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Rohit N Kulkarni

Publications and source records attributed to Rohit N Kulkarni.

At least 19 recordsLinked to original sources
PubMed2026

Proteomic analyses of human islets reveal potential markers of β cell dysfunction during prediabetes.

The mechanisms driving progressive β cell dysfunction in type 2 diabetes remain incompletely understood. This study aimed to identify pancreatic islet proteome changes that could predict diabetes onset. We isolated islets from individuals without diabetes undergoing partial pancreatectomy, previously characterized for glucose tolerance, insulin sensitivity, and insulin secretion, using laser capture microdissection, and analyzed them via high-performance liquid chromatography-mass spectrometry. Proteomic analysis revealed that individuals with impaired glucose tolerance (IGT) had reductions in proteins regulating glycolysis (PGK1, G3P), lipid metabolism (ACBP, ARF1), glucose transport (14-3-3B), and insulin secretion (STARD10, CAPDS) compared with normal glucose-tolerant (NGT) individuals. Additionally, IGT islets showed impaired expression of proteins involved in glucose- and incretin-stimulated insulin response (CREB1, IQGA1). Stratification by β cell glucose sensitivity (βGS) indicated that individuals with lower βGS exhibited reduced levels of insulin maturation (ERO1B) and antiapoptotic proteins (CASP8, PAK2, SKP1), along with increased SEL1L, a factor promoting endocrine precursor differentiation. These findings suggest that early defects in glucose metabolism and insulin secretion characterize IGT, while reduced βGS may trigger compensatory mechanisms, through enhanced β cell survival or neogenesis, to delay type 2 diabetes progression. Overall, proteomic alterations in prediabetic islets provide potential early predictive markers and targets for interventions aimed at preserving β cell function.

Humans↗
PubMed2006

Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3.

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a > 80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.

AMP-Activated Protein Kinases↗
PubMed2006

Orphan receptor small heterodimer partner is an important mediator of glucose homeostasis.

The orphan receptor small heterodimer partner (SHP; NROB2) is a transcriptional repressor that inhibits nuclear receptor signaling in diverse metabolic pathways. Here, we report that SHP(-/-) mice exhibited hypoinsulinemia with age, which was associated with increased peripheral insulin sensitivity and increased response of isolated islets to glucose stimulation, yet maintain normal levels of blood glucose. Deficiency in SHP function resulted in up-regulation of glucose transporter 4 mRNA and glucose uptake in muscles, and overexpression of SHP in C2C12 cells inhibited both basal and peroxisomal proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha-stimulated glucose transporter 4 expression and glucose uptake. SHP(-/-) hepatocytes showed markedly decreased basal glucose production in cultures, and SHP(-/-) livers had increased glycogen stores and were more sensitive to insulin inhibition of glucose output, which were concomitant with decreased expression for PPARgamma1, fatty acid translocase, glucose-6-phosphatase, and phosphoenol/pyruvate carboxykinase, and increased mRNAs for glucokinase and pyruvate kinase. In white fat, SHP deficiency resulted in up-regulation of genes involved in insulin sensitizing, including PPARgamma2 and adiponectin. We show that, at the transcriptional level, SHP directly represses adiponectin promoter activity by PPARgamma/liver receptor homolog-1. The results suggest that the increases in insulin sensitivity through multiple signaling pathways in muscle, liver, and fat, with an increase in islet secretory function, represent the complex mechanism whereby SHP deficiency leads to improvement in insulin sensitivity, secretion, and diabetes.

Adipose Tissue, White↗
PubMed2006

TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism.

During fasting, increased concentrations of circulating catecholamines promote the mobilization of lipid stores from adipose tissue in part by phosphorylating and inactivating acetyl-coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis. Here, we describe a parallel pathway, in which the pseudokinase Tribbles 3 (TRB3), whose abundance is increased during fasting, stimulates lipolysis by triggering the degradation of ACC in adipose tissue. TRB3 promoted ACC ubiquitination through an association with the E3 ubiquitin ligase constitutive photomorphogenic protein 1 (COP1). Indeed, adipocytes deficient in TRB3 accumulated larger amounts of ACC protein than did wild-type cells. Because transgenic mice expressing TRB3 in adipose tissue are protected from diet-induced obesity due to enhanced fatty acid oxidation, these results demonstrate how phosphorylation and ubiquitination pathways converge on a key regulator of lipid metabolism to maintain energy homeostasis.

