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E Van Obberghen

Publications and source records attributed to E Van Obberghen.

At least 19 recordsLinked to original sources
PubMed2006

Amino acids require glucose to enhance, through phosphoinositide-dependent protein kinase 1, the insulin-activated protein kinase B cascade in insulin-resistant rat adipocytes.

AIMS/HYPOTHESIS: Amino acids are well known to activate the mammalian target of the rapamycin (mTOR) pathway in synergy with insulin to regulate cell functions. Despite recent important advances, the mTOR signalling pathway is poorly understood. Our previous results revealed a new pathway in which amino acids permit insulin-induced activation of the protein kinase B (PKB)/mTOR pathway in freshly isolated adipocytes when phosphatidylinositol 3-kinase (PI3K) is inhibited. The aim of this study was to further investigate this pathway at the molecular level. METHODS: We studied the effect of amino acids on PKB phosphorylation in different cellular models or in freshly isolated adipocytes incubated in different buffers, after a time course of insulin and amino acids and in the presence of pharmacological inhibitors. To investigate the potential role of amino acids in insulin action, the effect on glucose transport in obese rat adipocytes following a high-fat diet was assessed. RESULTS: Insulin-induced PKB phosphorylation is restored by amino acids in the presence of wortmannin in adipose tissue explants and freshly isolated adipocytes, but not in cultured adipocytes or hepatocytes. Moreover, amino acids require the presence of glucose to phosphorylate PKB and to partially rescue glucose transport in a PI3K-independent manner. The results also suggest that the amino acids act through the phosphoinositide-dependent protein kinase 1. In addition, amino acids were seen to improve insulin-stimulated glucose transport in adipocytes from high-fat-fed rats. CONCLUSIONS/INTERPRETATION: This study suggests that amino acids could enhance adipocyte insulin signalling in pathophysiological situations such as insulin resistance associated with obesity.

Adipocytes↗
PubMed2006

Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

BACKGROUND: This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. METHODS: Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. RESULTS: The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. CONCLUSION: Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs.

Analysis of Variance↗
PubMed2006

[Thrombophilic factors in divers with undeserved decompression sickness].

UNLABELLED: In divers with a vascular disease in decompression sickness, who have not committed any technical error, thrombophilic risk factors were sought. Six cases of confirmed divers, without diving technical error, were investigated. Thrombophilic screening included proteins C, S, antithrombin III, and factor VIII assays, and circulating antibodies, Factor V Leiden, and mutation G20210A mutation in Factor II gene research. Total plasma homocysteine (Hcy), an atherosclerosis factor, even when slightly increased, nutitional factors: folate and vitamins B12 and B6, the cofactors of its metabolism, and inversely correlated with Hcy values, were assayed, and subjects were genotyped for mutation C677T in the MTHFR gene. RESULTS: In five divers, Hcy values were moderately increased, and in all the six, folate and/or B12 values were decreased. Three of them showed a genotype TT (mutation C677T), two, the genotype CT, and the sixth, an heterozygous Factor V Leiden. In these divers, a predisposition for vascular diseases, was detected, which was partially curable.

Adult↗
PubMed2006

Alterations in protein tyrosine kinase pathways in rat liver following normothermic ischemia-reperfusion.

The phosphoregulation of signal transduction pathways is a complex series of reactions that modulate the cellular response to ischemia-reperfusion (I-R). The aim of this study was to evaluate the effect of normothermic liver I-R on protein tyrosine phosphorylation, production of angiogenic growth factors, and activation of signal proteins in tyrosine kinase pathways. A segmental normothermic ischemia of the liver was induced in rats by occluding the blood vessels (including the bile duct) to the median and left lateral lobes for 120 minutes. Liver extracts from either ischemic or nonischemic lobes were prepared at 0, 1, 3, and 6 hours after reperfusion. Liver tyrosine phosphorylation of proteins was examined by Western blot analysis, whereas vascular endothelial growth factor (VEGF) mRNA was analyzed by Northern blot. In ischemic liver lobes, VEGF mRNA and total protein levels increased at 1 and 3 hours after reperfusion. Tyrosine phosphorylation of the VEGF receptor Flk-1 and the platelet-derived growth factor receptor (PDGF-R) was increased only at 1 hour after reperfusion, while c-Src tyrosine phosphorylation remained increased at 3 hours and remained up to 6 hours after reperfusion. In conclusion, 1-R led to alterations in protein tyrosine phosphorylation and increased expression of VEGF in rat liver.

