**Distinct Role for ARNT/HIF-1β in Pancreatic Beta-Cell Function, Insulin Secretion and Type 2 Diabetes**

Renjitha Pillai and Jamie W. Joseph *School of Pharmacy, University of Waterloo, Waterloo Canada* 

### **1. Introduction**

320 Biochemistry

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Diabetes mellitus is a common metabolic syndrome that has become an epidemic in modern society and is characterized by either a near-complete lack of insulin production due to autoimmune destruction of pancreatic beta-cells as in type 1 diabetes or abnormal insulin secretion, beta-cell dysfunction and insulin resistance as in type 2 diabetes (T2D). T2D is a complex heterogeneous disease that is characterized by elevated fasting and postprandial blood glucose levels that can result in severe complications including renal failure, cardiovascular disease, blindness and slow wound healing (Lin and Sun, 2010). Pancreatic islet beta-cells play a critical role in maintaining blood glucose levels by secreting the hormone insulin following a meal. Insulin maintains blood glucose levels in the normal physiological range by promoting glucose uptake in muscles, liver and adipose tissue, and by inhibiting hepatic glucose production. Therefore, any defect in insulin secretion in response to a meal or defects in insulin action in peripheral tissues can lead to increased blood glucose levels (Tripathy and Chavez, 2010; Muoio & Newgard, 2008).

Abnormal insulin secretion is a hallmark of T2D. Despite the central role of insulin in maintaining glucose homeostasis, the fundamental biochemical mechanism regulating nutrient-stimulated insulin secretion from pancreatic beta-cells is still incompletely understood. Insulin secretion from the pancreatic beta-cells is regulated by nutrients, neurotransmitters and hormones. Among these three factors, nutrients, particularly glucose is the most dominant stimulatory signal for insulin secretion. Insulin secretion is biphasic with a first acute phase occurring within 10 minutes after a glucose load and a second more sustained phase that reaches a plateau very quickly as seen in mice or more gradually as seen in rats and humans (Gerich, 2002). Numerous models have been proposed over the last several decades to explain the mechanism governing glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. The current model of GSIS holds that glucose enters betacells via the low affinity, high capacity glucose transporter 2 (GLUT2) and becomes phosphorylated by glucokinase (GK or hexokinase IV), which is the rate-limiting step in glycolysis. The glycolytic end product pyruvate then enters the tricarboxylic acid cycle (TCA), where oxidative phosphorylation occurs, leading to increased ATP production. The subsequent rise in the cytosolic ATP/ADP levels promotes closure of ATP-sensitive potassium channels (KATP channels) causing beta-cell membrane depolarization and activation of voltage-dependent Ca2+ channels (VDCC). The opening of VDCCs facilitates influx of extracellular Ca2+, leading to a rise in the beta-cell cytosolic Ca2+ levels, which triggers exocytosis of the insulin-containing secretory granules (Figure 1) (Jensen et al., 2008; Prentki & Matchinsky, 1987; Ashcroft & Rorsman, 1989; Newgard & Matchinsky, 2001; Newgard & McGarry, 1995). This so-called "KATP channel-dependent" mechanism appears to be particularly important for the first, acute phase of insulin release. However, in the second and more sustained phase of insulin secretion a "KATP channel-independent" pathway also appears to play a key role in the regulation of GSIS in conjunction with the KATP channel-dependent pathway (Henquin et al., 2003, Ravier et al., 2009). Important support for "KATP channelindependent" pathway of GSIS comes from studies showing that glucose still causes a significant increase in insulin secretion in conditions where KATP channels are held open by application of diazoxide followed by membrane depolarization with high K+, or in animals lacking functional KATP channels (Nenquin et al., 2004; Shiota et al., 2002; Szollozi et al., 2007; Ravier et al., 2009). These and more recent studies suggest that mitochondrial metabolism of glucose generates signals other than changes in the ATP/ADP ratio that are important for normal insulin secretion. Several molecules, including glutamate, malonyl-CoA/LC-CoA and NADPH, have been proposed as candidate coupling factors in GSIS (Maechler & Wollheim, 1999; Ivarsson et al., 2005; Corkey et al., 1989; Prentki et al., 1992).

Fig. 1. Current model of glucose stimulated insulin secretion (GSIS) from pancreatic betacells. Glucose equilibrates across the plasma membrane through glucose transporter GLUT2, which initiates glycolysis. Pyruvate produced by glycolysis preferentially enters the mitochondria and is metabolized in the TCA cycle, producing reducing equivalents in the form of NADH and FADH2. The transfer of electrons from these reducing equivalents through the mitochondrial electron transport chain is coupled with the pumping of protons from the mitochondrial matrix to the inter membrane space, leading to the generation of ATP. ATP is transferred to the cytosol through adenine nucleotide carrier (ANC), raising the ATP/ADP ratio. This results in the closure of the ATP sensitive K+ channels (KATP), which in turn leads to membrane depolarization, opening of the voltage-sensitive Ca2+ channels, promoting calcium entry and increase in cytoplasmic Ca2+ leading to exocytosis of insulin granules. Glucose also generates amplifying signals other than ATP, which plays a significant role in the secretion of insulin from pancreatic beta-cells.

