Biomedical subjects

Rohit N Kulkarni

Publications and source records attributed to Rohit N Kulkarni.

30 records · Page 2Linked to original sources

PubMed2003

TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

Insulin resistance is a major hallmark in the development of type II diabetes, which is characterized by the failure of insulin to promote glucose uptake in muscle and to suppress glucose production in liver. The serine-threonine kinase Akt (PKB) is a principal target of insulin signaling that inhibits hepatic glucose output when glucose is available from food. Here we show that TRB3, a mammalian homolog of Drosophila tribbles, functions as a negative modulator of Akt. TRB3 expression is induced in liver under fasting conditions, and TRB3 disrupts insulin signaling by binding directly to Akt and blocking activation of the kinase. Amounts of TRB3 RNA and protein were increased in livers of db/db diabetic mice compared with those in wild-type mice. Hepatic overexpression of TRB3 in amounts comparable to those in db/db mice promoted hyperglycemia and glucose intolerance. Our results suggest that, by interfering with Akt activation, TRB3 contributes to insulin resistance in individuals with susceptibility to type II diabetes.

Adenoviridae↗

PubMed2003

Characterization of multiple signaling pathways of insulin in the regulation of vascular endothelial growth factor expression in vascular cells and angiogenesis.

The effects of insulin on vascular endothelial growth factor (VEGF) expression in cultured vascular cells and in angiogenesis were characterized. Insulin increased VEGF mRNA levels in mouse aortic smooth muscle cells from 10(-9) to 10(-7) m with an initial peak of 3.7-fold increases at 1 h and a second peak of 2.8-fold after 12 h. The first peak of VEGF expression was inhibited by LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, and by the overexpression of dominant negative forms of p85 subunit of PI 3-kinase or Akt. Inhibitors of MEK kinase, PD98059, or overexpression of dominant negative forms of Ras was ineffective. In contrast, the chronic effect of insulin on VEGF expression was partially inhibited by both LY294002 or PD98059 as well as by the overexpression of dominant negatives of PI 3-kinase or Ras. The importance of PI 3-kinase-Akt pathway on VEGF expression was confirmed in mouse aortic smooth muscle cells isolated from insulin receptor substrate -1 knockout (IRS-1-/-) mice that showed parallel reductions of 46-49% in insulin-stimulated VEGF expression and PI 3-kinase-Akt activation. Insulin-induced activation of PI 3-kinase-Akt on hypoxia-induced VEGF expression and neovascularization was reduced by 40% in the retina of neonatal hypoxia model using IRS-1-/- mice. Thus, unlike other cells, insulin can regulate VEGF expression by both IRS-1/PI 3-kinase-Akt cascade and Ras-MAPK pathways in aortic smooth muscle cells. The in vivo results provide direct evidence that insulin can modulate hypoxia-induced angiogenesis via reduction in VEGF expression in vivo.

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PubMed2003

Lessons for human diabetes from experimental mouse models.

A precise knowledge of the defects underlying type 1 and type 2 diabetes is essential for designing appropriate therapeutic strategies. Because experiments in humans are limited, naturally occurring, and especially genetically engineered rodent models, have revolutionized research in diabetes. We review some of the models created recently and discuss their impact on human diabetes.

Animals↗

PubMed2003

Targeted elimination of peroxisome proliferator-activated receptor gamma in beta cells leads to abnormalities in islet mass without compromising glucose homeostasis.

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) 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 PPAR gamma can reduce cellular proliferation. We show here that activation of PPAR gamma 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 beta cells was eliminated (beta gamma KO mice), and these mice were found to have significant islet hyperplasia on a chow diet. Interestingly, the normal expansion of beta-cell mass that occurs in control mice in response to high-fat feeding is markedly blunted in these animals. Despite this alteration in beta-cell mass, no effect on glucose homeostasis in beta gamma KO mice was noted. Additionally, while thiazolidinediones enhanced insulin secretion from cultured wild-type islets, administration of rosiglitazone to insulin-resistant control and beta gamma KO mice revealed that PPAR gamma in beta cells is not required for the antidiabetic actions of these compounds. These data demonstrate a critical physiological role for PPAR gamma function in beta-cell proliferation and also indicate that the mechanisms controlling beta-cell hyperplasia in obesity are different from those that regulate baseline cell mass in the islet.

Animals↗

PubMed2003

Upregulation of insulin receptor substrate-2 in pancreatic beta 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.

Animals↗

PubMed2003

Impact of genetic background on development of hyperinsulinemia and diabetes in insulin receptor/insulin receptor substrate-1 double heterozygous mice.

