E. Van Obberghen

SOCS proteins causing trouble in insulin action.

First discovered as inhibitors of cytokine signalling, the suppressor of cytokine signalling (SOCS) proteins have appeared, over recent years, as potent repressors of other signalling pathways including the one induced by insulin. SOCS‐1 and SOCS‐3 have been extensively studied both in vitro and in vivo in the context of insulin action. It has been shown that these two SOCS members are able to inhibit the insulin signalling pathway by three different mechanisms: (1) inhibition of tyrosine phosphorylation of insulin receptor substrate (IRS) proteins because of competition at the docking site on the insulin receptor (IR), (2) induction of the proteasomal degradation of the IRS and (3) inhibition of the IR kinase. A key feature of the SOCS proteins is that they are induced regulators. Indeed, expression of SOCS proteins is virtually absent in basal conditions, but is rapidly and robustly induced in response to several stimuli such as hormones, cytokines and growth factors. A significant correlation between SOCS‐3 expression and insulin resistance has been demonstrated in vivo. Interestingly, the level of SOCS‐3 expression is strikingly enhanced in insulin‐sensitive tissues from both patients and animal models with type 2 diabetes and insulin resistance. While it remains to be established whether the increased expression of SOCS is a cause or a consequence of insulin resistance, a large body of observations supports a role for SOCS proteins in the disease process found in states with insulin resistance.

Methylglyoxal impairs insulin signalling and insulin action on glucose-induced insulin secretion in the pancreatic beta cell line INS-1E

Aims/hypothesisChronic hyperglycaemia aggravates insulin resistance, at least in part, by increasing the formation of advanced glycation end-products (AGEs). Methylglyoxal (MGO) is the most reactive AGE precursor and its abnormal accumulation participates in damage in various tissues and organs. Here we investigated the ability of MGO to interfere with insulin signalling and to affect beta cell functions in the INS-1E beta cell line.MethodsINS-1E cells were incubated with MGO and then exposed to insulin or to glucose. Western blotting was used to study signalling pathways, and real-time PCR to analyse gene expression; insulin levels were determined by radioimmunoassay.ResultsNon-cytotoxic MGO concentrations inhibited insulin-induced IRS tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway activation independently from reactive oxygen species (ROS) production. Concomitantly, formation of AGE adducts on immunoprecipitated IRS was observed. Aminoguanidine reversed MGO inhibitory effects and the formation of AGE adducts on IRS. Further, the insulin- and glucose-induced expression of Ins1, Gck and Pdx1 mRNA was abolished by MGO. Finally, MGO blocked glucose-induced insulin secretion and PI3K/PKB pathway activation. These MGO effects were abolished by LiCl, which inhibits glycogen synthase kinase-3 (GSK-3).Conclusions/interpretationMGO exerted major damaging effects on INS-1E cells impairing both insulin action and secretion. An important actor in these noxious MGO effects appears to be GSK-3. In conclusion, MGO participates not only in the pathogenesis of the debilitating complications of type 2 diabetes, but also in worsening of the diabetic state by favouring beta cell failure.

Search for variants of the gene-promoter and the potential phosphotyrosine encoding sequence of the insulin receptor substrate-2 gene: evaluation of their relation with alterations in insulin secretion and insulin sensitivity

Aims/hypothesis. The aim of this study was to screen part of the putative promoter sequence in addition to 14 potential phosphotyrosine residues of human IRS-2 for genetic variability which might cause changes in protein expression or function. Furthermore, the potential impact on insulin secretion and sensitivity of a previously identified IRS-2 variant (Gly1057Asp) was analysed Methods. The screenings were carried out by the SSCP-heteroduplex technique on DNA from Type II (non-insulin-dependent) diabetic patients. The impact of the Gly1057Asp variant was analysed in four glucose-tolerant Scandinavian study groups. Results. The results showed no nucleotide substitutions in the promoter sequence, however, a novel heterozygous amino acid variant was identified (Leu647Val). In an association study, the new variant was found in 3 of 413 diabetic patients and in none of 280 glucose tolerant subjects. The variant did not affect the binding of IRS-2 to the insulin receptor or p85α of phosphatidylinositol 3-kinase when measured in the yeast two-hybrid system. Examination of the common Gly1057Asp variant in 363 young healthy subjects and in 228 glucose tolerant offspring of one diabetic parent showed no differences in insulin secretion or insulin sensivity after an intravenous glucose tolerance test. Glucose tolerant middle-aged subjects homozygous for the polymorphism (n = 31), however, had on average a 25 % decrease in fasting serum insulin concentrations (p = 0.009) and 28 % (p = 0.01) and 34 % (p = 0.003) reductions in serum insulin concentrations at 30 and 60 min, respectively, during an OGTT compared with wildtype carriers (n = 107). In a cohort of 639 elderly Swedish men the amino acid variant did not have any detectable impact on insulin secretion after an OGTT. Conclusion/interpretation. No genetic variability was found in the IRS-2 promoter. A rare IRS-2 variant at codon 647 has been identified in Type II diabetic patients. The prevalent codon 1057 polymorphism had no consistent effect on insulin secretion or insulin sensitivity. [Diabetologia (1999) 42: 1244–1249]