activation of voltage-dependent Ca2+ channels (VDCC). The opening of VDCCs facilitates influx of extracellular Ca2+, leading to a rise in the beta-cell cytosolic Ca2+ levels, which triggers exocytosis of the insulin-containing secretory granules (Figure 1) (Jensen et al., 2008; Prentki & Matchinsky, 1987; Ashcroft & Rorsman, 1989; Newgard & Matchinsky, 2001; Newgard & McGarry, 1995). This so-called "KATP channel-dependent" mechanism appears to be particularly important for the first, acute phase of insulin release. However, in the second and more sustained phase of insulin secretion a "KATP channel-independent" pathway also appears to play a key role in the regulation of GSIS in conjunction with the KATP channel-dependent pathway (Henquin et al., 2003, Ravier et al., 2009). Important support for "KATP channelindependent" pathway of GSIS comes from studies showing that glucose still causes a significant increase in insulin secretion in conditions where KATP channels are held open by application of diazoxide followed by membrane depolarization with high K+, or in animals lacking functional KATP channels (Nenquin et al., 2004; Shiota et al., 2002; Szollozi et al., 2007; Ravier et al., 2009). These and more recent studies suggest that mitochondrial metabolism of glucose generates signals other than changes in the ATP/ADP ratio that are important for normal insulin secretion. Several molecules, including glutamate, malonyl-CoA/LC-CoA and NADPH, have been proposed as candidate coupling factors in GSIS (Maechler & Wollheim,

Fig. 1. Current model of glucose stimulated insulin secretion (GSIS) from pancreatic betacells. Glucose equilibrates across the plasma membrane through glucose transporter GLUT2,

which initiates glycolysis. Pyruvate produced by glycolysis preferentially enters the mitochondria and is metabolized in the TCA cycle, producing reducing equivalents in the form of NADH and FADH2. The transfer of electrons from these reducing equivalents through the mitochondrial electron transport chain is coupled with the pumping of protons from the mitochondrial matrix to the inter membrane space, leading to the generation of ATP. ATP is transferred to the cytosol through adenine nucleotide carrier (ANC), raising the ATP/ADP ratio. This results in the closure of the ATP sensitive K+ channels (KATP), which in turn leads to membrane depolarization, opening of the voltage-sensitive Ca2+ channels, promoting calcium entry and increase in cytoplasmic Ca2+ leading to exocytosis of insulin granules. Glucose also generates amplifying signals other than ATP, which plays a

significant role in the secretion of insulin from pancreatic beta-cells.

1999; Ivarsson et al., 2005; Corkey et al., 1989; Prentki et al., 1992).

Maintenance of a functional mature beta-cell phenotype requires optimal expression of key transcription factors. Transcription factors regulate a variety of pancreatic beta-cell processes including cell differentiation, proliferation, cell signaling and apoptosis. By regulating the expression of specific sets of genes, transcription factors determine the spatio-temporal specificity of gene expression in most organisms, including mammals. Numerous studies have shown that transcription factors act synergistically to achieve normal beta-cell development and function (Cerf, 2006; Mitchell & Frayling, 2002; Lyttle et al., 2008). Development of the endocrine pancreas is initiated from multipotent precursor cells, which differentiate to form five different cell types in the pancreatic islet namely the α-cells (glucagon), β-cells (insulin), -cells (somatostatin), PP (pancreatic polypeptide) cells and -cells (ghrelin) (Steiner et al., 2010). The development of the islet architecture is regulated by an ordered system of transcriptional events activated by a hierarchy of transcription factors. Some of the major transcription factors represented in islets include several homeodomain factors like pancreatic and duodenal homeobox-1 (Pdx-1), paired box gene (Pax) Pax 4, Pax 6, Nkx 2.1 and Nkx 6.1 which are expressed in both progenitor as well as differentiated beta-cells. Pdx-1 and Nkx 2.2 are required for both early beta-cell differentiation and maintenance of a mature beta-cell phenotype (Habner et al., 2005). In addition, other transcription factors are important for maintenance of a mature beta-cell phenotype and their impairment may account for various pathophysiological abnormalities observed in type 2 diabetics. Among these, Pdx-1, neurogenin differentiation (NeuroD/BETA-2), foxhead box protein (FoxO-1), sterol regulatory element binding protein (SREBP-1c), and musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) are the most studied (Johnson et al., 1994; Diraison et al., 2004; Kitamura et al., 2005).

In the context of T2D, it is a well-known fact that abnormal gene expression contributes to a myriad of beta-cell abnormalities. Support for this comes from studies of maturity-onset diabetes of the young (MODY), a monogenic form of T2D characterized by an early onset and defects in insulin secretion leading to hyperglycemia. With the exception of MODY-2, which is caused by a mutation in GK, MODY-1, 3, 4, 5 and 6 result from mutations in genes encoding transcription factors, hepatocyte nuclear factor (HNF) HNF-4α, HNF-1α, HNF-1β, Pdx-1, and NeuroD/BETA-2 respectively. These transcription factors regulate the expression of key genes involved in various aspects of beta-cell function (Stoffer & Zinkin, 1997; Habener et al., 1998; Fajans et al., 2001; Yamagata et al, 2003). Although there have been significant advancements in understanding the basic transcriptional network that exists in beta-cells, the exact mechanism of action of many of these factors still remains to be further defined. In this chapter we provide an overview of one of the recently described transcription factor in the context of impaired insulin secretion and beta-cell dysfunction, Aryl hydrocarbon receptor nuclear translocator (ARNT)/ hypoxia inducible factor 1β (HIF-1β), which is a master regulator of pancreatic beta-cell transcriptional network that regulates glucose metabolism and insulin secretion.
