**3.2 ARNT/HIF-1β is reduced in human diabetic hepatic cells**

In both rodents and humans, the liver plays a critical role in maintaining glucose and lipid homeostasis. During fasting, hepatic glucose production is critical for providing glucose for the brain, the kidneys and red blood cells. In liver, glucose is produced by glycogenolysis during the initial stages of fasting, however, after several hours of fasting, glucose production is primarily from gluconeogenesis, a process by which the liver produces glucose from precursors such as lactate and pyruvate (Michael et al., 2000; Saltiel & Kahn, 2001). Wang et al showed that ARNT/HIF-1β was severely reduced in the livers of human type 2 diabetics (Wang et al., 2009). Gene expression profiling of liver specimens from normal, obese and obese diabetic patients revealed a 30% reduction in the expression of ARNT/HIF-1β gene in obese diabetic individuals. The study demonstrated that the reduced expression of ARNT/HIF-1β in the livers of humans with T2D was associated with high glucose levels, high insulin levels, and insulin resistance. This study also suggested that insulin, not glucose regulates the expression of ARNT/HIF-1β gene and that ARNT/HIF-1β expression is reduced in both insulin-deficient and insulinresistant states.

Wang et al (2009) also looked at the effects of liver-specific deletion of ARNT/HIF-1β gene in mice (L-ARNT KO) and demonstrated that there was an increase in gluconeogenesis, lipogenesis and increased serum insulin levels, all characteristic of human type 2 diabetics. The increase in hepatic gluconeogenesis and lipogenesis in L-ARNT KO mice was associated with the upregulation of several important gluconeogenic and lipogenic genes including PEPCK, G6Pase, SCD1 and FAS. Expression of C/EBPα and SREBP-1C, was also induced by 2-folds in L-ARNT KO mice. C/EBPα plays a major role in kick-starting hepatic glucose production at birth, and disruption of the C/EBPα gene in mice is known to cause hypoglycemia associated with the impaired expression of the gluconeogenic enzymes PEPCK and G6Pase (Pedersen et al., 2007; Qiao et al., 2006). SREBP-1C, on the other hand is a major player in lipogenesis (Horton et al., 2002). ARNT/HIF-1β may act as an upstream regulator of these transcription factors and play a key role in maintaining whole body glucose and lipid homeostasis. However, as seen in pancreatic beta-cells, the exact pathways targeted by ARNT/HIF-1β in liver cells are not clearly understood and is complicated by the fact that ARNT/HIF-1β has multiple binding partners.

#### **3.3 ARNT/HIF-1β regulates glucose metabolism and insulin secretion in beta-cells**

The central role played by ARNT/HIF-1β/HIF-1α heterodimer in the regulation of glucose homeostasis, particularly glycolysis has been well studied (Dery et al., 2005; Semenza et al., 1994) with a focus in cancer cell metabolism (Song et al., 2009; Semenza et al., 2000; Semenza, 2003). It is widely accepted that ARNT/HIF-1β/HIF-1α heterodimer plays a role in the Warburg effect, where cancer cells undergo a high rate of anaerobic glycolysis compared to normal cells. It has been suggested that the observed increase in glycolytic enzymes in these cancer cells is associated with increased HIF-1 activity, thus aiding in tumor formation and progression. Studies conducted in ARNT/HIF-1β mutant clonal cells indicate that it is an essential component of the HIF-1α complex and that absence of ARNT/HIF-1β leads to reduced cellular responses to stimuli such as hypoxia (Woods et al., 1996).

In pancreatic beta-cells, metabolism of glucose through aerobic glycolysis and oxidative phosphorylation plays a significant role in maintaining a normal secretory capacity. Betacells sense glucose and secrete appropriate amounts of insulin to promote glucose uptake by muscles and adipose tissue. Insulin also inhibits hepatic glucose production. Abnormal insulin secretion is one of the earliest detectable defects at the onset of T2D and despite its relevance, the mechanisms underlying GSIS are not completely understood. The generally accepted model of GSIS holds that metabolism of glucose in the beta-cells leads to a rise in the cytosolic ATP/ADP levels, which promotes closure of the KATP channel, increased cytosolic Ca2+ and triggers exocytosis of insulin-containing secretory granules (Henquin et al., 2003; Jensen et al., 2008). In beta-cells glucose derived pyruvate is directed mostly towards TCA for the production of ATP, since both the pentose phosphate pathway and anaerobic glycolysis is relatively inactive (Schuit et al., 1997). This exceptionally high dependence of beta-cells on the TCA cycle suggests that hypoxia or mechanisms reducing the aerobic capacity of beta-cells would probably have profound effects on GSIS.