Liver HIF-1 Alpha Induction Precedes Apoptosis Following Normothermic Ischemia-Reperfusion in Rats

Apoptosis plays an important role in ischemia-reperfusion (I-R) injury during liver transplantation. The hypoxia-inducible factor alpha (HIF-1alpha) may trigger liver apoptosis following I-R through the induction of hypoxically regulated genes. The aim of this study was to evaluate the effect of normothermic liver I-R on HIF-1alpha expression and apoptosis in rats. Segmental normothermic ischemia of the liver was induced in rats for 120 minutes. Liver extracts from either ischemic or nonischemic lobes were prepared at 0, 1, 3, and 6 hours after reperfusion. Liver HIF-1alpha protein expression was examined by Western blot analysis. Liver apoptosis was quantified using terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling assay. Normothermic I-R resulted in a significant (P< .05) increase in liver HIF-1alpha protein levels 1 and 3 hours after reperfusion. Liver apoptosis was significantly (P< .005) increased at 3 and 6 hours after reperfusion. In conclusion, normothermic liver I-R leads to increased liver expression of HIF-1alpha and apoptosis.

Insulin receptors: Structure and function

The recent characterization of the human insulin receptor structure and its intrinsic tyrosine kinase activity represent major advances in our understanding of the mechanism of insulin action. It is reasonable to think that the insulin-induced autophosphorylation and activation of its receptor kinase represent an important event in the action of insulin on cell metabolism and growth. The fundamental research reviewed may be followed by the discovery of molecular receptor defects in clinical syndromes of insulin resistance.

Constitutive expression of suppressor of cytokine signalling-3 in skeletal muscle leads to reduced mobility and overweight in mice

Aims/hypothesisDue to their ability to regulate various signalling pathways (cytokines, hormones, growth factors), the suppressor of cytokine signalling (SOCS) proteins are thought to be promising therapeutic targets for metabolic and inflammatory disorders. Hence, their role in vivo has to be precisely determined.MethodsWe generated transgenic mice constitutively producing SOCS-3 in skeletal muscle to define whether the sole abundance of SOCS-3 is sufficient to induce metabolic disorders and whether SOCS-3 is implicated in physiological roles distinct from metabolism.ResultsWe demonstrate here that chronic expression of SOCS-3 in skeletal muscle leads to overweight in mice and worsening of high-fat diet-induced systemic insulin resistance. Counter-intuitively, insulin sensitivity in muscle of transgenic mice appears to be unaltered. However, following constitutive SOCS-3 production, several genes had deregulated expression, among them other members of the SOCS family. This could maintain the insulin signal into skeletal muscle. Interestingly, we found that SOCS-3 interacts with calcineurin, which has been implicated in muscle contractility. In Socs-3 transgenic muscle, this leads to delocalisation of calcineurin to the fibre periphery. Relevant to this finding, Socs-3 transgenic animals had dilatation of the sarcoplasmic reticulum associated with swollen mitochondria and decreased voluntary activity.Conclusions/interpretationOur results show that constitutive SOCS-3 production in skeletal muscle is not in itself sufficient to induce the establishment of metabolic disorders such as diabetes. In contrast, we reveal a novel role of SOCS-3, which appears to be important for muscle integrity and locomotor activity.

The suppressor of cytokine signalling 2 (SOCS2) is a key repressor of insulin secretion

Aims/hypothesisSuppressor of cytokine signalling (SOCS) proteins are powerful inhibitors of pathways involved in survival and function of pancreatic beta cells. Whereas SOCS1 and SOCS3 have been involved in immune and inflammatory processes, respectively, in beta cells, nothing is known about SOCS2 implication in the pancreas.MethodsTransgenic (tg) mice were generated that constitutively produced SOCS2 in beta cells (βSOCS2) to define whether this protein is implicated in beta cell functioning and/or survival.ResultsConstitutive production of SOCS2 in beta cells leads to hyperglycaemia and glucose intolerance. This phenotype is not a consequence of decreased beta cell mass or inhibition of insulin synthesis. However, insulin secretion to various secretagogues is profoundly altered in intact animals and isolated islets. Interestingly, constitutive SOCS2 production dampens the rise in cytosolic free calcium concentration induced by glucose, while glucose metabolism is unchanged. Moreover, tg islets have a depletion in endoplasmic reticulum Ca2+ stores, suggesting that SOCS2 interferes with calcium fluxes. Finally, in βSOCS2 mice proinsulin maturation is impaired, leading to an altered structure of insulin secretory granules and augmented levels of proinsulin. The latter is likely to be due to decreased production of prohormone convertase 1 (PC1/3), which plays a key role in proinsulin cleavage.Conclusions/InterpretationsSOCS2 was shown to be a potent regulator of proinsulin processing and insulin secretion in beta cells. While its constitutive production is insufficient to induce overt diabetes in this mouse model, it causes glucose intolerance. Thus, increased SOCS2 production could be an important event predisposing to beta cell failure.

Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells

Aims/hypothesisA high-fat diet and obesity are associated with increased risk of liver cancer. Because increased delivery of NEFA to the liver occurs in these conditions, we investigated the involvement of the unsaturated fatty acid oleate in hepatocarcinoma cell proliferation using human-derived hepatocarcinoma cell lines as model systems.MethodsWestern blotting, FACS analysis and [3H]thymidine incorporation were used to study the signalling pathways and the proliferation of cells cultured for up to 72xa0h with or without a concentration of oleate considered to be relevant to human pathophysiology (50xa0μmol/l) or a concentration considered elevated (1xa0mmol/l).ResultsIn HepG2 cells, proliferation was increased in the presence of 50xa0μmol/l oleate, but was decreased at 1xa0mmol/l. This differential effect was correlated with the activation of the mammalian target of rapamycin complex 1 (mTORC1) and with increased translation of cell cycle regulators. Oleate-mediated mTORC1 activation required phospholipase D activation, which produces phosphatidic acid and is known to render mTORC1 rapamycin resistant. Remarkably, rapamycin resistance was found to affect specifically the mTORC1/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) branch of the mTORC1 pathway in the presence of 50xa0μmol/l oleate. Furthermore, inhibition of phosphatidic acid production abolished oleate-induced increases in mTORC1 activity and cyclin A production. Importantly, the same differential effects of oleate on mTORC1 activation, cell cycle regulators and rapamycin resistance were found in SK-Hep1 cells.Conclusions/interpretationOleate stimulates mTORC1 activation and rapamycin resistance. We propose that rapamycin-derived mTOR inhibitors are likely to be of limited therapeutic use to restrain hepatic tumour growth, particularly in the context of associated obesity.

Spontaneous hypoglycaemia in the presence of both anti-insulin antibody and anti-insulin receptor antibody

Beside insulinoma, alternative causes of hyperinsulinaemic hypoglycaemia include the rare autoimmune syndrome related to spontaneous autoantibodies either to insulin or to insulin receptor. We describe a case of hypoglycaemia with high insulinemia in which insulinoma could not be evidenced. Surprisingly, we found in the patients serum both insulin autoantibodies and insulin receptor autoantibodies. Available data eventually supported the predominant role of insulin autoantibodies rather than insulin receptor autoantibodies in the mechanism of hypoglycaemia of this patient. Insulin antibodies were present in high titre. Most of the insulin in serum was bound to the insulin antibodies and free insulin was slightly increased. HLA typing displayed DR4 haplotype, known to be strongly linked to the insulin autoimmune syndrome. The patients serum was able to inhibit insulin binding to its receptor in a cultured cell line overexpressing insulin receptors both in experiments with native serum and with serum depleted from insulin antibodies. However, we could not demonstrate that the insulin receptor antibodies had insulin mimicking effect. We have no obvious explanation for the presence of these two antibodies in the same patient. Possible hypotheses might involve an idiotype-anti-idiotype mechanism or a poly-autoimmune disease.

IRS-4 mediated mitogenic signalling by insulin and growth hormone in LB cells, a murine T-cell lymphoma devoid of IGF-I receptors.

Insulin and growth hormone (GH) induce mitogenic and metabolic signals in cells, GH either directly or indirectly via IGF-I production. We have studied a spontaneous murine T-cell lymphoma (LB cells) devoid of IGF-1 receptors in which proliferation is maintained by insulin [Int. J. Cancer 50 (1992) 80], and show that GH is more potent than insulin, with both GH and insulin dose-response curves for thymidine incorporation being bell-shaped. Binding showed somatogenic rather than lactogenic GH receptors. Insulin stimulated phosphorylation of the insulin receptor and of a 160-kDa protein, identified as the IRS-4 protein. This phosphorylated IRS-4 associated with PI3-kinase, which was activated along with the downstream p70(S6) kinase, whereas the Ras-MAPK pathway was not. Using selective inhibitors, the PI3-kinase, but not p70(S6) kinase or MEK, was found to be involved in insulin-stimulated DNA synthesis. GH induced tyrosine phosphorylation of IRS-4 and nuclear translocation of STAT5. The LB cells constitute a new model for studying GH and insulin signalling without interference of IGF-1 receptors.