Insulin-mediated Signal Transduction Research Articles

Redox regulation of the insulin signalling pathway

The peptide hormone insulin is a key regulator of energy metabolism, proliferation and survival. Binding of insulin to its receptor activates the PI3K/AKT signalling pathway, which mediates fundamental cellular responses. Oxidants, in particular H2O2, have been recognised as insulin-mimetics. Treatment of cells with insulin leads to increased intracellular H2O2 levels affecting the activity of downstream signalling components, thereby amplifying insulin-mediated signal transduction. Specific molecular targets of insulin-stimulated H2O2 include phosphatases and kinases, whose activity can be altered via redox modifications of critical cysteine residues. Over the past decades, several of these redox-sensitive cysteines have been identified and their impact on insulin signalling evaluated. The aim of this review is to summarise the current knowledge on the redox regulation of the insulin signalling pathway.

Liver-specific expression of carboxylesterase 1g/esterase-x reduces hepatic steatosis, counteracts dyslipidemia and improves insulin signaling

Ces1g/Es-x deficiency in mice results in weight gain, insulin resistance, fatty liver and hyperlipidemia through upregulation of de novo lipogenesis and oversecretion of triacylglycerol (TG)-rich lipoproteins. Here, we show that restoration of Ces1g/Es-x expression only in the liver significantly reduced hepatic TG concentration accompanied by decreased size of lipid droplets, reduced secretion of very low-density lipoproteins and improved insulin-mediated signal transduction in the liver. Collectively, these results demonstrate that hepatic Ces1g/Es-x plays a critical role in limiting hepatic steatosis, very low-density lipoprotein assembly and in augmenting insulin sensitivity.

PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling.

In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis.

The role of the Nrf2/Keap1 pathway in obesity and metabolic syndrome.

Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key regulator of antioxidant signaling that may prevent the development of metabolic syndrome and related cardiovascular diseases. However, emerging evidence shows that lack of Nrf2 could ameliorate insulin resistance, adipogenesis and adipocyte differentiation. Consistent with this, overexpression of Nrf2 gene could also cause insulin resistance under certain conditions. Furthermore, an increasing number of studies indicate that redox balance can be a critical element that contributes to the contradictory effects of Nrf2 on insulin sensitivity and resistance. Reactive oxygen species can promote normal insulin-mediated signal transduction under physiological conditions but also induce insulin resistance under certain pathological conditions. Therefore, the contradictory effects of Nrf2 on insulin signaling pathways may be related to its regulation of redox homeostasis. This review attempts to summarize the latest developments in our understanding of the mechanisms of Nrf2-mediated signaling and its role in the modulation of metabolic homeostasis.

Dietary nitrite supplementation improves insulin resistance in type 2 diabetic KKAy mice

BackgroundBecause insulin signaling is essential for endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production, the loss of bioavailable NO might be a common molecular mechanism underlying the development of insulin resistance and endothelial dysfunction. Although dietary nitrite acts as a substrate for systemic NO generation, thereby serving as a physiological alternative source of NO for signaling, it is not precisely known how dietary nitrite affects type 2 diabetes mellitus. Here we report the therapeutic effects of dietary nitrite on the metabolic and histological features of KKAy diabetic mice. MethodsKKAy mice were divided into three groups (without nitrite, and with 50 mg/L and 150 mg/L nitrite in drinking water), and two groups of C57BL/6J mice served as controls (without nitrite and with 150 mg/L nitrite in drinking water). After 10 weeks, blood samples, visceral adipose tissues, and gastrocnemius muscles were collected after a 16-hour fast to assess the homeostasis model assessment of insulin resistance (HOMA-IR) levels, the histology of the adipose tissue, insulin-stimulated sequential signaling to glucose transporter 4 (GLUT4), and nitrite and nitrate contents in the muscle using an HPLC system. ResultsKKAy mice developed obesity with enhanced fasting plasma levels of glucose and insulin and exhibited increased HOMA-IR scores compared with the C57BL/6J control mice. Dietary nitrite dose-dependently reduced the size of the hypertrophic adipocytes and TNF-α transcription in the adipose tissue of KKAy diabetic mice, which also restored the insulin-mediated signal transduction, including p85 and Akt phosphorylation, and subsequently restored the GLUT4 expression in the skeletal muscles. ConclusionsThese results suggest that dietary nitrite provides an alternative source of NO, and subsequently improves the insulin-mediated signaling and the metabolic and histological features in KKAy diabetic mice.

