involvement is mainly implicated at low levels of GLP-1, whereas direct action of the hormone is predominant at high levels (Ahren et al, 2004).
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The defect in alpha cell function that occurs in type 2 diabetes reflects impaired glucose sensing. GLP-1 inhibits glucagon secretion in vitro and in vivo, and suppresses glucagon release in a glucose-dependant manner in healthy subjects. This effect is also evident in diabetic patients, but GLP-1 does not inhibit glucagon release in response to hypoglycaemia, and may even enhance it. GLP-1 can improve alpha cell glucose sensing in patients with type 2 diabetes.
The biologically active forms of GLP-1 are GLP-1 (7-37) and GLP-1(7-36) amide (Mojsov et al, 1987). GLP-1 and its agonist suppress glucagon secretion at euglycaemic and hyperglycaemic levels in healthy volunteers, but do not do so at hypoglycaemic levels. Thus, unlike other known modulators of glucagon release, GLP-1 appears to enhance α-cell glucose sensing (Dunning et al, 2005).
GLP-1 directly suppresses glucagon secretion in patients with type 2 diabetes, independently of any effects on insulin or gastric emptying. GLP-1 appears to improve alpha cell function, although further studies are needed to establish if this peptide can normalise the defects in alpha cell glucose sensing that are characteristic of type 2 diabetes (Dunning et al, 2005).
GLP-1 and GIP are responsible for 70% of postprandial insulin secretion. In diabetic patients, GLP-1 and GIP secretion and action are impaired, and the incretin effect is decreased to 30%. GLP-1 can enhance pancreatic beta cell mass through stimulation of beta cell proliferation and neogenesis in healthy and diabetic rodents (Farilla et al, 2002).
Treatment of isolated mouse islets with a PC1/3 expressing adenovirus induces GLP-1 release from alpha cells, increase glucose-stimulated insulin secretion and promote islet survival (Wideman et al, 2006). Thus, manipulation of proglucagon processing in the alpha cell to yield GLP-1, presents a means of enhancing islet function and survival. PC1/3 is not produced in adult islet alpha cells, precluding GLP-1 production, however, bioactive GLP-1 production has been detected in InRI-G9 and αTC-1 cells, and GLP-1 also was secreted at low levels in normal, nontransduced islets, consistent with several reports that showed that islet α cells do produce small amounts of fully processed GLP-1, which may act as a paracrine stimulus on beta cells.
Cholecystokinin is an intestinal satiation peptide. It is produced by I cells in the duodenal and jejunal mucosa, as well as the brain and enteric nervous system. Intestinal CCK is secreted in response to luminal nutrients, especially lipids and proteins. The CCK prepropeptide is processed by endoproteolytic cleavage into at least six peptides, ranging from 8 to 83 amino acids in length (Rehfeld et al, 2004). The multiple bioactive forms pertinent to feeding share a common carboxy-terminal octapeptide with an O-sulphated tyrosine. The major circulating moieties are CCK8, CCK22, CCK33, and CCK58. The C-terminal octapeptide CCK-8 is well conserved between species and is the smallest form that retains the full range of biological activities. The primary structure of CCK-8 is Asp-Tyr-(SO3H)Met-Gly-Trp-Met-Asp-Phe amide. Once released CCK-8 exerts its biological action on various target tissues within the body in a neurocrine, paracrine or endocrine manner.
Figure 2: Amino acid sequence of CCK8.
CCK peptides interact with two receptors expressed in the gut and brain. CCK receptor 1 (CCK1R) predominates the GI system, whereas CCK2R predominates the brain. Through endocrine and/or neural mechanisms, CCK regulates many GI functions, including satiation. In humans, the results of numerous studies support a role for CCK1R in regulation of a variety of physiological processes including; gall bladder contraction, insulin secretion, neurotransmission, sphincter of Oddi relaxation, stimulation of pancreatic secretion, inhibition of acid secretion, relaxation of lower esophegeal sphincter tone, slowing of colonic motility and regulation of satiety (Peter et al, 2006). When peripherally injected immediately before a meal, CCK decreases meal size in a dose-dependent manner without affecting water intake or causing illness (Gibbs et al, 1973).Typifying a short-acting satiation signal, the anorectic effects of CCK are very short lived and undetectable if the peptide is injected more than 30mins before meals. CCK1R is expressed on vagal afferents, and peripheral CCK administration increases vagal-afferent firing, as well as neuronal activity in the hindbrain region receiving visceral input (Moran et al, 2004).
Despite the role of CCK in terminating individual meals, its significance in long-term body-weight regulation and its potential as an antiobesity target are debatable. Chronic CCK administration in animals, with up to 20 peripheral injections per day, reduces meal size, but this is offset by increased meal frequency, leaving body weight unaffected (West et al, 1984). The most important role for CCK in body-weight regulation might be its synergistic interaction with long-term adiposity signals, such as leptin (Morton et al, 2006).
CCK stimulates pancreatic acinar secretion and gall bladder contraction. Two recent studies have suggested a role for CCK in islet cell proliferation, Kuntz and collaborators demonstrated that 8 days of injection of the mature eight-amino acid product of the CCK gene, CCK8, after streptozotocin treatment in rats resulted in reduced hyperglycaemia, increased plasma insulin, and increased β-cell proliferation in 2004. Additionally, Chen conducted a study in 2007 showing that CCK8 treatment of partially pancreatectomized rats stimulated islet cell proliferation.
