Terumasa Okada

Loss of ARNT/HIF1β Mediates Altered Gene Expression and Pancreatic-Islet Dysfunction in Human Type 2 Diabetes

beta cell dysfunction is a central component of the pathogenesis of type 2 diabetes. Using oligonucleotide microarrays and real-time PCR of pancreatic islets isolated from humans with type 2 diabetes versus normal glucose-tolerant controls, we identified multiple changes in expression of genes known to be important in beta cell function, including major decreases in expression of HNF4alpha, insulin receptor, IRS2, Akt2, and several glucose-metabolic-pathway genes. There was also a 90% decrease in expression of the transcription factor ARNT. Reducing ARNT levels in Min6 cells with small interfering RNA (siRNA) resulted in markedly impaired glucose-stimulated insulin release and changes in gene expression similar to those in human type 2 islets. Likewise, beta cell-specific ARNT knockout mice exhibited abnormal glucose tolerance, impaired insulin secretion, and changes in islet gene expression that mimicked those in human diabetic islets. Together, these data suggest an important role for decreased ARNT and altered gene expression in the impaired islet function of human type 2 diabetes.

Total insulin and IGF-I resistance in pancreatic β cells causes overt diabetes

An appropriate β cell mass is pivotal for the maintenance of glucose homeostasis. Both insulin and IGF-1 are important in regulation of β cell growth and function (reviewed in ref. 2). To define the roles of these hormones directly, we created a mouse model lacking functional receptors for both insulin and IGF-1 only in β cells (βDKO), as the hormones have overlapping mechanisms of action and activate common downstream proteins. Notably, βDKO mice were born with a normal complement of islet cells, but 3 weeks after birth, they developed diabetes, in contrast to mild phenotypes observed in single mutants. Normoglycemic 2-week-old βDKO mice manifest reduced β cell mass, reduced expression of phosphorylated Akt and the transcription factor MafA, increased apoptosis in islets and severely compromised β cell function. Analyses of compound knockouts showed a dominant role for insulin signaling in regulating β cell mass. Together, these data provide compelling genetic evidence that insulin and IGF-I–dependent pathways are not critical for development of β cells but that a loss of action of these hormones in β cells leads to diabetes. We propose that therapeutic improvement of insulin and IGF-I signaling in β cells might protect against type 2 diabetes.

Insulin receptors in β-cells are critical for islet compensatory growth response to insulin resistance

Insulin and insulin-like growth factor 1 (IGF1) are ubiquitous growth factors that regulate proliferation in most mammalian tissues including pancreatic islets. To explore the specificity of insulin receptors in compensatory β-cell growth, we examined two models of insulin resistance. In the first model, we used liver-specific insulin receptor knockout (LIRKO) mice, which exhibit hyperinsulinemia without developing diabetes due to a compensatory increase in β-cell mass. LIRKO mice, also lacking functional insulin receptors in β-cells (βIRKO/LIRKO), exhibited severe glucose intolerance but failed to develop compensatory islet hyperplasia, together leading to early death. In the second model, we examined the relative significance of insulin versus IGF1 receptors in islet growth by feeding high-fat diets to βIRKO and β-cell-specific IGF1 receptor knockout (βIGFRKO) mice. Although both groups on the high-fat diet developed insulin resistance, βIRKO, but not βIGFRKO, mice exhibited poor islet growth consistent with insulin-stimulated phosphorylation, nuclear exclusion of FoxO1, and reduced expression of Pdx-1. Together these data provide direct genetic evidence that insulin/FoxO1/Pdx-1 signaling is one pathway that is crucial for islet compensatory growth response to insulin resistance.