3T3-L1 Cells↗
PubMed2006

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

An appropriate beta cell mass is pivotal for the maintenance of glucose homeostasis. Both insulin and IGF-1 are important in regulation of beta 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 beta cells (betaDKO), as the hormones have overlapping mechanisms of action and activate common downstream proteins. Notably, betaDKO 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 betaDKO mice manifest reduced beta cell mass, reduced expression of phosphorylated Akt and the transcription factor MafA, increased apoptosis in islets and severely compromised beta cell function. Analyses of compound knockouts showed a dominant role for insulin signaling in regulating beta cell mass. Together, these data provide compelling genetic evidence that insulin and IGF-I-dependent pathways are not critical for development of beta cells but that a loss of action of these hormones in beta cells leads to diabetes. We propose that therapeutic improvement of insulin and IGF-I signaling in beta cells might protect against type 2 diabetes.

Animals↗
PubMed2006

Regulation of vascular endothelial growth factor expression and vascularization in the myocardium by insulin receptor and PI3K/Akt pathways in insulin resistance and ischemia.

OBJECTIVE: This study characterized the role of insulin receptors and resistance on vascular endothelial growth factor (VEGF) expression and myocardial vascularization in physiological conditions and after ischemia. METHODS AND RESULTS: Cardiac microvascular density was reduced by 30% in insulin-resistant Zucker fatty rats versus lean controls. This was associated with a parallel 40% inhibition of insulin-stimulated activation of both Akt and VEGF expression in the myocardium and cardiomyocytes. In contrast, the activation of Erk1/2 by insulin remained unchanged. In cultured cardiomyocytes, insulin or insulin-like growth factor (IGF)-1 increased VEGF mRNA and protein expression by 2-fold. Inhibition of PI3K/Akt, especially Akt2-mediated cascades but not the Ras/MEK/Erk pathway, using chemical inhibitors, dominant negative adenoviral constructs, or siRNA approaches suppressed VEGF mRNA expression by insulin. Ventricular tissues from muscle insulin receptor knockout (MIRKO) mice, which lack insulin receptors in the myocardium, have significant reductions in insulin but not IGF-1 signaling, VEGF expression, and vascular density before and after ischemia versus controls. CONCLUSIONS: Insulin regulates VEGF gene expression and vascularization in the myocardium specifically via insulin receptors and the activation of PI3K/Akt pathway. Selective inhibition of this pathway may lead to the decreases in VEGF expression and capillary density in the myocardium of patients with insulin resistance.

Androstadienes↗
PubMed2006

Molecular approaches to study control of glucose homeostasis.

Type 2 diabetes is a polygenic disease that can lead to severe complications in multiple tissues. Rodent models have been used widely for investigating the pathophysiology underlying type 2 diabetes and for examining the potential link with obesity, largely due to the limitations of invasive testing and of studying detailed molecular mechanisms in human tissues. Among rodents, the mouse model is especially popular because mice are easy to manipulate genetically, have a short generation time, and are relatively inexpensive. The most commonly used inbred mouse strains are reviewed in addition to several genetically engineered mouse models that have been generated to study type 2 diabetes in the context of obesity, with a focus on insulin, leptin, and peroxisome proliferator-activated receptor (PPAR) signaling pathways.

Animals↗
PubMed2005

Loss of ARNT/HIF1beta 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.

Animals↗
PubMed2005

Alterations in growth and apoptosis of insulin receptor substrate-1-deficient beta-cells.

Insulin and IGF-I activate antiapoptotic pathways via insulin receptor substrate (IRS) proteins in most mammalian cells, including beta-cells. IRS-1 knockout (IRS-1KO) mice show growth retardation, hyperinsulinemia, and hyperplastic but dysfunctional islets without developing overt diabetes, whereas IRS-2KOs develop insulin resistance and islet hypoplasia leading to diabetes. Because both models display insulin resistance, it is difficult to differentiate islet response to insulin resistance from islet defects due to loss of proteins in the islets themselves. We used a transplantation approach, as a means of separating host insulin resistance from islet function, to examine alterations in proteins in insulin/IGF-I signaling pathways that may contribute to beta-cell proliferation and/or apoptosis in IRS-1KO islets. Islets isolated from wild-type (WT) or IRS-1KO mice were transplanted into WT or insulin-resistant IRS-1KO males under the kidney capsule. The beta-cell mitotic rate in transplanted islets in IRS-1KO recipients was increased 1.5-fold compared with WT recipients and was similar to that in endogenous pancreases of IRS-1KOs, whereas beta-cell apoptosis was reduced by approximately 80% in IRS-1KO grafts in IRS-1KO recipients compared with WT recipients. Immunohistochemistry showed a substantial increase in IRS-2 expression in IRS-1KO islets transplanted into IRS-1KO mice as well as in endogenous islets from IRS-1KOs. Furthermore, enhanced cytosolic forkhead transcription factor (FoxO1) staining in IRS-1KO grafts suggests intact Akt/PKB activity. Together, these data indicate that, even in the absence of insulin resistance, beta-cells deficient in IRS-1 exhibit a compensatory increase in IRS-2, which is associated with islet growth and is characterized by both proliferative and antiapoptotic effects that likely occur via an insulin/IGF-I/IRS-2 pathway.