Animals↗
PubMed2006

Role of diabetes in atherosclerotic pathogenesis. What have we learned from animal models?

Diabetes mellitus is associated with a greater risk of developing atherosclerosis and its complications: stroke, myocardial infarction, and peripheral vascular disease. In patients with diabetes, atherosclerosis represents a complex multifactorial disease with increased lesion progression and severity compared to the nondiabetic population. Several risk factors have been proposed to explain the increased risk of cardiovascular disease with diabetes. They include: hyperglycaemia, dyslipidemia, accelerated formation of advanced glycation end-products (AGEs), increased oxidative stress, and genetic factors. It is difficult to precisely establish the elements leading to diabetes-accelerated atherosclerosis by means of epidemiological studies because all these factors coexist in diabetic patients. Thus, diabetic animal models that reproduce exacerbation of atherosclerosis would be helpful to understand why atherosclerosis is accelerated by diabetes, and to design appropriate treatments to limit its progression. This review analyzes most of the animal models developed to reproduce diabetes-accelerated atherosclerosis, and summarizes the effects of hyperglycaemia and lipid abnormalities on atherogenesis.

Animals↗
PubMed2005

Insulin signalling downstream of protein kinase B is potentiated by 5'AMP-activated protein kinase in rat hearts in vivo.

AIMS/HYPOTHESIS: 5'AMP-activated protein kinase (AMPK) and insulin stimulate glucose transport in heart and muscle. AMPK acts in an additive manner with insulin to increase glucose uptake, thereby suggesting that AMPK activation may be a useful strategy for ameliorating glucose uptake, especially in cases of insulin resistance. In order to characterise interactions between the insulin- and AMPK-signalling pathways, we investigated the effects of AMPK activation on insulin signalling in the rat heart in vivo. METHODS: Male rats (350-400 g) were injected with 1 g/kg 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or 250 mg/kg metformin in order to activate AMPK. Rats were administered insulin 30 min later and after another 30 min their hearts were removed. The activities and phosphorylation levels of components of the insulin-signalling pathway were subsequently analysed in individual rat hearts. RESULTS: AICAR and metformin administration activated AMPK and enhanced insulin signalling downstream of protein kinase B in rat hearts in vivo. Insulin-induced phosphorylation of glycogen synthase kinase 3 (GSK3) beta, p70 S6 kinase (p70S6K)(Thr389) and IRS1(Ser636/639) were significantly increased following AMPK activation. To the best of our knowledge, this is the first report of heightened insulin responses of GSK3beta and p70S6K following AMPK activation. In addition, we found that AMPK inhibits insulin stimulation of IRS1-associated phosphatidylinositol 3-kinase activity, and that AMPK activates atypical protein kinase C and extracellular signal-regulated kinase in the heart. CONCLUSIONS/INTERPRETATIONS: Our data are indicative of differential effects of AMPK on the activation of components in the cardiac insulin-signalling pathway. These intriguing observations are critical for characterisation of the crosstalk between AMPK and insulin signalling.

AMP-Activated Protein Kinases↗
PubMed2004

Modulation of insulin action.