Targeted Elimination of Peroxisome Proliferator-Activated Receptor γ in β Cells Leads to Abnormalities in Islet Mass without Compromising Glucose Homeostasis

ABSTRACT The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) is an important regulator of lipid and glucose homeostasis and cellular differentiation. Studies of many cell types in vitro and in vivo have demonstrated that activation of PPARγ can reduce cellular proliferation. We show here that activation of PPARγ is sufficient to reduce the proliferation of cultured insulinoma cell lines. We created a model with mice in which the expression of the PPARG gene in β cells was eliminated (βγKO mice), and these mice were found to have significant islet hyperplasia on a chow diet. Interestingly, the normal expansion of β-cell mass that occurs in control mice in response to high-fat feeding is markedly blunted in these animals. Despite this alteration in β-cell mass, no effect on glucose homeostasis in βγKO mice was noted. Additionally, while thiazolidinediones enhanced insulin secretion from cultured wild-type islets, administration of rosiglitazone to insulin-resistant control and βγKO mice revealed that PPARγ in β cells is not required for the antidiabetic actions of these compounds. These data demonstrate a critical physiological role for PPARγ function in β-cell proliferation and also indicate that the mechanisms controlling β-cell hyperplasia in obesity are different from those that regulate baseline cell mass in the islet.

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.

Melanin Concentrating Hormone Is a Novel Regulator of Islet Function and Growth

Melanin concentrating hormone (MCH) is a hypothalamic neuropeptide known to play a critical role in energy balance. We have previously reported that overexpression of MCH is associated with mild obesity. In addition, mice have substantial hyperinsulinemia and islet hyperplasia that is out of proportion with their degree of obesity. In this study, we further explored the role of MCH in the endocrine pancreas. Both MCH and MCHR1 are expressed in mouse and human islets and in clonal β-cell lines as assessed using quantitative real-time PCR and immunohistochemistry. Mice lacking MCH (MCH-KO) on either a C57Bl/6 or 129Sv genetic background showed a significant reduction in β-cell mass and complemented our earlier observation of increased β-cell mass in MCH-overexpressing mice. Furthermore, the compensatory islet hyperplasia secondary to a high-fat diet, which was evident in wild-type controls, was attenuated in MCH-KO. Interestingly, MCH enhanced insulin secretion in human and mouse islets and rodent β-cell lines in a dose-dependent manner. Real-time PCR analyses of islet RNA derived from MCH-KO revealed altered expression of islet-enriched genes such as glucagon, forkhead homeobox A2, hepatocyte nuclear factor (HNF)4α, and HNF1α. Together, these data provide novel evidence for an autocrine role for MCH in the regulation of β-cell mass dynamics and in islet secretory function and suggest that MCH is part of a hypothalamic-islet (pancreatic) axis.

Altered Insulin Receptor Signalling and β-Cell Cycle Dynamics in Type 2 Diabetes Mellitus

Insulin resistance, reduced β-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced β-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in β-cells – which also exhibits reduced β-cell mass, the β-cell-specific insulin receptor knockout (βIRKO). Freshly isolated islets and β-cell lines derived from βIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in βIRKO β-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in β-cell growth. These data provide evidence that human β- and α-cells can enter the cell-cycle, but proliferation of β-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in β-cell-cycle progression are attractive therapeutic targets to enhance β-cell regeneration in the treatment of T2DM.

Insulin Signaling Regulates Mitochondrial Function in Pancreatic β-Cells

Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in β-cells that included glucokinase (GK), and the pro-apoptotic protein, BADS. Mitochondria isolated from β-cells derived from β-cell specific insulin receptor knockout (βIRKO) mice exhibited reduced BADS, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in βIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BADS. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in βIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for β-cell dysfunction in type 2 diabetes.

Tissue-specific targeting of the insulin receptor gene

The techniques to study the mechanisms that underlie the pathogenesis of disease processes have been revolutionized by the development of methods that allow spatiotemporal control of gene deletion or gene expression in transgenic and knockout animals. The ability to interfere with the function of a single protein in a specific tissue allows unprecedented flexibility for exploring gene function in both health and disease. The present review will summarize some of the different knockouts and transgenics generated recently to study type 2 diabetes and critically evaluate the techniques used to examine the function of the insulin receptor in two nonclassical insulin target tissues—the pancreatic islet and the central nervous system.