1.1.1
Basic Anatomy
The name pancreas is derived from the Greek word, ‘pan’ meaning ‘all’ and ‘creas’ meaning ‘flesh’ (Slack 1995). In humans, the pancreas weighs around 70-150 grams and measures 15-25 cm in length (Slack 1995) draining to the duodenum by the ampulla of Vater, which is where the main pancreatic duct is connected with common bile duct (Slack 1995) (Figure 1.2). The pancreas is comprised of two different types of glandular tissue, the exocrine pancreas; and the focus of this thesis, the endocrine pancreas. Exocrine function comprises secretion of digestive enzymes (proteases, lipases, amylases and nucleases) from acinar/duct cells. The Islets of Langerhans are the endocrine component of the pancreas containing alpha (α)- cells secreting glucagon (Nadal et al., 1999) beta (β) cells secreting insulin (Quesada et al., 2006) somatostatin releasing delta cells, PP cells secreting polypeptides and more recently identified epsilon (ε) - cells producing ghrelin (Prado et al., 2004). All of these endocrine cells collectively account for only 1-2% of total pancreatic tissue (Bouwens and Rooman, 2005).
1.1.2
Pancreas- function
The regulation of blood glucose concentrations (endocrine) and digestion (exocrine) are the key functions performed by the pancreas (Gesina et al., 2004). Circulating glucose concentrations must be maintained in a precise range independent of dietary ingestion. This process of blood glucose homeostasis involves the liver, adipose tissue, brain, skeletal muscle and endocrine pancreas (Fritsche et al., 2008). Insulin secretion remains at low levels during fasting because of low plasma glucose concentrations. During this stage the compensatory hormone glucagon, along with corticosteroids and adrenalin aid in promoting the production of glucose from hepatic storage, and subsequent release into the circulation. Glucagon, a counter regulatory hormone to insulin, acts in response to insulin-induced hypoglycemia by raising glucose concentrations (Freychet et al., 1988) and thereby maintains blood glucose homeostasis. Glucagon is secreted in a pulsatile fashion by the α-cells of the islets (Opara et al.,1988) inducing hepatic glucose output via glycogenolysis and gluconeogenesis processes (Weigle and Goodner, 1986). Glucagon activates PKA phosphorylation, which then activates glycogen phosphorylase kinase, which phosphorylates (serine- 14) glycogen phosphorylase. The phosphorylation of glycogen phosphorylase leads to increased glycogen breakdown (glycogenolysis) and produces glucose 6-phosphate (G-6-P), which is then converted into glucose by glucose-6-phosphatase (G-6-Pase), increasing the glucose pool for hepatic output (Johnson et al.,1997). Glucagon also regulates glucose concentration by stimulating hepatic gluconeogenesis mechanism acting via phosphoenolpyruvate carboxykinase (PEPCK) and CREB cycle finally leading to glucose secretion (Larsson and Ahrén, 2000; Okar and Lange, 1999). Whilst insulin is required for much of the peripheral uptake of glucose, it is imperative that circulating glucose concentrations are maintained within strict limits, as neuronal tissue glucose uptake is not insulin mediated and thus neuronal glucose regulation is a reflection of circulating concentrations. Insulin acts in a paracrine fashion and inhibits glucagon release via activation of the insulin receptor- phosphatidylinositol 3-kinase (PI3K) pathway (Kaneko
et al., 1999) and also increases KATP channel activity in rat α-cells via
membrane hyperpolarization, thus inducing an inhibitory effect on glucagon production (Franklin et al., 2005).
During the fed stage, where glucose concentrations rise due to absorption from the gut, insulin secretion from pancreatic beta cells is increased which then suppresses the glucose level in the body by promoting glucose uptake via muscles and adipocytes (insulin mediated glucose disposal (IMGD). Insulin also prevents liver hepatocytes from producing glucose by inhibiting the processes of gluconeogenesis and glucogenolysis (Fritsche et al., 2008). Increased translation of pre-pro-insulin transcript regulates the synthesis of insulin in response to acute glucose stimulation, whilst prolonged exposure to glucose results in the synthesis of insulin through insulin gene transcription (Poitout et al., 2006. β-cells are electrically excitable, playing a key role in regulation of secretion (Drews et al., 2010). The final insulin secretory response in β-cells is in turn driven by oscillations of membrane potential via Ca2+ influx (Rorsman et al., 2000).
