Stimulation Of Insulin Receptor Research Articles

Insulin-Responsive Transcription Factors.

The hormone insulin executes its function via binding and activating of the insulin receptor, a receptor tyrosine kinase that is mainly expressed in skeletal muscle, adipocytes, liver, pancreatic β-cells, and in some areas of the central nervous system. Stimulation of the insulin receptor activates intracellular signaling cascades involving the enzymes extracellular signal-regulated protein kinase-1/2 (ERK1/2), phosphatidylinositol 3-kinase, protein kinase B/Akt, and phospholipase Cγ as signal transducers. Insulin receptor stimulation is correlated with multiple physiological and biochemical functions, including glucose transport, glucose homeostasis, food intake, proliferation, glycolysis, and lipogenesis. This review article focuses on the activation of gene transcription as a result of insulin receptor stimulation. Signal transducers such as protein kinases or the GLUT4-induced influx of glucose connect insulin receptor stimulation with transcription. We discuss insulin-responsive transcription factors that respond to insulin receptor activation and generate a transcriptional network executing the metabolic functions of insulin. Importantly, insulin receptor stimulation induces transcription of genes encoding essential enzymes of glycolysis and lipogenesis and inhibits genes encoding essential enzymes of gluconeogenesis. Overall, the activation or inhibition of insulin-responsive transcription factors is an essential aspect of orchestrating a wide range of insulin-induced changes in the biochemistry and physiology of insulin-responsive tissues.

Immediate-early transcriptional response to insulin receptor stimulation

Insulin binding to the insulin receptor triggers intracellular signaling cascades involving the activation of protein and lipid kinases. As a result, multiple biological functions of the cells are changed. Here, we analyzed the regulation and signaling cascades leading to insulin-induced activation of the stimulus-responsive transcription factors. For the analyses, we used chromatin-embedded reporter genes having a cellular nucleosomal organisation, and fibroblasts expressing human insulin receptors (HIRcB cells). The results show that stimulation of the insulin receptor induced the expression of the transcription factor Egr-1. Attenuation of Egr-1 promoter activation was observed following expression of a dominant-negative mutant of the ternary complex factor Elk-1. These data were corroborated by experiments showing that insulin receptor stimulation increased the transcriptional activation potential of Elk-1. In addition, the transcriptional activity of AP-1 was significantly elevated in insulin-stimulated HIRcB cells. Expression of the dominant-negative mutant of Elk-1 reduced insulin-induced activation of AP-1, indicating that Elk-1 controls both serum response element and AP-1-regulated transcription. Moreover, we show that stimulation of the insulin receptor activates cyclic AMP response element (CRE)-controlled transcription, involving the transcription factor CREB. Insulin-induced transcription of Elk-1 and CREB-controlled reporter genes was attenuated by overexpression of MAP kinase phosphatase-1 or a constitutively active mutant of calcineurin A, indicating that both phosphatases are part of a negative feedback loop for reducing insulin-mediated gene transcription. Finally, we show that expression of the adenoviral protein E1A selectively reduced CRE-mediated transcription following stimulation of the insulin receptor. These data indicate that insulin-regulated transcription of CRE-containing genes is under epigenetic control.

Difficult to Heal Wounds Intralesional Insulin Therapy Trial

Introduction-Insulin exerts metabolic and growth-stimulating actions both through its own receptors and the receptors of its homologous factors (IGFs), although with different degrees of affinity. The A receptor of insulin acts more intensely on the cell membrane, with a metabolic response, whereas the B receptor is quickly internalized, stimulates cell growth, can be directed to the treatment of wounds difficult to heal. Objective-we proposes to evaluate the potential of insulin as an inducing agent in the regeneration of wounds that are difficult to heal as a therapeutic option. Methodology-Simultaneous intra and perilesional injections of 0.01ml of the solution containing 5UI of NPH single-component insulin diluted in 1 ml of lidocaine into various planes and wound sites. Clinical, glycemic and photographic evaluations of the patients were performed. Results-The cicatricial response occured in all patients and observed since the first applications. Withouth glycemic changes. Comments - The responses obtained can be attributed to both the stimulation of insulin receptors and that of their counterparts. Conclusions-The method was effective and without adverse effects. Different intervals between applications did not change the results. The availability of insulin and safety for its use indicate the method as a therapeutic option in difficult to heal wounds.