Past, Present and Future of Insulin Gene and Its Related Genes In Relation To Polycystic Ovary Syndrome

Polycystic ovary syndrome is the most common endocrinal disorder and is associated with infertility. Insulin resistance is a key component in the pathogenesis of PCOS and the predisposition to Type 2 diabetes mellitus. The existing literature supports a strong basis of PCOS clusters in families. However, due to the genetic and phenotypic heterogeneity of PCOS and the lack of large cohort studies and identification of specific contributing genes to date have yielded only few conclusive results. Although several loci have been proposed as PCOS genes including CYP11A, the insulin gene, and a region near the insulin receptor, the strongest case can be made for the region near the insulin receptor gene. Insulin receptor substrates proteins (IRS) are critical for insulin mediated signal transduction in insulin target tissues. Several studies have shown that IRS-1 Gly972Arg polymorphism might be the factor causing susceptibility to Type 2 diabetes mellitus (T2DM) and are associated with phenotypic features of PCOS. In this article we reviewed the current status of the genetic analysis of insulin (INS), insulin receptor (INSR), insulin receptor substrate (IRS) in relation to polycystic ovary syndrome, an infertility disorder in females.

Hpyerglycemic effect of a mixture of sea cucumber and cordyceps sinensis in streptozotocin-induced diabetic rat

Sea cucumber and cordyceps sinensis are used as both food and traditional medicines in Asia. This study was carried out in order to investigate the hpyerglycemic effect of a mixture of sea cucumber (Apostichopus japonicas) and cordyceps sinensis (Cordyceps militaris) (SCC) in diabetic rat and explore the mechanism underlining such an effect. The diabetic model rat was induced with intraperitoneal injection of streptozotocin (STZ). The diabetic model rats were randomly divided into control group (0.9% NaCl), low dose group (300 mg SCC·(kg body weight)−1) and high dose group (1200 mg SCC (kg body weight)−1). Sodium chloride and SCC were intragastrically administered once a day for 35 d. Changes in fasting serum glucose and serum insulin content, oral glucose tolerance and liver and muscle glycogen content were routinely evaluated. Pancreas tissue and β-cells of islets were observed under both optical and transmission electronic microscope, respectively. The abundance of glucose metabolism-relating genes in gastrocnemius and epididymal adipose tissue was determined with either reverse transcription PCR (RT-PCR) or western blotting. Results showed that SCC significantly decreased fasting serum glucose content, improved glucose tolerance and increased serum insulin and glycogen content; repaired STZ-injured β-cells of diabetic rat, and increased the expression of phosphatidylinositol 3 kinase (PI(3)K), protein kinase B (PKB) and glucose transporter 4 (Glut4) encoding protein in both gastrocnemius and adipose tissue, and Glut4 encoding gene in peripheral tissue. Our findings demonstrated that SCC exerted an anti-hyperglycemic effect by repairing β-cells and promoting insulin-mediated signal transduction pathway in insulin-sensitive gastrocnemius and adipose tissue.

Molecular characterization of a novel p.R118C mutation in the insulin receptor gene from patients with severe insulin resistance

Mutations of the insulin receptor gene (INSR) can cause genetic syndromes associated with severe insulin resistance. We aimed to analyse INSR mutations in Saudi patients with severe insulin resistance. Ten patients with Type A insulin resistance syndrome from five unrelated Saudi families were investigated. The entire coding region of INSR was sequenced. The founder effect was assessed by microsatellite haplotype analysis. The functional effect of the mutation was investigated by in vitro functional assays. A novel biallelic c.433 C>T (p.R118C) mutation was detected in all patients. The c.433 C>T (p.R118C) sequence variation was not found in 100 population controls. The arginine residue at position 118 is located in the ligand-binding domain of INSR and is highly conserved across species. Microsatellite haplotype analysis of these patients indicated that p.R118C was a founder mutation created approximately 2900 years ago. The wild-type and mutant (R118C) INSR were cloned and expressed in CHO cells for functional analysis. Specific insulin binding to the mutant receptor was reduced by 83% as compared to wild-type (WT), although the mutant receptor was processed and expressed on the cell surface. Insulin-mediated receptor autophosphorylation was also significantly reduced in CHO(R118C) cells. Biallelic c.433 C>T (p.R118C) mutation of INSR causes significant damage to insulin binding and insulin-mediated signal transduction. p.R118C is a founder mutation frequently present in the Saudi patients with severe insulin resistance.