1.1.3 Glucose stimulated Insulin Synthesis (GSIS) in
pancreatic beta- cells
Insulin is secreted in response to glucose in a biphasic manner (Barbosa et
al., 1998). Glucose transporter 2 (GLUT2) mediates the entry of glucose into
beta cells through facilitated diffusion (Jiang et al., 2008), conferring regulation of insulin secretion at a cellular level. In response to increased glucose concentrations, glucokinase (GK) from the nucleus is released into the cytosol pool (Agius et al., 1995; van Schaftingen et al., 1997). The result being that glucose in the β-cell is now phosphorylated to glucose-6- phosphate (Matschinsky et al 1998) (Figure 1.3). In this way, GK is considered as a pancreatic beta cell glucosensor and plays a critical role in glucose stimulated insulin secretion (Iynedjian 2009). Pancreatic β-cells express low levels of lactate dehydrogenase (Schuit et al., 1997; Sekine et
meaning pyruvate is synthesized by glycolysis in the β-cells and later enters the Kreb’s cycle (Schuit et al., 1997). Following glucose phosphorylation by GK, adenosine triphosphate (ATP) is generated both by glycolysis and Kreb’s cycle in the mitochondria (mitochondrial metabolism) leading to closure of ATP-sensitive K+ channels (KATP channels) due to an increase in both
intracellular diadenosine polyphosphates (DPs) (Ashcroft, 2006) and ATP/ADP ratio (Ashcroft et al.,1984). As a consequence, depolarization of the plasma membrane occurs due to the closure of ATP sensitive K+ channels, leading to extracellular calcium influx (Ashcroft, 2006).
This electrical activity regulates the secretory response in β-cells that consists of oscillations of the membrane potential ranging from electrical silent periods to Ca2+ action potential originating depolarizing plateaus (Rorsman et al., 2000). The increased carbon dioxide (CO2) influx produced from glucose into
the Krebs cycle leads to production of intermediates such as malate (Brun et
al., 1996; Schuit et al., 1997) glutamate (Maechler and Wollheim, 1999) and
citrate (Brun et al., 1996; Schuit et al., 1997), which leave the mitochondria and accumulate into the cytosol stimulating insulin release (Prentki et al., 1997). Malate efflux promotes electron transfer from cytosolic NADH to NADPH (MacDonald, 1995), whilst citrate produces acetyl esters using malonyl coenzyme A (CoA) as a precursor and finally the combination of ATP and proton dependent step, the glutamate efflux from the mitochondria leads to the uptake of glutamate by the secretory vesicles, also referred to as large dense-core vesicles, which leads to exocytosis of insulin secretory granules (Maechler and Wollheim, 1999) (Figure 1.3).
Cytosolic Ca2+ induced insulin-containing secretory granules act
synergistically with the cyclic adenine monophosphate (cAMP) pathway (Wang and Iynedjian, 1997) on the exocytosis process, transporting the insulin secretory granules to the plasma membrane of β-cells and finally releasing insulin into the circulation (Flatt 1996).
Figure 1.3 Schematic representation of glucose stimulated insulin secretion
(GSIS) in pancreatic β-cells.
In brief, after the glucose uptake by Glut-2, glucokinase acts as a rate limiter by converting glucose to glucose-6-phosphate. This increase in glucose metabolism leads to production of ATP via glycolysis, pyruvate oxidation and reducing equivalent shuttles, resulting in increased ATP/ADP ratio, leading to inhibition of KATP channels and membrane depolarization. This in turn leads to activation of voltage gated Ca2+ channels and in combination with coupling factors exocytosis of insulin granules is activated resulting in insulin secretion.