E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte.

Background/PurposeType 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism.MethodsWe have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes.ResultsUsing the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events.ConclusionWe conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

Abstract P4-06-03: Targeting the insulin receptor with a small peptide (S961) in cancer

Abstract The insulin-like growth factor (IGF)/insulin receptors are members of the superfamily of growth factor receptor tyrosine kinases. The insulin and type I IGF-I receptors (IGF-1R) have very similar structure and share high homology (∼84%) in the kinase domain. Their dimeric structure allows for the formation of either holo or hybrid receptors. Thus, targeting of any individual receptor has been a challenge. We have previously shown that the insulin receptor is expressed in tamoxifen resistant breast cancer cell lines. Therefore, specific targeting might be a useful strategy to block this pathway. The antagonist S961 is a single chain peptide that binds insulin receptor and has been reported to have partial antagonist activity. The affinity of S961 for the insulin receptor is comparable to that of insulin and the selectivity of the insulin receptor versus IGF1 is higher than that of insulin itself (Sturis et al). To determine if S961 has activity in breast cancer cells, we tested its ability to disrupt insulin and IGF-I signaling and growth in a panel of cancer cell lines (MCF-7L, MCF-7L TamR, MDA-231, and HepG2 cells). MCF-7L cells express high levels of IGF1, while HepG2 express mostly insulin receptor. Pre-incubation with S961 significantly suppressed p-Akt and p-MAPK after insulin stimulation in the HepG2, but not in MCF-7L. In contrast, insulin receptor stimulation in MCF-7L TamR cells, which do not express IGF1R, was inhibited by S961. Even a 1000-fold increase in S961 was unable to suppress insulin receptor activation in cells expressing both insulin receptor and IGF1R. S961 inhibits insulin-stimulate cell cycle progression in MCF7L TamR cells. In these TamR cells, S961 also suppresses colony formation in anchorage independent growth assays. These data suggest S961 is an effective inhibitor of insulin receptor activation but only when little IGF1R is expressed. Thus, insulin receptor targeting might be useful in the management of endocrine resistant breast cancer. Citation Format: Kelly LaPara, Douglas Yee. Targeting the insulin receptor with a small peptide (S961) in cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-06-03.

Vitamin D levels and microvascular complications in type 2 diabetes.

Background:Vitamin D has important actions on glucose metabolism. These include improved insulin exocytosis, direct stimulation of insulin receptor, improved uptake of glucose by peripheral tissues, improving insulin resistance. It has got various pleiotropic effects like suppression of cell mediated immunity, regulation of cell proliferation, stimulation of neurotropic factors such as nerve growth factor, Glial cell line-derived neurotrophic factor, neurotropin, suppression of RAAS, reduction of albuminuria, immunomodulatory effects, and anti-inflammatory effects. Thus, vitamin D is implicated in many ways in the pathogenesis of retinopathy, neuropathy and nephropathy.Objectives:To study the correlation of vitamin D levels with microvascular complications in type 2 diabetes.Materials and Methods:Cross-sectional case-control study of 18 patients (18-70 years), who met the American Diabetes Association 2011 criteria for type 2 diabetes, was conducted. Age and sex matched healthy controls were taken. Subjects were evaluated for the presence of microvascular complications by clinical evaluation, urine examination, fundus examination, nerve conduction studies, and various biochemical tests. 25-OH cholecalciferol levels were done for each. Cut off level for vitamin D deficiency was 20 ng/ml.Results:Mean vitamin D was lower in type 2 diabetics than healthy subjects (19.046 vs. 27.186 ng/ml). Prevalence of vitamin D deficiency and insufficiency was found to significantly higher in diabetics when compared to healthy subjects (P = 0.0001). Vitamin D deficiency was found to be significantly associated with neuropathy (χ2 = 5.39, df = 1, P = 0.020), retinopathy, (χ2 = 6.6, df = 1, P = 0.010) and nephropathy (χ2 = 10. 52, df = 1, P = 0.001). Lower levels of vitamin D were found to be associated with increasing prevalence of combinations of microvascular complications namely neuropathy with retinopathy (P = 0.036), neuropathy with nephropathy (P = 0.029), retinopathy with nephropathy (P = 0.022) and neuropathy with retinopathy with nephropathy (P = 0.0001).

