**3.5 Regulation of ARNT/HIF-1β in beta-cells**

Since ARNT/HIF-1β appears to be a major player in the pathogenesis of T2D, several attempts have been made to identify the upstream regulators of the gene in beta-cells. In 2008, Dror *et al.*, showed that glucose and endoplasmic reticulum Ca 2+ channels regulate the expression of ARNT/HIF-1β in beta-cells via presinilin. Presinilin is a protein that has been implicated in the cellular response to reduced metabolic activity (Koo and Koppen, 2004). Overexpression of presenilin-1 in clonal Min6 beta-cells increased ARNT/HIF-1β suggesting that ARNT/HIF-1β may be a downstream target of presenilin (Dror et al., 2008). They demonstrated that this pathway is controlled by Ca2+ flux through intracellular channels. ARNT/HIF-1β has also recently been shown to be regulated by the carbohydrate-responsive element-binding protein (ChREBP), which is a transcription factor shown to regulate carbohydrate metabolism in the liver and pancreatic beta-cells in response to elevated glucose concentrations (Noordeen et al., 2009). In a genome-wide approach using highdensity oligonucleotide arrays, the study showed that ChREBP binds directly to ARNT/HIF-1β promoter in Min6 clonal beta-cells. Accordingly, knockdown of ChREBP using siRNA resulted in an increase ARNT/HIF-1β mRNA levels whereas overexpression of ChREBP resulted in a decrease in ARNT/HIF-1β mRNA levels in rat. They also showed that incubating INS-1 (832/13) cells with glucose led to a substantial decrease in ARNT/HIF-1β mRNA levels.

Fig. 7. ARNT/HIF-1β regulates the expression of several key genes involved in

Glucose-6-phosphatase (G6Pase), Fructose-1,6-biphosphatase (FBP1), Steroyl–CoAdesaturase (SCD1), Fatty acid synthase (FAS), CCAAT enhancer binding protein α

(FXR). Adapted from Wang *et al.* (2009).

ARNT/HIF-1β mRNA levels.

**3.5 Regulation of ARNT/HIF-1β in beta-cells** 

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),

(C/EBPα), sterol regulatory element binding protein (SREBP-1C) and Farsenoid X receptor

Since ARNT/HIF-1β appears to be a major player in the pathogenesis of T2D, several attempts have been made to identify the upstream regulators of the gene in beta-cells. In 2008, Dror *et al.*, showed that glucose and endoplasmic reticulum Ca 2+ channels regulate the expression of ARNT/HIF-1β in beta-cells via presinilin. Presinilin is a protein that has been implicated in the cellular response to reduced metabolic activity (Koo and Koppen, 2004). Overexpression of presenilin-1 in clonal Min6 beta-cells increased ARNT/HIF-1β suggesting that ARNT/HIF-1β may be a downstream target of presenilin (Dror et al., 2008). They demonstrated that this pathway is controlled by Ca2+ flux through intracellular channels. ARNT/HIF-1β has also recently been shown to be regulated by the carbohydrate-responsive element-binding protein (ChREBP), which is a transcription factor shown to regulate carbohydrate metabolism in the liver and pancreatic beta-cells in response to elevated glucose concentrations (Noordeen et al., 2009). In a genome-wide approach using highdensity oligonucleotide arrays, the study showed that ChREBP binds directly to ARNT/HIF-1β promoter in Min6 clonal beta-cells. Accordingly, knockdown of ChREBP using siRNA resulted in an increase ARNT/HIF-1β mRNA levels whereas overexpression of ChREBP resulted in a decrease in ARNT/HIF-1β mRNA levels in rat. They also showed that incubating INS-1 (832/13) cells with glucose led to a substantial decrease in Interestingly, it has also been shown that HIF-1α, the highly regulated binding partner for ARNT/HIF-1β, may be active under normoxic conditions in mouse and human beta-cells (Cheng et al., 2010). Coimmunoprecipitation studies demonstrated that HIF-1α was bound to ARNT/HIF-1β at the promoter region providing evidence for an interaction between HIF-1α and ARNT/HIF-1β in beta-cells. Treatment of diabetic mice with deferasirox (DFS), an agent that increases HIF-1α protein levels, improved glucose tolerance, normalized the expression of ARNT/HIF-1β and its target genes in human T2D islets. The same study also showed that HIF-2α, but not AhR, is another possible binding partner for ARNT/HIF-1β in pancreatic beta-cells. These studies provide a novel mechanism to regulate ARNT/HIF-1β gene expression in beta-cells.

Three studies published from independent laboratories studying the impact of increasing HIF-1α levels in beta-cells indicate that one has to be extremely cautious when using pharmacological agents, such as DFS, to activate HIF-1α in the islets (Puri et al., 2008; Zehetner et al., 2008; Cantley et al., 2009). These studies used the Cre-loxP system to conditionally delete VHL gene in beta-cells and showed that there were adverse effects associated with an increase in HIF-1α levels on beta-cell function. In all the three studies, increased HIF-1α levels were accompanied by severely impaired GSIS and increased lactate production, indicating a switch from aerobic to anaerobic glycolysis. Thus there appears to be a dose-response curve for the affects HIF-1α protein levels on beta-cell function (Cheng *et al.*, 2010). Although complete lack of HIF-1α seems deleterious to GSIS in mice and Min6 cells, milder increases are beneficial for beta-cell function. As seen in VHL knockout mice, very high levels of HIF-1α are detrimental for normal beta-cell function. Therefore, before we begin to develop a novel treatment regime that enhances HIF-1α or ARNT/HIF-1β activity in human diabetic islets, it is imperative that we understand the expected outcomes of such changes to avoid any detrimental effects.