Insulin is a key hormone regulating the control of metabolism and the maintenance of normoglycaemia and normolipidaemia. Insulin acts by binding to its cell surface receptor, thus activating the receptor's intrinsic tyrosine kinase activity, resulting in receptor autophosphorylation and phosphorylation of several substrates. Tyrosine phosphorylated residues on the receptor itself and on subsequently bound receptor substrates provide docking sites for downstream signalling molecules, including adapters, protein serine/threonine kinases, phosphoinositide kinases and exchange factors. Collectively, those molecules orchestrate the numerous insulin-mediated physiological responses. A clear picture is emerging of the way in which insulin elicits several intracellular signalling pathways to mediate its physiologic functions. A further challenge, being pursued by several laboratories, is to understand the molecular mechanisms that underlie insulin action at the peripheral level, deregulation of which ultimately leads to hyperglycaemia and Type 2 diabetes. We review how circulating factors such as insulin itself, TNF-alpha, interleukins, fatty acids and glycation products influence insulin action through insulin signalling molecules themselves or through other pathways ultimately impinging on the insulin-signalling pathway. Understanding how the mechanism by which molecular insulin action is modulated by these factors will potentially provide new targets for pharmacological agents, to enable the control of altered glucose and lipid metabolism and diabetes.

AMP-Activated Protein Kinases↗
PubMed2004

The molecular basis of succinic semialdehyde dehydrogenase deficiency in one family.

Succinic semialdehyde dehydrogenase (SSADH) deficiency has predominantly neurological consequences, affecting psychomotor, speech and language development. Recently, two clinical reviews summarized the features of this disease and their relative frequency [Neurology 60 (2003) 1413; Ann. Neurol. 54 (2003) S73]. The molecular genetics of SSADH deficiency is still being explored. We describe the molecular basis of this defect in a Tunisian female child presenting with a mild phenotype. A small scale deletion in exon 10 of the gene led to a frameshift that predicts premature termination of the resulting putative protein. The parents were shown to be heterozygotes for this deletion, supporting its causative role.

Aldehyde Oxidoreductases↗
PubMed2003

Expression of myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase in muscle cell lines: involvement of PtdIns(3)P in insulin-stimulated glucose transport.

X-linked myotubular myopathy is a muscle disorder caused by mutations on the myotubular myopathy-1 (MTM-1) gene, coding for myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A myotubularin.[32P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)-domain-containing PtdIns(3)P binding protein early endosomal antigen 1 as well as of a gluathione-S-transferase-FYVE probe directed to PtdIns(3)P. Expression of wt myotubularin, although not affecting activation of proximal insulin signal transduction targets such as protein kinase B and MAPK, induced a decrease in insulin-induced glucose uptake, whereas basal glucose uptake was augmented by expression of D278A (DA) and C375S (CS) mutants. Moreover, overexpression of myotubularin in 3T3-L1 adipocytes impaired insulin-induced translocation at the plasma membrane of green fluorescent protein-tagged glucose transporter 4. These data indicate that PtdIns(3)P is required to direct glucose transporter 4 to insulin-responsive compartments and/or to allow the translocation of the latter at the plasma membrane. We conclude that myotubularin, by modulating the intracellular levels of PtdIns(3)P, plays a role in the control of vesicular traffic related to glucose transport, by counteracting the activities of the PtdIns(3)P-producing phosphatidylinositol 3-kinases.

Adenoviridae↗
PubMed2003

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.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Modulators of insulin action and their role in insulin resistance.

Insulin is a key anabolic hormone that plays a crucial role in growth, differentiation and metabolism. Insulin action is initiated by the binding of the hormone to its tyrosine kinase cell surface receptor, leading to the multisite autophosphorylation of the receptor. This results in the activation of the receptor kinase and subsequent tyrosine phosphorylation of insulin receptor substrates, most of which are docking proteins for signaling molecules. For the last several years, our laboratory has been interested in the mechanisms that lead to the modulation of insulin signal transduction, and hence might be involved in insulin resistance found in obesity and type II diabetes. For this review, we have focused on three 'modulators' of insulin action: hyperinsulinemia, suppressor of cytokine signaling proteins and advanced glycation end products.

Cytokines↗
PubMed2002

[Clinical relevance of homocysteine monitoring in the diabetic patient].

