Kim Cheng

Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1alpha protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1alpha is required for normal beta cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1alpha protein is present at low levels in mouse and human normoxic beta cells and islets. Decreased levels of HIF-1alpha impaired glucose-stimulated ATP generation and beta cell function. C57BL/6 mice with beta cell-specific Hif1a disruption (referred to herein as beta-Hif1a-null mice) exhibited glucose intolerance, beta cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1alpha levels by inhibiting its degradation through iron chelation markedly improved insulin secretion and glucose tolerance in control mice fed a high-fat diet but not in beta-Hif1a-null mice. Increasing HIF-1alpha levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1alpha was bound to the Arnt promoter in a mouse beta cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1alpha in beta cell reserve and regulation of ARNT expression and demonstrate that HIF-1alpha is a potential therapeutic target for the beta cell dysfunction of T2D.

Vitamin D Signaling Regulates Proliferation, Differentiation, and Myotube Size in C2C12 Skeletal Muscle Cells

Vitamin D deficiency is linked to a range of muscle disorders including myalgia, muscle weakness, and falls. Humans with severe vitamin D deficiency and mice with transgenic vitamin D receptor (VDR) ablation have muscle fiber atrophy. However, molecular mechanisms by which vitamin D influences muscle function and fiber size remain unclear. A central question is whether VDR is expressed in skeletal muscle and is able to regulate transcription at this site. To address this, we examined key molecular and morphologic changes in C2C12 cells treated with 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D). As well as stimulating VDR expression, 25(OH)D and 1,25(OH)(2)D dose-dependently increased expression of the classic vitamin D target cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1), demonstrating the presence of an autoregulatory vitamin D-endocrine system in these cells. Luciferase reporter studies demonstrated that cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1) was functional in these cells. Both 25OHD and 1,25(OH)(2)D altered C2C12 proliferation and differentiation. These effects were related to the increased expression of genes involved in G(0)/G(1) arrest (retinoblastoma protein [Rb], 1.3-fold; ATM, 1.5-fold, both P < .05), downregulation of mRNAs involved in G(1)/S transition, including myc and cyclin-D1 (0.7- and 0.8-fold, both P < .05) and reduced phosphorylation of Rb protein (0.3-fold, P < .005). After serum depletion, 1,25(OH)(2)D (100nM) suppressed myotube formation with decreased mRNAs for key myogenic regulatory factors (myogenin, 0.5-fold; myf5, 0.4-fold, P < .005) but led to a 1.8-fold increase in cross-sectional size of individual myotubes associated with markedly decreased myostatin expression (0.2-fold, P < .005). These data show that vitamin D signaling alters gene expression in C2C12 cells, with effects on proliferation, differentiation, and myotube size.

Novel links between HIFs, type 2 diabetes, and metabolic syndrome

Hypoxia inducible factors (HIFs) are master-regulators of cellular responses to hypoxia, and thus are crucial for survival. HIFs also play a role in regulating cellular processes in β-cells, liver, muscle, and adipose tissue, have effects on the regulation of weight, and play a role in type 2 diabetes (T2D). Indeed, in people with T2D the HIF pathway is dyregulated in major metabolic tissues involved in the pathogenesis of diabetes. This review covers the contrasting, complementary and conflicting effects of decreasing and increasing HIFs in various tissues, and shows that a delicate balance exists between HIF levels and optimal metabolic function. We propose that increasing the activity of HIFs might be a potential therapeutic strategy for treating T2D.

Hypoxia-inducible factor-1α (HIF-1α) potentiates β-cell survival after islet transplantation of human and mouse islets.

A high proportion of β-cells die within days of islet transplantation. Reports suggest that induction of hypoxia-inducible factor-1α (HIF-1α) predicts adverse transplant outcomes. We hypothesized that this was a compensatory response and that HIF-1α protects β-cells during transplantation. Transplants were performed using human islets or murine β-cell-specific HIF-1α-null (β-HIF-1α-null) islets with or without treatment with deferoxamine (DFO) to increase HIF-1α. β-HIF-1α-null transplants had poor outcomes, demonstrating that lack of HIF-1α impaired transplant efficiency. Increasing HIF-1α improved outcomes for mouse and human islets. No effect was seen in β-HIF-1α-null islets. The mechanism was decreased apoptosis, resulting in increased β-cell mass posttransplantation. These findings show that HIF-1α is a protective factor and is required for successful islet transplant outcomes. Iron chelation with DFO markedly improved transplant success in a HIF-1α-dependent manner, thus demonstrating the mechanism of action. DFO, approved for human use, may have a therapeutic role in the setting of human islet transplantation.