Type 2 diabetes is a complex disease in which genetic and environmental factors interact to produce alterations in insulin action and insulin secretion, leading to hyperglycemia. To evaluate the influence of genetic background on development of diabetes in a genetically susceptible host, we generated mice that are double heterozygous (DH) for knockout of the insulin receptor and insulin receptor substrate-1 on three genetic backgrounds (C57BL/6 [B6], 129Sv, and DBA). Although DH mice on all backgrounds showed insulin resistance, their phenotypes were dramatically different. B6 DH mice exhibited marked hyperinsulinemia and massive islet hyperplasia and developed early hyperglycemia, with 85% overtly diabetic by 6 months. By contrast, 129Sv DH mice showed mild hyperinsulinemia and minimal islet hyperplasia, and < 2% developed diabetes. DBA mice had slower development of hyperglycemia, intermediate insulin levels, and evidence of islet degeneration, with 64% developing diabetes. Thus, mice carrying the same genetic defects on different backgrounds exhibited the full spectrum of abnormalities observed in humans with type 2 diabetes, which allowed for identification of potential loci that promote development of the diabetic phenotype.

Animals↗

PubMed2003

Identification of interactive loci linked to insulin and leptin in mice with genetic insulin resistance.

Mice double heterozygous (DH) for deletion of insulin receptor and insulin receptor substrate-1 are lean, insulin resistant, and have a phenotype that strongly depends on the genetic background of the mouse. On the C57BL/6 (B6) background, DH mice develop marked hyperinsulinemia and diabetes, whereas on the 129S6 background, DH mice exhibit only mild elevations of insulin and remain free of diabetes. F2 male mice created by an intercross between these two strains exhibit a 60% incidence of diabetes and a bell-shaped distribution of insulin levels as related to glucose, reminiscent of that in humans with type 2 diabetes. These mice also exhibit a wide range of leptin levels as related to body weight. A genome-wide scan of F2 mice reveals a quantitative trait locus (QTL) related to hyperinsulinemia on chromosome 14 (D14Mit55) with a peak logarithm of odds (LOD) score of 5.6, accounting for up to 69% of this trait. A QTL with a LOD score of 3.7 related to hyperleptinemia is present on chromosome 7 at D12Mit38 (a marker previously assigned to chromosome 12) in the area of the uncoupling protein 2/3 gene cluster. This locus also interacts synergistically with D14Mit55 in development of hyperinsulinemia and with a QTL on chromosome 12 (D12Mit231) related to hyperglycemia. These data demonstrate how multiple genetic modifiers can interact and influence the development of diabetes and the phenotype of animals with genetically programmed insulin resistance and provide evidence as to the location and nature of these genes.

Animals↗

PubMed2002

Lipoatrophic diabetes in Irs1(-/-)/Irs3(-/-) double knockout mice.

Based on the phenotypes of knockout mice and cell lines, as well as pathway-specific analysis, the insulin receptor substrates IRS-1, IRS-2, IRS-3, and IRS-4 have been shown to play unique roles in insulin signal transduction. To investigate possible functional complementarity within the IRS family, we generated mice with double knockout of the genes for IRS-1/IRS-3 and IRS-1/IRS-4. Mice with a combined deficiency of IRS-1 and IRS-4 showed no differences from Irs1(-/-) mice with respect to growth and glucose homeostasis. In contrast, mice with a combined deficiency of IRS-1 and IRS-3 developed early-onset severe lipoatrophy associated with marked hyperglycemia, hyperinsulinemia, and insulin resistance. However, in contrast to other models of lipoatrophic diabetes, there was no accumulation of fat in liver or muscle. Furthermore, plasma leptin levels were markedly decreased, and adenovirus-mediated expression of leptin in liver reversed the hyperglycemia and hyperinsulinemia. The results indicate that IRS-1 and IRS-3 play important complementary roles in adipogenesis and establish the Irs1(-/-)/Irs3(-/-) double knockout mouse as a novel model of lipoatrophic diabetes.

Adenoviridae↗

PubMed2002

Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function.