Role of GALNT2 in the modulation of ENPP1 expression, and insulin signaling and action: GALNT2: A novel modulator of insulin signaling

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling and action. Understanding the mechanisms underlying ENPP1 expression may help unravel molecular mechanisms of insulin resistance. Recent data suggest a role of ENPP1-3′untraslated region (UTR), in controlling ENPP1 expression. We sought to identify trans-acting ENPP1-3′UTR binding proteins, and investigate their role on insulin signaling.By RNA pull-down, 49 proteins bound to ENPP1-3′UTR RNA were identified by mass spectrometry (MS). Among these, in silico analysis of genome wide association studies and expression profile datasets pointed to N-acetylgalactosaminyltransferase 2 gene (GALNT2) for subsequent investigations. Gene expression levels were evaluated by RT-PCR. Protein expression levels, IRS-1 and Akt phosphorylation were evaluated by Western blot. Insulin receptor (IR) autophosphorylation was evaluated by ELISA.GALNT2 down-regulation increased while GALNT2 over-expression reduced ENPP1 expression levels. In addition, GALNT2 down-regulation reduced insulin stimulation of IR, IRS-1 and Akt phosphorylation and insulin inhibition of phosphoenolpyruvate carboxykinase (PEPCK) expression, a key neoglucogenetic enzyme.Our data point to GALNT2 as a novel factor involved in the modulation of ENPP1 expression as well as insulin signaling and action in human liver HepG2 cells.

Novel roles for insulin receptor (IR) in adipocytes and skeletal muscle cells via new and unexpected substrates.

The insulin signaling pathway regulates whole-body glucose homeostasis by transducing extracellular signals from the insulin receptor (IR) to downstream intracellular targets, thus coordinating a multitude of biological functions. Dysregulation of IR or its signal transduction is associated with insulin resistance, which may culminate in type 2 diabetes. Following initial stimulation of IR, insulin signaling diverges into different pathways, activating multiple substrates that have roles in various metabolic and cellular processes. The integration of multiple pathways arising from IR activation continues to expand as new IR substrates are identified and characterized. Accordingly, our review will focus on roles for IR substrates as they pertain to three primary areas: metabolism/glucose uptake, mitogenesis/growth, and aging/longevity. While IR functions in a seemingly pleiotropic manner in many cell types, through these three main roles in fat and skeletal muscle cells, IR multi-tasks to regulate whole-body glucose homeostasis to impact healthspan and lifespan.

Insulin and its analogues and their affinities for the IGF1 receptor

Insulin analogues have been developed in an attempt to achieve a more physiological replacement of insulin and thereby a better glycaemic control. However, structural modification of the insulin molecule may result in altered binding affinities and activities to the IGF1 receptor (IGF1R). As a consequence, insulin analogues may theoretically have an increased mitogenic action compared to human insulin. In view of the lifelong exposure and large patient populations involved, insulin analogues with an increased mitogenic effect in comparison to human insulin may potentially constitute a major health problem, since these analogues may possibly induce the growth of pre-existing neoplasms. This hypothesis has been evaluated extensively in vitro and also in vivo by using animal models. In vitro, all at present commercially available insulin analogues have lower affinities for the insulin receptor (IR). Although it has been suggested that especially insulin analogues with an increased affinity for the IGF1R (such as insulin glargine) are more mitogenic when tested in vitro in cells expressing a high proportion of IGF1R, the question remains whether this has any clinical consequences. At present, there are several uncertainties which make it very difficult to answer this question decisively. In addition, recent data suggest that insulin (or insulin analogues)-mediated stimulation of IRs may play a key role in the progression of human cancer. More detailed information is required to elucidate the exact mechanisms as to how insulin analogues may activate the IR and IGF1R and how this activation may be linked to mitogenesis.