High Passage MIN6 Cells Have Impaired Insulin Secretion with Impaired Glucose and Lipid Oxidation

Type 2 diabetes is a metabolic disorder characterized by the inability of beta-cells to secrete enough insulin to maintain glucose homeostasis. MIN6 cells secrete insulin in response to glucose and other secretagogues, but high passage (HP) MIN6 cells lose their ability to secrete insulin in response to glucose. We hypothesized that metabolism of glucose and lipids were defective in HP MIN6 cells causing impaired glucose stimulated insulin secretion (GSIS). HP MIN6 cells had no first phase and impaired second phase GSIS indicative of global functional impairment. This was coupled with a markedly reduced ATP content at basal and glucose stimulated states. Glucose uptake and oxidation were higher at basal glucose but ATP content failed to increase with glucose. HP MIN6 cells had decreased basal lipid oxidation. This was accompanied by reduced expressions of Glut1, Gck, Pfk, Srebp1c, Ucp2, Sirt3, Nampt. MIN6 cells represent an important model of beta cells which, as passage numbers increased lost first phase but retained partial second phase GSIS, similar to patients early in type 2 diabetes onset. We believe a number of gene expression changes occurred to produce this defect, with emphasis on Sirt3 and Nampt, two genes that have been implicated in maintenance of glucose homeostasis.

First Phase Insulin Secretion and Type 2 Diabetes

Type 2 diabetes (T2D) is a metabolic disorder characterised by the inability of β-cells to secrete enough insulin to maintain glucose homeostasis. Pancreatic β-cells secrete insulin in a biphasic manner, first and second phase insulin secretion, and loss of first phase insulin secretion is an independent predictor of T2D onset. Restoration of first phase insulin secretion has been shown to improve blood glucose in T2D by suppressing hepatic glucose production and priming insulin sensitive tissue to more readily take up glucose and has thus prompted numerous studies into its regulation. First phase insulin secretion is initiated primarily by the classical triggering pathway, a complex system comprised of multiple stimulatory signals. Recent studies have identified a number of novel regulatory factors that are crucial for first phase insulin secretion and glucose homeostasis. These include, among others, hypoxia inducible factor 1α, von Hippel-Lindau, factor inhibiting HIF, nicotinamide phospho-ribosyl-transferase, and the sirtuin family. This review will outline how first phase insulin secretion is initiated and detail some of the recent findings in its regulation.

Synergistic effects of genetic beta cell dysfunction and maternal glucose intolerance on offspring metabolic phenotype in mice

Aims/hypothesisDiabetes in pregnancy is linked to development of obesity in the offspring, but the mechanisms are not fully understood. Gestational diabetes mellitus (GDM) occurs when beta cells are unable to compensate for the normal insulin resistance of late pregnancy. In this study, we used a murine model of beta cell dysfunction to examine the effects of maternal GDM on phenotype in male offspring with and without an inherited predisposition for beta cell dysfunction.MethodsBeta cell-specific aryl-hydrocarbon receptor nuclear translocator-null (βArnt) mice develop GDM from beta cell dysfunction. βArnt and control female mice were used to induce GDM and non-diabetic pregnancies, respectively.ResultsOffspring from GDM pregnancies became spontaneously obese on a normal-chow diet. They were heavier than offspring from non-diabetic pregnancies, with increased body fat. Respiratory exchange ratio (RER) was higher, indicating decreased capacity to switch to lipid oxidation. Metabolic rate in GDM offspring was decreased prior to onset of obesity. The phenotype was more pronounced in βArnt GDM offspring than in GDM offspring of control genotype, demonstrating an interaction between genotype and pregnancy exposure. βArnt GDM offspring had increased hypothalamic neuropeptide Y (Npy) and decreased pro-opiomelanocortin (Pomc) expression. Weight, body fat, insulin sensitivity and RER in all mice, and hypothalamic Npy in βArnt mice were significantly correlated with AUC of maternal late pregnancy glucose tolerance tests (p < 0.01), but not with litter size, maternal weight, triacylglycerol or pre-pregnancy glycaemia.Conclusions/interpretationIn βArnt mice, exposure to GDM and inheritance of genetic beta cell dysfunction had additive effects on male offspring obesity; severity of the offspring phenotype correlated with maternal glycaemia.