The insulin/insulin-like growth factor-1 (IGF-1) signalling pathways are present in most mammalian cells and play important roles in the growth and metabolism of tissues. Most proteins in these pathways have also been identified in the beta-cells of the pancreatic islets. Tissue-specific knockout of the insulin receptor (betaIRKO) or IGF-1 receptor (betaIGFRKO) in pancreatic beta-cells leads to altered glucose-sensing and glucose intolerance in adult mice, and betaIRKO mice show an age-dependent decrease in islet size and beta-cell mass. These data indicate that these receptors are important for differentiated function and are unlikely to play a major role in the early growth and/or development of the pancreatic islets. Conventional insulin receptor substrate-1 (IRS-1) knockouts manifest growth retardation and mild insulin resistance. The IRS-1 knockouts also display islet hyperplasia, defects in insulin secretory responses to multiple stimuli both in vivo and in vitro, reduced islet insulin content and an increased number of autophagic vacuoles in the beta-cells. Re-expression of IRS-1 in cultured beta-cells is able to partially restore the insulin content indicating that IRS-1 is involved in the regulation of insulin synthesis. Taken together, these data provide evidence that insulin and IGF-1 receptors and IRS-1, and potentially other proteins in the insulin/IGF-1 signalling pathway, contribute to the regulation of islet hormone secretion and synthesis and therefore in the maintenance of glucose homeostasis.

Animals↗

PubMed2002

Knockout models are useful tools to dissect the pathophysiology and genetics of insulin resistance.

OBJECTIVE: The development of type 2 diabetes is linked to insulin resistance coupled with a failure of pancreatic beta-cells to compensate by adequate insulin secretion. DESIGN: Here, we review studies obtained from genetically engineered mice that provide novel insights into the pathophysiology of insulin resistance. RESULTS: Knockout models with monogenic impairment in insulin action have highlighted the potential role for insulin signalling molecules in insulin resistance at a tissue-specific level. Polygenic models have strengthened the idea that minor defects in insulin secretion and insulin action, when combined, can lead to diabetes, emphasizing the importance of interactions of different genetic loci in the production of diabetes. Knockout models with tissue-specific alterations in glucose or lipid metabolism have dissected the individual contributions of insulin-responsive organs to glucose homeostasis. They have demonstrated the central role of fat as an endocrine tissue in the maintenance of insulin sensitivity and the development of insulin resistance. Finally, these models have shown the potential role of impaired insulin action in pancreatic beta-cells and brain in the development of insulin deficiency and obesity.

Adipose Tissue↗

PubMed2002

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.

Animals↗

PubMed2002

Effect of the insulin mimetic L-783,281 on intracellular Ca2+ and insulin secretion from pancreatic beta-cells.

L-783,281, an antidiabetic fungal metabolite that has previously been shown to activate insulin signaling in CHO cells, was tested for its effect on intracellular Ca(2+) ([Ca(2+)](i)) and insulin secretion in single mouse pancreatic beta-cells. Application of 10 micromol/l L-783,281 for 40 s to isolated beta-cells in the presence of 3 mmol/l glucose increased [Ca(2+)](i) to 178 +/- 10% of basal levels (n = 18) as measured by fluo-4 fluorescence. L-767,827, an inactive structural analog of the insulin mimetic, had no effect on beta-cell [Ca(2+)](i). The L-783,281-evoked [Ca(2+)](i) increase was reduced by 82 +/- 4% (n = 6, P < 0.001) in cells incubated with 1 micromol/l of the SERCA (sarco/endoplasmic reticulum calcium ATPase) pump inhibitor thapsigargin and reduced by 33 +/- 6% (n = 6, P < 0.05) in cells incubated with 20 micromol/l of the L-type Ca(2+)-channel blocker nifedipine. L-783,281-stimulated [Ca(2+)](i) increases were reduced to 31 +/- 3% (n = 9, P < 0.05) and 48 +/- 10% (n = 6, P < 0.05) of control values by the phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 (25 micromol/l) and wortmannin (100 nmol/l), respectively. In beta-cells from IRS-1-/- mice, 10 micromol/l L-783,281 had no significant effect on [Ca(2+)](i) (n = 5). L-783,281 also resulted in insulin secretion at single beta-cells. Application of 10 micromol/l L-783,281 for 40 s resulted in 12.2 +/- 2.1 (n = 14) exocytotic events as measured by amperometry, whereas the inactive structural analog had no stimulatory effect on secretion. Virtually no secretion was evoked by L-783,281 in IRS-1-/- beta-cells. LY294002 (25 micromol/l) significantly reduced the effect of the insulin mimetic on beta-cell exocytosis. It is concluded that L-783,281 evokes [Ca(2+)](i) increases and exocytosis in beta-cells via an IRS-1/PI3-K-dependent pathway and that the [Ca(2+)](i) increase involves release of Ca(2+) from intracellular stores.

Animals↗