A Pathobiological Role of the Insulin Receptor in Chronic Lymphocytic Leukemia

The chromosomal deletion 11q affects biology and clinical outcome in chronic lymphocytic leukemia (CLL) but del11q-deregulated genes remain incompletely characterized. We have employed integrated genomic profiling approaches on CLL cases with and without del11q to identify 11q-relevant genes. We have identified differential expression of the insulin receptor (INSR) in CLL, including high-level INSR expression in the majority of CLL with del11q. High INSR mRNA expression in 11q CLL (∼10-fold higher mean levels than other genomic categories) was confirmed by quantitative PCR in 247 CLL cases. INSR protein measurements in 257 CLL cases through flow cytometry, compared with measurements in normal CD19(+) B cells and monocytes, confirmed that a subset of CLL aberrantly expresses high INSR levels. INSR stimulation by insulin in CLL cells ex vivo resulted in the activation of canonical INSR signaling pathways, including the AKT-mTOR and Ras/Raf/Erk pathways, and INSR activation partially abrogated spontaneous CLL cell apoptosis ex vivo. Higher INSR levels correlated with shorter time to first therapy and shorter overall survival (OS). In bivariate analysis, INSR expression predicted for rapid initial disease progression and shorter OS in ZAP-70-low/negative CLL. Finally, in multivariate analysis (ZAP-70 status, IgV(H) status, and INSR expression), we detected elevated HRs and trends for short OS for CLL cases with high INSR expression (analyzed inclusive or exclusive of cases with del11q). Our aggregate biochemical and clinical outcome data suggest biologically meaningful elevated INSR expression in a substantial subset of all CLL cases, including many cases with del11q.

A Pathobiological Role of the Insulin Receptor in Chronic Lymphocytic Leukemia.

Abstract 2324Poster Board II-301The presence of genomic aberrations in CLL, including del17p and del11q, affects CLL patient survival, and thus an in-depth understanding of the genes influenced through these deletions is important. Further, genes affecting CLL disease progression that is observed in patients with recurrent genomic deletions, including del11q, remain incompletely characterized. Focusing therefore on del11q in CLL, we employed comparative expression array profiling (Affymetrix 133 2.0 plus arrays) of RNA from sorted CD19+ cells from 10 cases with and nine cases without del11q to identify stable transcriptome differences associated with del11q. Through these efforts we have identified for the first time differential expression of the insulin receptor (INSR) in CLL and predominant INSR expression in CLL with del11q. We have validated this finding in a cohort of 219 CLL cases. Our aggregate data suggest biologically and clinically meaningful INSR expression in a substantial subset of all CLL cases. INSR stimulation by insulin in CLL cells expressing the INSR resulted in the activation of canonical INSR signaling pathways, including the AKT-mTOR and Ras/Raf/Erk pathways, and INSR activation partially abrogated CLL cell apoptosis ex vivo. Clinically, CLL cases with INSR expression displayed larger lymphnodes and higher Rai stage at study enrollment and the disease progressed faster requiring repeat therapy earlier than CLL cases with low or absent INSR expression. This novel data provides the first description of a pathobiological role of the insulin receptor in CLL biology and disease progression, thus identifying a potential mediator and mechanism for del11q-associated CLL phenotypes. The identification of a transmembrane tyrosine kinase receptor with direct effects on CLL biology and clinical behavior offers opportunities for CLL prognostication and targeted therapy development and further strengthens the case for involvement of the INSR in cancer biology. Disclosures:Malek:Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding.

The Drosophila SH2B Family Adaptor Lnk Acts in Parallel to Chico in the Insulin Signaling Pathway

Insulin/insulin-like growth factor signaling (IIS) plays a pivotal role in the regulation of growth at the cellular and the organismal level during animal development. Flies with impaired IIS are developmentally delayed and small due to fewer and smaller cells. In the search for new growth-promoting genes, we identified mutations in the gene encoding Lnk, the single fly member of the SH2B family of adaptor molecules. Flies lacking lnk function are viable but severely reduced in size. Furthermore, lnk mutants display phenotypes reminiscent of reduced IIS, such as developmental delay, female sterility, and accumulation of lipids. Genetic epistasis analysis places lnk downstream of the insulin receptor (InR) and upstream of phosphoinositide 3-kinase (PI3K) in the IIS cascade, at the same level as chico (encoding the single fly insulin receptor substrate [IRS] homolog). Both chico and lnk mutant larvae display a similar reduction in IIS activity as judged by the localization of a PIP3 reporter and the phosphorylation of protein kinase B (PKB). Furthermore, chico; lnk double mutants are synthetically lethal, suggesting that Chico and Lnk fulfill independent but partially redundant functions in the activation of PI3K upon InR stimulation.

