**3.4 ARNT/HIF-1β regulates beta-cell and hepatic transcriptional networks**

Studies in ARNT/HIF-1β deficient beta-cells suggest that it plays a crucial role in the regulation of key genes involved in glucose metabolism and insulin secretion (Gunton et al., 2005, Pillai et al., 2011). ARNT/HIF-1β target genes in beta-cells include the MODY1 and MODY3 genes HNF4α and HNF1α, glucose metabolism genes GK, G6PI, PFK, aldolase, PC, PDH, MEc, DIC, OGC and insulin signaling genes IR, IRS2 and AKT2. In non-beta-cells it

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

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.

**3.4 ARNT/HIF-1β regulates beta-cell and hepatic transcriptional networks** 

induced anaplerosis, which provides crucial signals for GSIS.

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 glucose-

Studies in ARNT/HIF-1β deficient beta-cells suggest that it plays a crucial role in the regulation of key genes involved in glucose metabolism and insulin secretion (Gunton et al., 2005, Pillai et al., 2011). ARNT/HIF-1β target genes in beta-cells include the MODY1 and MODY3 genes HNF4α and HNF1α, glucose metabolism genes GK, G6PI, PFK, aldolase, PC, PDH, MEc, DIC, OGC and insulin signaling genes IR, IRS2 and AKT2. In non-beta-cells it

glucose-stimulated anaplerosis and insulin release.

has been shown that ARNT/HIF-1β is essential for the normal function of HIF-1α, HIF2α, and AhR. These heterodimeric complexes are required for cellular responses to hypoxia (HIF proteins) and environmental toxins (AhR), respectively (Kozak et al., 1997; Kewley et al., 2004). It has been estimated that there are more than 13,000 putative ARNT/HIF-1β binding sites in promoters in the human genome (Gunton et al., 2005). Many of the target gene promoters have multiple potential binding sites. Thus it is reasonable to estimate that a substantial decrease in ARNT/HIF-1β would affect the expression of a large number of genes in humans. Although there is a lack of direct biochemical evidence, many of the genes found to be altered in association with decreased ARNT/HIF-1β gene expression have putative ARNT/HIF-1β-dimer consensus binding sites in their promoters (including HNF4α, HNF1α, Akt2, G6PI, PFK, and aldolase), suggesting a direct role for ARNT/HIF-1β containing dimers in the regulation of their expression (Figure 6).

Fig. 6. Transcriptional network in pancreatic beta-cells regulated by ARNT/HIF-1β. ARNT/HIF-1β regulates several key genes involved in glucose metabolism, insulin signaling and MODY. Beta-cell specific knockout of ARNT/HIF-1β in mice leads to reduced expression of a number of important beta-cell genes including HNF-4α, HNF-1α, insulin receptor (IR), insulin receptor substrate-2 (IRS2), protein kinase b (Akt2), glucokinase (GK), glucose-6-phosphoisomerase (G6PI), phosphofructokinase (PFK), aldolase (ALDO), pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH).

In liver cells ARNT/HIF-1β has been shown to regulate the expression of several genes involved in glucose and lipid homeostasis. Support for the involvement of ARNT/HIF-1β in liver glucose homeostasis was provided by experiments showing that basal and insulin-induced expression of GLUT1, GLUT3, ALDO, PGK and VEGF were significantly reduced in ARNT/HIF-1β-defective HepG2 cells (Salceda et al., 1996). Wang *et al* (2009) demonstrated that a reduction of ARNT/HIF-1β in liver cells was associated with an increase in the expression of several important gluconeogenic and lipogenic genes including PEPCK, G6Pase, SCD1, FXR, C/EBPα, SREBP-1C, FBP-1 and FAS. The discovery that ARNT/HIF-1β may contribute to the regulation of beta-cell and hepatic genes suggests an essential role for this transcription factor in the regulation of glucose and lipid homeostasis (Figure 7).

Fig. 7. ARNT/HIF-1β regulates the expression of several key genes involved in gluconeogenesis, lipogenesis and ketogenesis in the liver cells. Liver-specific knockout of ARNT/HIF-1β in mice leads to increased hepatic gluconeogensis and lipogenesis with a corresponding increase in the expression of phosphoenolpyruvate carboxykinase (PEPCK), Glucose-6-phosphatase (G6Pase), Fructose-1,6-biphosphatase (FBP1), Steroyl–CoAdesaturase (SCD1), Fatty acid synthase (FAS), CCAAT enhancer binding protein α (C/EBPα), sterol regulatory element binding protein (SREBP-1C) and Farsenoid X receptor (FXR). Adapted from Wang *et al.* (2009).
