Deborah J. Burks

Disruption of IRS-2 causes type 2 diabetes in mice

Human type 2 diabetes is characterized by defects in both insulin action and insulin secretion. It has been difficult to identify a single molecular abnormality underlying these features. Insulin-receptor substrates (IRS proteins) may be involved in type 2 diabetes: they mediate pleiotropic signals initiated by receptors for insulin and other cytokines. Disruption of IRS-1 in mice retards growth, but diabetes does not develop because insulin secretion increases to compensate for the mild resistance to insulin,. Here we show that disruption of IRS-2 impairs both peripheral insulin signalling and pancreatic β-cell function. IRS-2-deficient mice show progressive deterioration of glucose homeostasis because of insulin resistance in the liver and skeletal muscle and a lack of β-cell compensation for this insulin resistance. Our results indicate that dysfunction of IRS-2 may contribute to the pathophysiology of human type 2 diabetes.

Irs-2 coordinates Igf-1 receptor-mediated β-cell development and peripheral insulin signalling

Insulin receptor substrates (Irs proteins) mediate the pleiotropic effects of insulin and Igf-1 (insulin-like growth factor-1), including regulation of glucose homeostasis and cell growth and survival. We intercrossed mice heterozygous for two null alleles (Irs1+/– and Irs2+/–) and investigated growth and glucose metabolism in mice with viable genotypes. Our experiments revealed that Irs-1 and Irs-2 are critical for embryonic and post-natal growth, with Irs-1 having the predominant role. By contrast, both Irs-1 and Irs-2 function in peripheral carbohydrate metabolism, but Irs-2 has the major role in β-cell development and compensation for peripheral insulin resistance. To establish a role for the Igf-1 receptor in β-cells, we intercrossed mice heterozygous for null alleles of Igf1r and Irs2. Our results reveal that Igf-1 receptors promote β-cell development and survival through the Irs-2 signalling pathway. Thus, Irs-2 integrates the effects of insulin in peripheral target tissues with Igf-1 in pancreatic β-cells to maintain glucose homeostasis.

Tissue-specific insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2

Type 2 diabetes is characterized by abnormalities of insulin action in muscle, adipose tissue, and liver and by altered beta-cell function. To analyze the role of the insulin signaling pathway in these processes, we have generated mice with combined heterozygous null mutations in insulin receptor (ir), insulin receptor substrate (irs-1), and/or irs-2. Diabetes developed in 40% of ir/irs-1/irs-2(+/-), 20% of ir/irs-1(+/-), 17% of ir/irs-2(+/-), and 5% of ir(+/-) mice. Although combined heterozygosity for ir/irs-1(+/-) and ir/irs-2(+/-) results in a similar number of diabetic mice, there are significant differences in the underlying metabolic abnormalities. ir/irs-1(+/-) mice develop severe insulin resistance in skeletal muscle and liver, with compensatory beta-cell hyperplasia. In contrast, ir/irs-2(+/-) mice develop severe insulin resistance in liver, mild insulin resistance in skeletal muscle, and modest beta-cell hyperplasia. Triple heterozygotes develop severe insulin resistance in skeletal muscle and liver and marked beta-cell hyperplasia. These data indicate tissue-specific differences in the roles of IRSs to mediate insulin action, with irs-1 playing a prominent role in skeletal muscle and irs-2 in liver. They also provide a practical demonstration of the polygenic and genetically heterogeneous interactions underlying the inheritance of type 2 diabetes.

IRS-2 pathways integrate female reproduction and energy homeostasis

Severe dietary restriction, catabolic states and even short-term caloric deprivation impair fertility in mammals. Likewise, obesity is associated with infertile conditions such as polycystic ovary syndrome. The reproductive status of lower organisms such as Caenorhabditis elegans is also modulated by availability of nutrients. Thus, fertility requires the integration of reproductive and metabolic signals. Here we show that deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/insulin-like growth factor-1 signalling cascade, causes female infertility. Mice lacking IRS-2 have small, anovulatory ovaries with reduced numbers of follicles. Plasma concentrations of luteinizing hormone, prolactin and sex steroids are low in these animals. Pituitaries are decreased in size and contain reduced numbers of gonadotrophs. Females lacking IRS-2 have increased food intake and obesity, despite elevated levels of leptin. Our findings indicate that insulin, together with leptin and other neuropeptides, may modulate hypothalamic control of appetite and reproductive endocrinology. Coupled with findings on the role of insulin-signalling pathways in the regulation of fertility, metabolism and longevity in C. elegans and Drosophila, we have identified an evolutionarily conserved mechanism in mammals that regulates both reproduction and energy homeostasis.

Bisphenol A Exposure during Pregnancy Disrupts Glucose Homeostasis in Mothers and Adult Male Offspring