Effect of IGF-1R inhibitor BMS-754807 on tumor glucose metabolism and proliferation in Sal-IGF xenografts by FDG and FLT- PET imaging

e14501 Background: The type 1 insulin-like growth factor receptor (IGF-1R) plays a critical role in mitogenesis and survival in a variety of human tumor cells. IGF-1R is also closely related to the insulin receptor (IR). It is becoming increasingly evident that stimulation of IR or IR/IGF-1R hybrid receptors by insulin or IGF-1/2 may contribute to cancer growth, suggesting that both IGF-1R and IR may be targets for cancer therapy. BMS-754807 (‘807) is an oral IGF-1R tyrosine kinase antagonist under development that also blocks IR activity. This study explores the feasibility of fluorodeoxyglucose (FDG) and fluorothymidine (FLT) positron emission tomography (PET) as pharmacodynamic imaging biomarkers in a xenograft model. Methods: Mice were implanted with fragments of Sal-IGF, a transgenic salivary gland tumor expressing constitutively active IGF-1R. Once tumors reached desired size, the mice were divided into 3 treatment groups (vehicle, 6.25 mg/kg ‘807, & 50 mg/kg ‘807 on days 1–4). FDG-PET scans were performed on day -2 (prior to treatment), days 1 & 2 (max/min concentrations post day 1 dose), and days 4 & 5 (max/min day 4 dose). FLT-PET scans were performed on days -2, 2 (min day 1 dose), & 5 (min day 4 dose). Results: With FDG-PET imaging, a 66% reduction (P<0.05, paired t-test) in tumor SUVmean was observed at the end of the 50 mg/kg therapy (day 4) and sustained for 24h (day 5) with a concomitant reduction of 37% in tumor burden by day 5 (P<0.05). FLT-PET studies demonstrated a 45% reduction (P<0.05) in tumor SUVmean observed 24h after the end of 50 mg/kg therapy (day 5) with a concomitant reduction of 50% in tumor burden (day 5, P<0.05). Conclusions: Correlation with tumor growth inhibition suggests feasibility of FDG- PET despite IR blockade. These results support the use of both FDG and FLT PET imaging in clinical trials with ‘807. [Table: see text]

Getting Radical About Obesity

Visceral obesity has become the most frequent preventable condition accounting for diseases such as hypertension and diabetes, two of the major risk factors for coronary artery disease, stroke, and chronic renal failure.1–3 Despite the awareness aiming to reduce the prevalence of obesity, the number of obese patients continues to rise and now includes even developing countries, and increasingly children are affected.4,5 In fact, if this trend continues, 26 million children in Europe will be overweight or obese by the year 2010, rising by approximately 1.3 million per year.5,6 The annual health costs related to obesity have been estimated close to 100 billion USD in the United States,7,8 illustrating the enormous economic burden the disease obesity carries. See accompanying article in ATVB 2009;29:239–245. What are the mechanisms contributing to the high cardiovascular risk brought about by obesity? Obesity is a low-grade inflammatory condition9 and typically characterized by increase in visceral adipose tissue. Visceral fat is both a source and target of inflammatory cytokines and growth factors, which directly may affect preadipocyte differentiation via signaling cascades implicated in cell growth such as Ras-Raf-ERK-1/2.10 Adipose tissue also contains inflammatory cells, including cytokine-producing macrophages.9 In addition, adipocytes express a fully functional NADPH oxidase …

Carbohydrate intake, glycemic index, glycemic load, and risk of postmenopausal breast cancer in a prospective study of French women