Adaptation, Physiological↗
PubMed2005

Roles for leptin receptor/STAT3-dependent and -independent signals in the regulation of glucose homeostasis.

Leptin activates the long form of the leptin receptor (LRb) to control feeding and neuroendocrine function and thus regulate adiposity. While adiposity influences insulin sensitivity, leptin also regulates glucose homeostasis independently of energy balance. Disruption of the LRb/STAT3 signal in s/s mice results in hyperphagia, neuroendocrine dysfunction, and obesity similar to LRb null db/db mice. Insulin resistance and glucose intolerance are improved in s/s compared to db/db animals, however, suggesting that LRb/STAT3-independent signals may contribute to the regulation of glucose homeostasis by leptin. Indeed, caloric restriction normalized glycemic control in s/s animals, but db/db mice of similar weight and adiposity remained hyperglycemic. These differences in glucose homeostasis were not attributable to differences in insulin production between s/s and db/db animals but rather to decreased insulin resistance in s/s mice. Thus, in addition to LRb/STAT3-mediated adiposity signals, non-LRb/STAT3 leptin signals mediate an important adiposity-independent role in promoting glycemic control.

Amino Acid Substitution↗
PubMed2004

Glucose homeostasis and tissue transcript content of insulin signaling intermediates in four inbred strains of mice: C57BL/6, C57BLKS/6, DBA/2, and 129X1.

Transgenic mice phenotypes generally depend on the background strains used in their creation. To examine the effects of genetic background on insulin signaling, we analyzed glucose homeostasis in four inbred strains of mice [C57BL/6 (B6), C57BLKS/6 (KLS), DBA/2 (DBA), and 129X1] and quantitated mRNA content of insulin receptor (IR) and its substrates in insulin-responsive tissues. At 2 months, the male B6 mouse is the least glucose-tolerant despite exhibiting similar insulin sensitivity and first-phase insulin secretion as the other strains. The 129X1 male mouse islet contains less insulin and exhibits a higher threshold for glucose-stimulated first-phase insulin secretion than the other strains. Female mice generally manifest better glucose tolerance than males, which is likely due to greater insulin sensitivity in liver and adipose tissue, a robust first-phase insulin secretion in B6 and KLS females, and improved insulin sensitivity in muscle in DBA and 129X1 females. At 6 months, although males exhibit improved first-phase insulin secretion, their physiology was relatively unchanged, whereas female B6 and KLS mice became less insulin sensitive. Gene expression of insulin signaling intermediates in insulin-responsive tissues was generally not strain dependent with the cell content of IR mRNA being highest. IR substrate (IRS)-1 and IRS-2 mRNA are ubiquitously expressed and IRS-3 and IRS-4 mRNA were detected in significant amounts in fat and brain tissues, respectively. These data indicate strain-, gender-, and age-dependent tissue sensitivity to insulin that is generally not associated with transcript content of IR or its substrates and should be taken into consideration during phenotypic characterization of transgenic mice.

Adipose Tissue↗
PubMed2004

The islet beta-cell.

The beta-cell is one of four major types of cells present in the islets of Langerhans, which are islands of cells distributed throughout the endocrine pancreas in most mammals. The beta-cell synthesizes and secretes the hormone insulin mainly in response to glucose but also in response to several nutrients, hormones and nervous stimuli. In adult rodents the beta-cell has a slow mitotic rate. Recent studies provide novel insights into the functions of the beta-cell. The presence of functional insulin-like growth factor-1 and insulin receptors and components of their signaling pathway indicate an important role for insulin/IGF-1 signaling in the regulation of beta-cell function. Further, the recent discovery of glucokinase (GK) and the ATP-dependent potassium channels on insulin secretory granules, the detection of AMP-protein kinase in the beta-cell and the identification of a new beta-cell transcription factor, mMafA, are some exciting new areas of research currently underway to further understand the complex pathways that regulate the functions of beta-cells.

Animals↗
PubMed2004

PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance.