Beta-Cell ARNT Is Required for Normal Glucose Tolerance in Murine Pregnancy

AIMS Insulin secretion increases in normal pregnancy to meet increasing demands. Inability to increase beta-cell function results in gestational diabetes mellitus (GDM). We have previously shown that the expression of the transcription factor ARNT (Aryl-hydrocarbon Receptor Nuclear Translocator) is reduced in the islets of humans with type 2 diabetes. Mice with a beta-cell specific deletion of ARNT (β-ARNT mice) have impaired glucose tolerance secondary to defective insulin secretion. We hypothesised that ARNT is required to increase beta-cell function during pregnancy, and that β-ARNT mice would be unable to compensate for the beta-cell stress of pregnancy. The aims of this study were to investigate the mechanisms of ARNT regulation of beta-cell function and glucose tolerance in pregnancy. METHODS β-ARNT females were mated with floxed control (FC) males and FC females with β-ARNT males. RESULTS During pregnancy, β-ARNT mice had a marked deterioration in glucose tolerance secondary to defective insulin secretion. There was impaired beta-cell proliferation in late pregnancy, associated with decreased protein and mRNA levels of the islet cell-cycle regulator cyclinD2. There was also reduced expression of Irs2 and G6PI. In contrast, in control mice, pregnancy was associated with a 2.1-fold increase in ARNT protein and a 1.6-fold increase in cyclinD2 protein, and with increased beta-cell proliferation. CONCLUSIONS Islet ARNT increases in normal murine pregnancy and beta-cell ARNT is required for cyclinD2 induction and increased beta-cell proliferation in pregnancy.

Mice deficient in GEM GTPase show abnormal glucose homeostasis due to defects in beta-cell calcium handling

Aims and Hypothesis Glucose-stimulated insulin secretion from beta-cells is a tightly regulated process that requires calcium flux to trigger exocytosis of insulin-containing vesicles. Regulation of calcium handling in beta-cells remains incompletely understood. Gem, a member of the RGK (Rad/Gem/Kir) family regulates calcium channel handling in other cell types, and Gem over-expression inhibits insulin release in insulin-secreting Min6 cells. The aim of this study was to explore the role of Gem in insulin secretion. We hypothesised that Gem may regulate insulin secretion and thus affect glucose tolerance in vivo. Methods Gem-deficient mice were generated and their metabolic phenotype characterised by in vivo testing of glucose tolerance, insulin tolerance and insulin secretion. Calcium flux was measured in isolated islets. Results Gem-deficient mice were glucose intolerant and had impaired glucose stimulated insulin secretion. Furthermore, the islets of Gem-deficient mice exhibited decreased free calcium responses to glucose and the calcium oscillations seen upon glucose stimulation were smaller in amplitude and had a reduced frequency. Conclusions These results suggest that Gem plays an important role in normal beta-cell function by regulation of calcium signalling.

Hepatic Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) regulates metabolism in mice

Background & aims Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) and its partners hypoxia-inducible factors (HIF)-1α and HIF-2α are candidate factors for the well-known link between the liver, metabolic dysfunction and elevation in circulating lipids and glucose. Methods: Hepatocyte-specific ARNT-null (LARNT), HIF-1α-null (LHIF1α) and HIF-2α-null (LHIF2α) mice were created. Results LARNT mice had increased fasting glucose, impaired glucose tolerance, increased glucose production, raised post-prandial serum triglycerides (TG) and markedly lower hepatic ATP versus littermate controls. There was increased expression of G6Pase, Chrebp, Fas and Scd-1 mRNAs in LARNT animals. Surprisingly, LHIF1α and LHIF2α mice exhibited no alterations in any metabolic parameter assessed. Conclusions These results provide convincing evidence that reduced hepatic ARNT can contribute to inappropriate hepatic glucose production and post-prandial dyslipidaemia. Hepatic ARNT may be a novel therapeutic target for improving post-prandial hypertriglyceridemia and glucose homeostasis.