BackgroundDiets high in carbohydrates may result in chronically elevated insulin concentrations and may affect breast cancer risk by stimulation of insulin receptors or through insulin-like growth factor I (IGF-I)–mediated mitogenesis. Insulin response to carbohydrate intake is increased in insulin-resistant states such as obesity. ObjectiveWe sought to evaluate carbohydrate intake, glycemic index (GI), and glycemic load (GL) and subsequent overall and hormone-receptor-defined breast cancer risk among postmenopausal women. DesignA prospective cohort analysis of dietary carbohydrate and fiber intakes was conducted among 62 739 postmenopausal women from the E3N French study who had completed a validated dietary history questionnaire in 1993. During a 9-y period, 1812 cases of pathology-confirmed breast cancer were documented through follow-up questionnaires. Nutrients were categorized into quartiles and energy-adjusted with the regression-residual method. Cox model–derived relative risks (RRs) were adjusted for known determinants in breast cancer. ResultsDietary carbohydrate and fiber intakes were not associated with overall breast cancer risk. Among overweight women, we observed an association between GI and breast cancer (RRQ1–Q4: 1.35; 95% CI: 1.00, 1.82; P for trend = 0.04). For women in the highest category of waist circumference, the RRQ1–Q4 was 1.28 (95% CI: 0.98, 1.67; P for trend = 0.10) for carbohydrates, 1.35 (95% CI: 1.04, 1.75; P for trend = 0.01) for GI, and 1.37 (95% CI: 1.05, 1.77; P for trend = 0.003) for GL. We also observed a direct association between carbohydrate intake, GL, and estrogen receptor–negative breast cancer risk. ConclusionsRapidly absorbed carbohydrates are associated with postmenopausal breast cancer risk among overweight women and women with large waist circumference. Carbohydrate intake may also be associated with estrogen receptor–negative breast cancer.

The Role of Membrane Glycoprotein Plasma Cell Antigen 1/Ectonucleotide Pyrophosphatase Phosphodiesterase 1 in the Pathogenesis of Insulin Resistance and Related Abnormalities

Insulin resistance is a major feature of most patients with type 2 diabetes mellitus (T2D). A number of laboratories have observed that PC-1 (membrane [corrected] glycoprotein plasma cell antigen 1; also termed [corrected] ectonucleotide pyrophosphatase phosphodiesterase 1 or ENPP1) [corrected] is either overexpressed or overactive in muscle, adipose tissue, fibroblasts, and other tissues of insulin-resistant individuals, both nondiabetic and diabetic. Moreover, PC-1 (ENPP1) overexpression [corrected] in cultured cells in vitro and in transgenic mice in vivo, [corrected] impairs insulin stimulation of insulin receptor (IR) activation and downstream signaling. PC-1 binds to the connecting domain of the IR alpha-subunit that is located in residues 485-599. The connecting domain transmits insulin binding in the alpha-subunit to activation of tyrosine kinase activation in the beta-subunit. When PC-1 is overexpressed, it inhibits insulin [corrected]induced IR beta-subunit tyrosine kinase activity. In addition, a polymorphism of PC-1 (K121Q) in various ethnic populations is closely associated with insulin resistance, T2D, and cardio [corrected] and nephrovascular diseases. The product of this polymorphism has a 2- to 3-fold increased binding affinity for the IR and is more potent than the wild-type PC-1 protein (K121K) in inhibiting the IR. These data suggest therefore that PC-1 is a candidate protein that may play a role in human insulin resistance and T2D by its overexpression, its overactivity, or both.