Inadequate compensatory beta cell hyperplasia in insulin-resistant states triggers the development of overt diabetes. The mechanisms that underlie this crucial adaptive response are not fully defined. Here we show that the compensatory islet-growth response to insulin resistance in 2 models--insulin receptor (IR)/IR substrate-1 (IRS-1) double heterozygous mice and liver-specific IR KO (LIRKO) mice--is severely restricted by PDX-1 heterozygosity. Six-month-old IR/IRS-1 and LIRKO mice both showed up to a 10-fold increase in beta cell mass, which involved epithelial-to-mesenchymal transition. In both models, superimposition of PDX-1 haploinsufficiency upon the background of insulin resistance completely abrogated the adaptive islet hyperplastic response, and instead the beta cells showed apoptosis resulting in premature death of the mice. This study shows that, in postdevelopmental states of beta cell growth, PDX-1 is a critical regulator of beta cell replication and is required for the compensatory response to insulin resistance.

Aging↗
PubMed2004

Islet secretory defect in insulin receptor substrate 1 null mice is linked with reduced calcium signaling and expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2b and -3.

Mice with deletion of insulin receptor substrate (IRS)-1 (IRS-1 knockout [KO] mice) show mild insulin resistance and defective glucose-stimulated insulin secretion and reduced insulin synthesis. To further define the role of IRS-1 in islet function, we examined the insulin secretory defect in the knockouts using freshly isolated islets and primary beta-cells. IRS-1 KO beta-cells exhibited a significantly shorter increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) than controls when briefly stimulated with glucose or glyceraldehyde and when l-arginine was used to potentiate the stimulatory effect of glucose. These changes were paralleled by a lower number of exocytotic events in the KO beta-cells in response to the same secretagogues, indicating reduced insulin secretion. Furthermore, the normal oscillations in intracellular Ca(2+) and O(2) consumption after glucose stimulation were dampened in freshly isolated KO islets. Semiquantitative RT-PCR showed a dramatically reduced islet expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b and -3 in the mutants. These data provide evidence that IRS-1 modulation of insulin secretion is associated with Ca(2+) signaling and expression of SERCA-2b and -3 genes in pancreatic islets and provides a direct link between insulin resistance and defective insulin secretion.

Animals↗
PubMed2003

Reduced beta-cell mass and altered glucose sensing impair insulin-secretory function in betaIRKO mice.

Pancreatic beta-cell-restricted knockout of the insulin receptor results in hyperglycemia due to impaired insulin secretion, suggesting that this cell is an important target of insulin action. The present studies were undertaken in beta-cell insulin receptor knockout (betaIRKO) mice to define the mechanisms underlying the defect in insulin secretion. On the basis of responses to intraperitoneal glucose, approximately 7-mo-old betaIRKO mice were either diabetic (25%) or normally glucose tolerant (75%). Total insulin content was profoundly reduced in pancreata of mutant mice compared with controls. Both groups also exhibited reduced beta-cell mass and islet number. However, insulin mRNA and protein were similar in islets of diabetic and normoglycemic betaIRKO mice compared with controls. Insulin secretion in response to insulin secretagogues from the isolated perfused pancreas was markedly reduced in the diabetic betaIRKOs and to a lesser degree in the nondiabetic betaIRKO group. Pancreatic islets of nondiabetic betaIRKO animals also exhibited defects in glyceraldehyde- and KCl-stimulated insulin release that were milder than in the diabetic animals. Gene expression analysis of islets revealed a modest reduction of GLUT2 and glucokinase gene expression in both the nondiabetic and diabetic mutants. Taken together, these data indicate that loss of functional receptors for insulin in beta-cells leads primarily to profound defects in postnatal beta-cell growth. In addition, altered glucose sensing may also contribute to defective insulin secretion in mutant animals that develop diabetes.

Animals↗
PubMed2003

cAMP promotes pancreatic beta-cell survival via CREB-mediated induction of IRS2.

The incretin hormone GLP1 promotes islet-cell survival via the second messenger cAMP. Here we show that mice deficient in the activity of CREB, caused by expression of a dominant-negative A-CREB transgene in pancreatic beta-cells, develop diabetes secondary to beta-cell apoptosis. Remarkably, A-CREB severely disrupted expression of IRS2, an insulin signaling pathway component that is shown here to be a direct target for CREB action in vivo. As induction of IRS2by cAMP enhanced activation of the survival kinase Akt in response to insulin and IGF-1, our results demonstrate a novel mechanism by which opposing pathways cooperate in promoting cell survival.

Animals↗