In human islet cells, overexpression of intracellular signalling pathway components or transcription factors, including constitutively active Akt, protein kinase C-ζ, p8, mouse pax4, and Nkx6.1, stimulates human islet or beta cell proliferation. Strikingly, in human islets CCK overexpression can stimulate a robust 15- to 20-fold increase in β-cell proliferation (Lavine et al, 2008). Two peptide growth factors, TFF3 and HGF, also trigger human islet cell proliferation. CCK receptors have been shown to colocalize with α- and β-cells (CCK1R receptor). It has been found that islets from CCK receptor-deficient mice were as responsive as wild-type islets in response to CCK overexpression. Additionally, human islet cells treated with CCK receptor antagonists were also responsive to CCK overexpression as vehicle-treated islets. Because the CCK deletions and antagonists affect all islet cells, it was concluded that α- and β-cell proliferation is CCK receptor independent. The 3 possible explanations for this are: 1) CCK signals through a third, yet-to-be-identified CCK receptor; 2) overexpression of CCK stimulates another receptor pathway; 3) CCK signals independently of any receptor (Lavine et al, 2008).
CCK dramatically enhances the effect of leptin on c-Fos synthesis in the paraventricular nucleus of the hypothalamus (Barrachina et al, 1997) as well as in brain stem areas (Emond et al., 1999) involved in food intake regulation. The synergy between leptin and CCK is not only linked to a decrease in food consumption, but also to the activation of metabolic pathways (Merino et al., 2008).
A study by Ahren et al., has shown that in both healthy subjects and subjects with type 2 diabetes, iv administration of CCK-8 reduces glucose levels and increases insulin levels after meal ingestion without significantly affecting the postprandial levels of GIP, GLP-1, or glucagon. The study, therefore, suggests that CCK may be explored for future use as a treatment for diabetes, in parallel with the antidiabetogenic action of GLP-1 (Ahren et al, 2000).
In a study conducted by Shimizu et al, exploring CCK producing cells in rat pancreatic islets, CCK-LI (cholecystokinin-like immunoreactivity) was detected in the central portion of the islets by immunohistochemical studies. Analysis of mirror section and double staining clearly demonstrated that CCK and insulin immunoreactivity coexisted in the same cells. Glucagon, somatostatin, and PP cells, on the other hand, were located in the periphery of the islets. There was no gastrin immunoreactivity in the whole islets. CCK antiserum saturated with CCK showed no immunoreactivity in the immunohistochemical study, and CCK antiserum incubated with rat gastrin failed to abolish CCK-LI in the islets.
Examination of the rat pancreas by in situ hybridization and RT-PCR revealed that the CCK gene was expressed in the islets of adult rats. The CCK gene was expressed in the islets, corresponding to those observed with mRNA from the duodenal mucosa by RT-PCR. The restriction digestion pattern and Southern blot analysis of the PCR amplified cDNA from the islet and the duodenum strongly suggest that this is the product of authentic CCK mRNA (Shimizu et al, 1998).
Grube et al. also demonstrated colocalization of CCK-LI with glucagon in A-cells in adult rat and human pancreatic islets. These observations suggest that CCK is synthesized in A cells, but that is inconsistent with Shimizu's findings. Although CCK-LI was found in the intrapancreatic nerve fibers or A cells in the pancreas, it has never been reported the presence of CCK in B cells. The discrepancy between both observations and previous other studies might depend on the species, age, and antibody used. In the Shimizu's study, neither CCK-containing nerve fibre nor ganglia was found in the rat pancreas.
Beta cells in Pregnancy:
Apart from adaptation to an increase in body weight and therefore an increased demand for insulin, the beta cell mass can also compensate for the increased demand for insulin during pregnancy. The failure of beta cell compensation is thought to lead to gestational diabetes. Gestational diabetes occurs in ~3-5% of pregnancies, and represents one of the major health concerns relating to pregnancy. Although gestational diabetes usually only lasts the duration of the pregnancy, these mothers are at higher risk of developing type 2 diabetes in the future.
In a study using rats, in pregnancy the beta-cell mass is increased around 2.5-fold compared with non-pregnant females (Van Assche et al, 1978). This results from both cellular hypertrophy and an increase in beta cell number. Enlargement of islets and beta-cell hyperplasia have also been noted in autopsies from pregnant humans (Van Assche et al, 1980). However, when streptozotocin induces diabetes in rats, the endocrine pancreas does not have the same capacity to compensate during pregnancy (Van Assche et al, 1980). Post pregnancy, a rapid decrease of the beta-cell mass occurs in postpartum rats (Marynissen et al, 1983). This is associated with a decrease in beta-cell replication and beta-cell size, and by increased apoptosis (Blondeau et al, 1999).
Aims and objectives:
To look at the effect of stable GLP-1 on islets
To look at the adaptation of islets in obesity and pregnancy
To see if GLP-1 and CCK can directly be seen in islets
To do some indirect functional studies using DPPIV inhibitors
To look at GLP-1 receptor knockout mice
Generic skills training (completed)
Learning techniques (completed)
Radiation training (completed)
Animal handling training (completed)
Histology (obesity and pregnancy)
Effect of Liragulatide
Effect of DPPIV inhibitors on GLP-1 in islets
GLP-1 measurement in islets
Effects on GLP-1 receptor knockout mice
GLP-1 receptor knockout mice
CCK measurement in islets
In vivo: Body weight/ food intake
Basal glucose/ insulin
Glucose Tolerance Test
Circulating GLP-1 and CCK levels
In vitro: Isolated islets
Western Blot/ PCR
Morphology of islets
Morphology of intestine