Protein kinase C-dependent antilipolysis by insulin in rat adipocytes

Recently, we have shown that protein kinase C (PKC) activated by phorbol 12-myristate 13-acetate (PMA) attenuates the β1-adrenergic receptor (β1-AR)-mediated lipolysis in rat adipocytes. Stimulation of cells by insulin, angiotensin II, and α1-AR agonist is known to cause activation of PKC. In this study, we found that lipolysis induced by the β1-AR agonist dobutamine is decreased and is no longer inhibited by PMA in adipocytes that have been treated with 20 nM insulin for 30 min followed by washing out insulin. Such effects on lipolysis were not found after pretreatment with angiotensin II and α1-AR agonists. The rate of lipolysis in the insulin-treated cells was normalized by the PKCα- and β-specific inhibitor Gö 6976 and PKCβ-specific inhibitor LY 333531. In the insulin-treated cells, wortmannin increased lipolysis and recovered the lipolysis-attenuating effect of PMA. Western blot analysis revealed that insulin slightly increases membrane-bound PKCα, βI, and δ, and wortmannin decreases PKCβI, βII, and δ in the membrane fraction. These results indicate that stimulation of insulin receptor induces a sustained activation of PKC-dependent antilipolysis in rat adipocytes.

Glucose and Insulin Synergistically Activate Phosphatidylinositol 3-Kinase to Trigger Oscillations of Phosphatidylinositol 3,4,5-Trisphosphate in β-Cells

In insulin-secreting beta-cells, activation of phosphatidylinositol 3'-OH-kinase with resulting formation of phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) has been implicated in the regulation of ion channels, insulin secretion, and gene transcription as well as in cell growth and survival, but the kinetics of PIP(3) signals following physiological stimulation of insulin secretion is unknown. Using evanescent wave microscopy and a green fluorescent protein-tagged PIP(3)-binding protein domain for real-time monitoring of plasma membrane PIP(3) concentration in single MIN6 beta-cells, we now demonstrate that glucose stimulation of insulin secretion results in pronounced PIP(3) oscillations via autocrine stimulation of insulin receptors. Glucose lacked effect when insulin secretion was prevented with the hyperpolarizing agent diazoxide, but the sugar dose dependently enhanced the PIP(3) response to maximal insulin stimulation without affecting the rate of PIP(3) degradation. We conclude that glucose is an important co-activator of phosphatidylinositol-3'-OH-kinase and that the plasma membrane PIP(3) concentration in beta-cells undergoes oscillations due to pulsatile release of insulin.

Tumor Necrosis Factor α Produces Insulin Resistance in Skeletal Muscle by Activation of Inhibitor κB Kinase in a p38 MAPK-dependent Manner

Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-alpha caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-alpha could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-alpha. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-alpha was still produced. Moreover, TNF-alpha produced inhibitor kappaB kinase (IKK)-beta activation and inhibitor kappaB-beta and -alpha degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-alpha produced serine phosphorylation of IR and IRS-1 (total and on Ser(307) residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-alpha, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.

Efficient adenovirus transduction of 3T3-L1 adipocytes stably expressing coxsackie-adenovirus receptor

3T3-L1 adipocytes have proven difficult to transfect with plasmid-encoded cDNAs or even infect with virally-derived cDNAs. We have developed and characterized a 3T3-L1 adipocyte cell line stably expressing the truncated receptor for coxsackievirus and adenovirus receptor (CAR) for its ability to be infected with adenoviruses at a low multiplicity of infection (m.o.i.). Using green fluorescent protein driven by the cytomegalovirus promoter in adenovirus fiber type 5 we compared infection efficiencies of CAR adipocytes versus the parental 3T3-L1 adipocytes. As assessed by immunofluorescence, CAR adipocytes were infected at ∼100-fold greater efficiency than regular 3T3-L1 adipocytes. The efficiency of transduction for the CAR adipocytes was >90% at multiplicities of infection of 50 whereas standard adipocytes were poorly transduced even at an m.o.i. of 2000. Since many investigators studying insulin action use 3T3-L1 adipocytes, we compared CAR adipocytes versus regular adipocytes and showed that the two cell lines were similar with respect to insulin stimulation of insulin receptor, MAPK, and Akt phosphorylation and basal- and insulin-stimulated glucose transport. In addition, CAR adipocytes accumulated GLUT4 and SCD1 proteins during the adipogenesis program with the same time course as regular 3T3-L1 adipocytes. Lastly, CAR adipocytes produced and secreted the adipose-specific hormone Acrp30. These data suggest 3T3-L1CARΔ1 adipocytes are virtually indistinguishable from their parental cells, but demonstrate a significant advantage with improved efficiency of adenoviral transduction for gain or deletion of function studies.