ChemInform Abstract: Synthetic Glycosylphosphatidylinositol (GPI) Anchors: How these Complex Molecules Have Been Made

AbstractReview: 116 refs.

Synthetic glycosylphosphatidylinositol (GPI) anchors: how these complex molecules have been made

Covering: up to the end of 2009 Glycosylphosphatidylinositols (GPIs) are a class of natural glycosylphospholipids that anchor proteins, glycoproteins and lipophosphoglycans to the membrane of eukaryotic cells. GPI anchors are widely present in parasitic protozoa, where GPI-anchored mucins and phosphoglycans are abundant and form a dense protective layer (glycocalyx) on the surface of the parasites. This type of anchor appears to be present in these organisms with a much higher frequency than in higher eukaryotes. Since the first full assignment of a GPI structure in 1988, more than 50 glycosylphosphatidylinositols have been structurally characterised. The functions of GPI anchors (in addition to the clear one of linking the above biopolymers to membranes) have been extensively discussed. The high lateral mobility of GPIs and GPI-anchored polymers seems to actively facilitate the selective release of molecules from the cell surface and the exchange of membrane proteins between cells. There is also evidence that GPIs and/or their metabolites can act as secondary messengers, modulating biological events including insulin production, insulin-mediated signal transduction, cellular proliferation and cell–cell recognition. Their discovered role as mediators of regulatory processes makes the chemical preparation of these compounds and their analogues of great interest. This comprehensive review highlights the progress in the chemical synthesis of GPI anchors and related glycoconjugate structures from protozoan parasites, yeast and mammals in the last two decades. The synthesis of a structurally related prokaryotic glycoconjugate of Mycobacterium tuberculosis is also discussed.

Up-Regulating the Hemeoxygenase System Enhances Insulin Sensitivity and Improves Glucose Metabolism in Insulin-Resistant Diabetes in Goto-Kakizaki Rats

Insulin-mediated signal transduction is positively correlated to adiponectin, adenosine monophosphate-activated protein kinase (AMPK), and glucose-transporter-4 (GLUT4) but negatively to oxidative/inflammatory mediators such as nuclear factor-kappaB, activating-protein (AP)-1, AP-2, and c-Jun-N-terminal-kinase. Although hemeoxygenase (HO) suppresses oxidative insults, its effects on insulin-sensitizing agents like AMPK and GLUT4 remains unclear and were investigated using Goto-Kakizaki rats (GK), a nonobese insulin-resistant type-2 diabetic model. HO was induced with hemin or inhibited with chromium mesoporphyrin (CrMP). The application of hemin to GK rats evoked a 3-month antidiabetic effect, whereas the HO-inhibitor, CrMP, exacerbated hyperglycemia and nullified insulin-signaling/glucose metabolism. Interestingly, the antidiabetic was accompanied by a paradoxical increase of insulin alongside the potentiation of insulin-sensitizing agents such as adiponectin, AMPK, and GLUT4 in the gastrocnemius muscle. Furthermore, hemin enhanced mediators/regulators of insulin signaling like cGMP and cAMP and suppressed oxidative insults by up-regulating HO-1, HO activity, superoxide dismutase, catalase, and the total antioxidant capacity in the gastrocnemius muscle. Accordingly, oxidative markers/mediators including nuclear factor-kappaB, AP-1, AP-2, c-Jun-N-terminal-kinase, and 8-isoprostane were abated, whereas CrMP annulled the cytoprotective and antidiabetic effects of hemin. Correspondingly, ip glucose tolerance, insulin tolerance, and homeostasis model assessment insulin resistance analyses revealed improved glucose tolerance, reduced insulin intolerance, enhanced insulin sensitivity, and reduced insulin resistance in hemin-treated GK rats. In contrast, CrMP, abolished the insulin-sensitizing effects and restored and/or exacerbated insulin resistance. Our study unveils a 3-month enduring antidiabetic effect of hemin and unmasks the synergistic interaction among the HO system, adiponectin, AMPK, and GLUT4 that could be explored to enhance insulin signaling and improve glucose metabolism in insulin-resistant diabetes.