It has been shown that down regulation of ARNT/HIF-1β in pancreatic beta-cells leads to loss of GSIS (Gunton et al., 2005; Pillai et al 2011). Our group has demonstrated that beta-cells with reduced ARNT/HIF-1β expression levels exhibit a 31% reduction in glycolytic flux without significant changes in glucose oxidation or the ATP/ADP ratio. Metabolomics analysis revealed that clonal beta-cells (832/13) treated with siRNAs against the ARNT/HIF-1β gene have lower levels of glycolytic, TCA cycle and fatty acid intermediates (Figure 3). It was also shown that the reduced levels of glycolysis, TCA and fatty acid intermediates were associated with a corresponding decrease in the expression of key genes in all three metabolic pathways including GLUT2, GK, PC, PDH, MEc, CIC DIC, CPT1a and FAS (Figure 4). The novel finding that reducing ARNT/HIF-1β levels leads to a profound reduction in PC, DIC, and OGC expression levels and a reduction in glycoslysis and TCA metabolites, even though glucose oxidation and ATP production were unaltered, is an unexpected result. These collective changes in metabolite levels suggest that the oxidative entry of pyruvate into the TCA cycle is preserved in the absence of ARNT/HIF-1β at the expense of a loss of anaplerosis. A key role for anaplerosis in insulin secretion is supported by the finding that pyruvate flows into mitochondrial metabolic pathways, in roughly equal proportions, through the anaplerotic (PC) and oxidative (PDH) entry points. Glucose carbon entering through the PC reaction leads to an increase in TCA intermediates (called anaplerosis) (Schuit et al., 1997; Khan et al., 1996). In addition, beta-cells contain enzymes that allow "cycling" of pyruvate via its PC-catalyzed conversion to oxaloacetate (OAA), metabolism of OAA to malate, citrate, or isocitrate in the TCA cycle, and subsequent recycling of these metabolites to pyruvate via several possible

The central role played by ARNT/HIF-1β/HIF-1α heterodimer in the regulation of glucose homeostasis, particularly glycolysis has been well studied (Dery et al., 2005; Semenza et al., 1994) with a focus in cancer cell metabolism (Song et al., 2009; Semenza et al., 2000; Semenza, 2003). It is widely accepted that ARNT/HIF-1β/HIF-1α heterodimer plays a role in the Warburg effect, where cancer cells undergo a high rate of anaerobic glycolysis compared to normal cells. It has been suggested that the observed increase in glycolytic enzymes in these cancer cells is associated with increased HIF-1 activity, thus aiding in tumor formation and progression. Studies conducted in ARNT/HIF-1β mutant clonal cells indicate that it is an essential component of the HIF-1α complex and that absence of ARNT/HIF-1β leads to

In pancreatic beta-cells, metabolism of glucose through aerobic glycolysis and oxidative phosphorylation plays a significant role in maintaining a normal secretory capacity. Betacells sense glucose and secrete appropriate amounts of insulin to promote glucose uptake by muscles and adipose tissue. Insulin also inhibits hepatic glucose production. Abnormal insulin secretion is one of the earliest detectable defects at the onset of T2D and despite its relevance, the mechanisms underlying GSIS are not completely understood. The generally accepted model of GSIS holds that metabolism of glucose in the beta-cells leads to a rise in the cytosolic ATP/ADP levels, which promotes closure of the KATP channel, increased cytosolic Ca2+ and triggers exocytosis of insulin-containing secretory granules (Henquin et al., 2003; Jensen et al., 2008). In beta-cells glucose derived pyruvate is directed mostly towards TCA for the production of ATP, since both the pentose phosphate pathway and anaerobic glycolysis is relatively inactive (Schuit et al., 1997). This exceptionally high dependence of beta-cells on the TCA cycle suggests that hypoxia or mechanisms reducing

**3.3 ARNT/HIF-1β regulates glucose metabolism and insulin secretion in beta-cells** 

reduced cellular responses to stimuli such as hypoxia (Woods et al., 1996).

the aerobic capacity of beta-cells would probably have profound effects on GSIS.