Background Bisphenol A (BPA) is a widespread endocrine-disrupting chemical used as the base compound in the manufacture of polycarbonate plastics. In humans, epidemiological evidence has associated BPA exposure in adults with higher risk of type 2 diabetes and heart disease. Objective We examined the action of environmentally relevant doses of BPA on glucose metabolism in mice during pregnancy and the impact of BPA exposure on these females later in life. We also investigated the consequences of in utero exposure to BPA on metabolic parameters and pancreatic function in offspring. Methods Pregnant mice were treated with either vehicle or BPA (10 or 100 μg/kg/day) during days 9–16 of gestation. Glucose metabolism experiments were performed on pregnant mice and their offspring. Results BPA exposure aggravated the insulin resistance produced during pregnancy and was associated with decreased glucose tolerance and increased plasma insulin, triglyceride, and leptin concentrations relative to controls. Insulin-stimulated Akt phosphorylation was reduced in skeletal muscle and liver of BPA-treated pregnant mice relative to controls. BPA exposure during gestation had long-term consequences for mothers: 4 months postpartum, treated females weighed more than untreated females and had higher plasma insulin, leptin, triglyceride, and glycerol levels and greater insulin resistance. At 6 months of age, male offspring exposed in utero had reduced glucose tolerance, increased insulin resistance, and altered blood parameters compared with offspring of untreated mothers. The islets of Langerhans from male offspring presented altered Ca2+ signaling and insulin secretion. BrdU (bromodeoxyuridine) incorporation into insulin-producing cells was reduced in the male progeny, yet β-cell mass was unchanged. Conclusions Our findings suggest that BPA may contribute to metabolic disorders relevant to glucose homeostasis and that BPA may be a risk factor for diabetes.

Upregulation of insulin receptor substrate-2 in pancreatic β cells prevents diabetes

The insulin receptor substrate-2 (Irs2) branch of the insulin/IGF signaling system coordinates peripheral insulin action and pancreatic beta cell function, so mice lacking Irs2 display similarities to humans with type 2 diabetes. Here we show that beta cell-specific expression of Irs2 at a low or a high level delivered a graded physiologic response that promoted beta cell growth, survival, and insulin secretion that prevented diabetes in Irs2-/- mice, obese mice, and streptozotocin-treated mice; and that upon transplantation, the transgenic islets cured diabetes more effectively than WT islets. Thus, pharmacological approaches that promote Irs2 expression in beta cells, especially specific cAMP agonists, could be rational treatments for beta cell failure and diabetes.

Pdx1 restores β cell function in Irs2 knockout mice

The homeodomain transcription factor Pdx1 is required for pancreas development, including the differentiation and function of beta cells. Mutations in Pdx1 or upstream hepatocyte nuclear factors cause autosomal forms of early-onset diabetes (maturity-onset diabetes of the young [MODY]). In mice, the Irs2 branch of the insulin/Igf signaling system mediates peripheral insulin action and pancreatic beta cell growth and function. To investigate whether beta cell failure in Irs2(-/-) mice might be related to dysfunction of MODY-related transcription factors, we measured the expression of Pdx1 in islets from young Irs2(-/-) mice. Before the onset of diabetes, Pdx1 was reduced in islets from Irs2(-/-) mice, whereas it was expressed normally in islets from wild-type or Irs1(-/-) mice, which do not develop diabetes. Whereas male Irs2(-/-)Pdx1(+/+) mice developed diabetes between 8 and 10 weeks of age, haploinsufficiency for Pdx1 caused diabetes in newborn Irs2(-/-) mice. By contrast, transgenic expression of Pdx1 restored beta cell mass and function in Irs2(-/-) mice and promoted glucose tolerance throughout life, as these mice survived for at least 20 months without diabetes. Our results suggest that dysregulation of Pdx1 might represent a common link between ordinary type 2 diabetes and MODY.

Hypoxia Promotes Efficient Differentiation of Human Embryonic Stem Cells to Functional Endothelium

Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O2) as somatic cells. We hypothesized that O2 levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O2) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio‐ and vasculogenesis including vascular endothelial growth factor and angiopoitein‐like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O2 levels; after 24 hours at 5% O2, more than 50% of cells were CD34+. Transplantation of resulting endothelial‐like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction. STEM CELLS 2010;28:407–418

An important role for CDK2 in G1 to S checkpoint activation and DNA damage response in human embryonic stem cells.

A precise understanding of mechanisms used by human embryonic stem cells (hESCs) to maintain genomic integrity is very important for their potential clinical applications. The G1 checkpoint serves to protect genomic integrity and prevents cells with damaged DNA from entering S‐phase. Previously, we have shown that downregulation of cyclin‐dependent kinase 2 (CDK2) in hESC causes G1 arrest, loss of pluripotency, upregulation of cell cycle inhibitors p21 and p27 and differentiation toward extraembryonic lineages. In this study, we investigate in detail the role of CDK2 in cellular processes, which are crucial to the maintenance of genomic stability in hESC such as G1 checkpoint activation, DNA repair, and apoptosis. Our results suggest that downregulation of CDK2 triggers the G1 checkpoint through the activation of the ATM‐CHK2‐p53‐p21 pathway. Downregulation of CDK2 is able to induce sustained DNA damage and to elicit the DNA damage response (DDR) as evidenced by the formation of distinct γ‐H2.AX and RAD52‐BRCA1 foci in hESC nuclei. CDK2 downregulation causes high apoptosis at the early time points; however, this is gradually decreased overtime as the DDR is initiated. Our mass spectrometry analysis suggest that CDK2 does interact with a large number of proteins that are involved in key cellular processes such as DNA replication, cell cycle progression, DNA repair, chromatin modeling, thus, suggesting a crucial role for CDK2 in orchestrating a fine balance between cellular proliferation, cell death, and DNA repair in hESC. STEM Cells 2011;29:651–659

Inhibition of PTP1B Restores IRS1-Mediated Hepatic Insulin Signaling in IRS2-Deficient Mice

OBJECTIVE Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2−/− mice. RESEARCH DESIGN AND METHODS We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2−/− and IRS2−/−/PTP1B−/− mice. Additionally, hepatic insulin signaling was assessed in control and IRS2−/− mice treated with resveratrol, an antioxidant present in red wine. RESULTS In livers of hyperglycemic IRS2−/− mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2−/− mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action.