Exercise training increases insulin-stimulated glucose disposal and GLUT4 (SLC2A4) protein content in patients with type 2 diabetes

Exercise enhances insulin-stimulated glucose transport in skeletal muscle through changes in signal transduction and gene expression. The aim of this study was to assess the impact of acute and short-term exercise training on whole-body insulin-mediated glucose disposal and signal transduction along the canonical insulin signalling cascade. A euglycaemic-hyperinsulinaemic clamp, with vastus lateralis skeletal muscle biopsies, was performed at baseline and 16 h after an acute bout of exercise and short-term exercise training (7 days) in obese non-diabetic (n=7) and obese type 2 diabetic (n=8) subjects. Insulin-mediated glucose disposal was unchanged following acute exercise in both groups. Short-term exercise training increased insulin-mediated glucose disposal in obese type 2 diabetic (p<0.05), but not in obese non-diabetic subjects. Insulin activation of (1) IRS1, (2) IRS2, (3) phosphotyrosine-associated phosphatidylinositol-3 kinase activity and (4) the substrate of phosphorylated Akt, AS160, a functional Rab GTPase activating protein important for GLUT4 (now known as solute carrier family 2 [facilitated glucose transporter], member 4 [SLC2A4]) translocation, was unchanged after acute or chronic exercise in either group. GLUT4 protein content was increased in obese type 2 diabetic subjects (p<0.05), but not in obese non-diabetic subjects following chronic exercise. Exercise training increased whole-body insulin-mediated glucose disposal in obese type 2 diabetic patients. These changes were independent of functional alterations in the insulin-signalling cascade and related to increased GLUT4 protein content.

Nitric oxide agents impair insulin-mediated signal transduction in rat skeletal muscle

BackgroundEvidence demonstrates that exogenously administered nitric oxide (NO) can induce insulin resistance in skeletal muscle. We have investigated the modulatory effects of two NO donors, S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) on the early events in insulin signaling in rat skeletal myocytes.ResultsSkeletal muscle cells from 6–8 week old Sprague-Dawley rats were treated with SNAP or GSNO (25 ng/ml) in the presence or absence of glucose (25 mM) and insulin (100 nM). Cellular insulin receptor-β levels and tyrosine phosphorylation in IRS-1 were significantly reduced, while serine phosphorylation in IRS-1 was significantly increased in these cells, when compared to the insulin-stimulated control. Reversal to near normal levels was achieved using the NO scavenger, 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO).ConclusionThese data suggest that NO is a potent modulator of insulin-mediated signal transduction and may play a significant role in the pathogenesis of type 2 diabetes mellitus.

A High-Throughput Microfluidic Assay for SH2 Domain-Containing Inositol 5-Phosphatase 2

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.

Exogenous nitric oxide inhibits IRS-1 expression in rat hepatocytes and skeletal myocytes

Accumulative evidence has supported the role of nitric oxide (NO) in a variety of normal physiological functions as well as many pathological conditions. In this study, we examined the possible diabetogenicity of NO by measuring the expression of the insulin receptor substrate (IRS)-1 in rat hepatocytes and skeletal myocytes. IRS-1 is important in the insulin-mediated signal transduction pathway in both liver and skeletal muscle. Exogenous NO donated by S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) resulted in significant reduction in levels of IRS-1 in both cells, when compared to the insulin-stimulated control (p<0.001). Reversal to near normal levels was achieved using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). SNAP was the more potent drug, and the skeletal myocytes were the more sensitive cells to the inhibitory effects of NO released from the drugs. These results provide further evidence that exogenous NO is a potent modulator of insulin-mediated signal transduction and may play a significant role in the pathogenesis of type 2 diabetes mellitus.

Insulin Dynamically Regulates Calmodulin Gene Expression by Sequential O-Glycosylation and Phosphorylation of Sp1 and Its Subcellular Compartmentalization in Liver Cells

O-glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (approximately 30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression.