It has been shown that down regulation of ARNT/HIF-1β in pancreatic beta-cells leads to loss of GSIS (Gunton et al., 2005; Pillai et al 2011). Our group has demonstrated that beta-cells with reduced ARNT/HIF-1β expression levels exhibit a 31% reduction in glycolytic flux without significant changes in glucose oxidation or the ATP/ADP ratio. Metabolomics analysis revealed that clonal beta-cells (832/13) treated with siRNAs against the ARNT/HIF-1β gene have lower levels of glycolytic, TCA cycle and fatty acid intermediates (Figure 3). It was also shown that the reduced levels of glycolysis, TCA and fatty acid intermediates were associated with a corresponding decrease in the expression of key genes in all three metabolic pathways including GLUT2, GK, PC, PDH, MEc, CIC DIC, CPT1a and FAS (Figure 4). The novel finding that reducing ARNT/HIF-1β levels leads to a profound reduction in PC, DIC, and OGC expression levels and a reduction in glycoslysis and TCA metabolites, even though glucose oxidation and ATP production were unaltered, is an unexpected result. These collective changes in metabolite levels suggest that the oxidative entry of pyruvate into the TCA cycle is preserved in the absence of ARNT/HIF-1β at the expense of a loss of anaplerosis. A key role for anaplerosis in insulin secretion is supported by the finding that pyruvate flows into mitochondrial metabolic pathways, in roughly equal proportions, through the anaplerotic (PC) and oxidative (PDH) entry points. Glucose carbon entering through the PC reaction leads to an increase in TCA intermediates (called anaplerosis) (Schuit et al., 1997; Khan et al., 1996). In addition, beta-cells contain enzymes that allow "cycling" of pyruvate via its PC-catalyzed conversion to oxaloacetate (OAA), metabolism of OAA to malate, citrate, or isocitrate in the TCA cycle, and subsequent recycling of these metabolites to pyruvate via several possible

Fig. 3. Summary of the effects of siRNA mediated suppression of ARNT/HIF-1β in betacells. Several key metabolites in both glycolysis and TCA cycle were negatively affected by the knockdown of ARNT/HIF-1β. TCA, tricarboxylic acid cycle; DHAP, dihydroxyacetone phosphate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase.

Fig. 4. Effects of siRNA mediated suppression of ARNT/HIF-1β on key genes involved in the metabolic regulation of β-cell function in 832/13 cells. Gene expression is expressed as a percentage of the target gene from siControl treated cells and corrected for by an internal control gene cyclophilin E (Cyp). There was no significant difference seen between treatment groups for Cyp (n=5). HNF4a, hepatocyte nuclear factor-4α; HNF1a, hepatocyte nuclear factor-1α; HIF1a, hypoxia inducible factor 1α; GK, glucokinase; GLUT2, glucose transporter-2; PLGg1, phospholipase γ-1; PC, pyruvate carboxylase; PDHa1, pyruvate dehydrogenase (α1 subunit); MEc, cytosolic malic enzyme; ICDc, cytosolic isocitrate dehydrogenase; CIC, citrate carrier; DIC, dicarboxylate carrier; OGC, α-ketoglutarate carrier; FAS, fatty acid synthase; CPT1a, Carnitine palmitoyl transferase 1α. \* P<0.05, \*\* P<0.01, \*\*\* P<0.001 siControl vs siARNT1.

combinations of cytosolic and mitochondrial pathways (MacDonald et al., 1995). Numerous groups have shown that both pyruvate cycling and anaplerosis are important to maintain normal secretory capacity of beta-cells (Lu et al., 2002; Joseph et al., 2006; MacDonald et al., 2005; Ronnebaum et al., 2006). Since the amount of pyruvate is substantially lower in ARNT/HIF-1β depleted beta-cells, our data also suggests that this gene may play an important role in maintaining pyruvate cycling. However a direct link between ARNT/HIF-1β and pyruvate cycling has not yet been established. Figure 5 shows the diagrammatic representation of the transcriptional network regulated by ARNT/HIF-1β and its involvement glucose-stimulated anaplerosis and insulin release.

Fig. 5. Schematic of the transcriptional network regulated by ARNT/HIF-1β and its involvement in glucose-stimulated anaplerosis and insulin release from beta-cells. ARNT/HIF-1β regulates key genes in glycolysis and TCA cycle (shown in blue), including key metabolite carriers such as DIC and OGC. MODY genes regulated by ARNT/HIF-1β are shown in green. Interestingly, ARNT/HIF-1β does not seem to play a significant role in the regulation ATP production in beta-cells, however, it seems to be very important for glucoseinduced anaplerosis, which provides crucial signals for GSIS.