Engineering eukaryotic signal transduction with RNAi: EnhancingDrosophila S2 cell growth and recombinant protein synthesis via silencing ofTSC1

RNAi has been useful in the study of biochemical pathways, but has not been widely used as a tool in metabolic engineering. The work described here makes use of double-stranded RNA (dsRNA) for the post-transcriptional gene silencing of TSC1 in Drosophila S2 cells. TSC1 downregulates the insulin-mediated signal transduction pathway, and serves as a metabolic control to guard against cellular overproliferation and tumorogenesis in both flies and mammals. By silencing TSC1 with in vitro-synthesized dsRNA, we have created a tunable and specific metabolic "throttle" that, like insulin, apparently increases the specific growth rate of S2 cells in a dose-dependent manner. This "throttle," augments the benefits of insulin addition while apparently avoiding deleterious and pleiotropic effects which can lead to lysis. During the period wherein dsRNA was active, cell growth rate was increased by 11% by the addition of 15 microg/mL dsTSC1 and by over 20% by the addition of 30 microg/mL dsTSC1. Additionally, synthesis of recombinant green fluorescent protein (GFP) was increased nearly 50% in a stable S2 cell line inducibly expressing GFP. Accordingly, we have "tuned" a normally tumorogenic pathway in animals into an advantage for both growth and recombinant product synthesis in cell culture. Potential applications for improving eukaryotic cell culture are anticipated.

A Nonspecific Phosphotyrosine Phosphatase Inhibitor, Bis(maltolato)oxovanadium(IV), Improves Glucose Tolerance and Prevents Diabetes in Zucker Diabetic Fatty Rats

The molecular basis of insulin resistance, a major risk factor for development of Type II diabetes, involves defective insulin signaling. Insulin-mediated signal transduction is negatively regulated by the phosphotyrosine phosphatase, PTP1B, and numerous studies have demonstrated that organo-vanadium compounds, which are nonselective phosphotyrosine phosphatase inhibitors, have insulin-mimetic properties. However, whether or not vanadium compounds can prevent the transition from insulin resistance to overt diabetes is unknown. We compared the ability of bis(maltolato)oxovanadium(IV) (BMOV), an orally bioavailable organo-vanadium compound, and rosiglitazone maleate (RSG), a known insulin sensitizer, to prevent development of diabetes in Zucker diabetic fatty (ZDF) rats. Treatment began at 6 weeks of age when animals are insulin resistant and hyperinsulinemic, but not yet hyperglycemic, and ended at 12 weeks of age, which is 4 weeks after ZDF rats typically develop overt diabetes. BMOV-treated ZDF rats did not develop hyperglycemia, showed significant improvement in insulin sensitivity, and retained normal pancreatic islet morphology and endocrine cell distribution, similar to RSG-treated animals. BMOV and RSG treatment also prevented the hyper-phagia and polydipsia present in untreated ZDF rats; however, BMOV-treated ZDF rats gained much less weight than did RSG-treated animals. Circulating levels of adiponectin decreased in untreated ZDF rats compared to lean controls, but these levels remained normal in BMOV-treated ZDF rats. In contrast, in RSG-treated ZDF rats, plasma adiponectin levels were nearly 4-fold higher than in lean control rats, primarily as a result of a large increase in the amount of low-molecular weight forms of adiponectin in circulation. These data demonstrate that phosphatase inhibition offers a new approach to diabetes prevention, one that may have advantages over current approaches.

NAADP on the up in pancreatic beta cells-a sweet message?

Pancreatic beta cells secrete insulin in response to elevated plasma glucose levels in a Ca(2+)-dependent fashion. Released insulin may act on the beta cell itself to promote further insulin synthesis and release. Recent studies by Johnson and Misler,1 Masgrau et al.2 and Mitchell et al.3 provide strong evidence (1) for the existence of intracellular Ca(2+) stores sensitive to NAADP, a potent Ca(2+)-mobilizing messenger, and (2) that these Ca(2+) stores are involved in both glucose- and insulin-mediated signal transduction. NAADP may therefore play an important role in controlling secretion of insulin from pancreatic beta cells.

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC ζ activation and insulin-induced tyrosine phosphorylation of Giα to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01∼1 ng/ml PTX for 2 hours, followed by stimulation with 10–100 nM insulin or 1 μM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [ 3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [ 3H]2-DOG uptake, without affecting basal [ 3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC ζ translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [ 125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa α-subunit of G-protein (Giα-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Giα-2 antibody. Insulin-induced tyrosine phosphorylation of Giα-2 was found by immunoblot analysis with anti-Giα-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Giα-2 with GTP and PI 3-kinase-PKC ζ signalling by releasing of Gβγ via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.