Accelerated atherosclerosis is common in diabetes mellitus, although its extent is not always related to its strong association with classical cardiovascular risk factors. Diabetic patients, especially with type 2 diabetes, are prone to cardiovascular disease which is the leading cause of death in this population. Recent clinical studies among general population have shown that an even mild increase of homocysteinemia play an important role in the progression of atherosclerosis, either in coronary or peripheral arteries. An increasing amount of in vitro data is providing evidence that excess of homocysteine has a toxic effect on the arterial wall. This aminoacid thus appears to be not only a risk marker but also an emerging cardiovascular risk factor. The measurement of plasma homocysteine contributes to the identification, among the diabetic population, of patients at high cardio-vascular risk, with the aim of improving their global management. Moreover the addition of group B vitamins provides an easy and low-cost treatment to lower hyperhomocysteinemia.

Diabetes Complications↗
PubMed2001

SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice.

SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr(960) of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-alpha (TNF-alpha), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-alpha induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-alpha target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.

3T3 Cells↗
PubMed2001

Regulation of vascular endothelial growth factor expression by advanced glycation end products.

Advanced glycation end products (AGEs) are generated during long term diabetes and are correlated with the development of diabetic complications, such as retinopathy. Diabetic retinopathy is characterized by an increased retinal neovascularization due to the action of the angiogenic factor, vascular endothelial growth factor (VEGF). In this report, we show that injection of insulin and glycated albumin (Alb-AGE) to mice increases VEGF mRNA expression in eyes. Insulin and Alb-AGE stimulate VEGF mRNA and protein expression in retinal epithelial cells (ARPE-19). Alb-AGE-induced VEGF expression is not modulated by the use of antioxidants, N-acetyl-l-cysteine or pyrrolidinedithiocarbamate, or by an inhibitor of phosphatidylinositol 3-kinase (PI3K), wortmannin. However, using an inhibitor of ERK activation, U0126, we show that Alb-AGE stimulates VEGF expression through an ERK-dependent pathway. Accordingly, we found that Alb-AGE activated mitogen-activate protein kinase, ERK1/2, JNK1/2, but not p38, and that Alb-AGE did not activate PI3K and PKB. Moreover, Alb-AGE activated the transcription factor, hypoxia inducible factor-1 (HIF-1) DNA binding activity. This activation is mediated by an increase in accumulation of the HIF-1alpha protein through an ERK-dependent pathway. Thus, stimulation of VEGF expression by Alb-AGE, through the activation of HIF-1, could play an important role in the development of diabetic retinopathy.

Albumins↗
PubMed2001

Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells.

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed2001

The adapter protein, Grb10, is a positive regulator of vascular endothelial growth factor signaling.

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and angiogenesis. Activation of VEGF receptors leads to the recruitment of SH2 containing proteins which link the receptors to the activation of signaling pathways. Here we report that Grb10, an adapter protein of which the biological role remains unknown, is tyrosine phosphorylated in response to VEGF in endothelial cells (HUVEC) and in 293 cells expressing the VEGF receptor KDR. An intact SH2 domain is required for Grb10 tyrosine phosphorylation in response to VEGF, and this phosphorylation is mediated in part through the activation of Src. In HUVEC, VEGF increases Grb10 mRNA level. Expression of Grb10 in HUVEC or in KDR expressing 293 cells results in an increase in the amount and in the tyrosine phosphorylation of KDR. In 293 cells, this is correlated with the activation of signaling molecules, such as MAP kinase. By expressing mutants of Grb10, we found that the positive action of Grb10 is independent of its SH2 domain. Moreover, these Grb10 effects on KDR seem to be specific since Grb10 has no effect on the insulin receptor, and Grb2, another adapter protein, does not mimic the effect of Grb10 on KDR. In conclusion, we propose that VEGF up-regulates Grb10 level, which in turn increases KDR molecules, suggesting that Grb10 could be involved in a positive feedback loop in VEGF signaling.

Amino Acid Sequence↗
PubMed2001

Insulin induces suppressor of cytokine signaling-3 tyrosine phosphorylation through janus-activated kinase.

Suppressor of cytokine signaling (SOCS) proteins were originally described as cytokine-induced molecules involved in negative feedback loops. We have shown that SOCS-3 is also a component of the insulin signaling network (). Indeed, insulin leads to SOCS-3 expression in 3T3-L1 adipocytes. Once produced, SOCS-3 binds to phosphorylated tyrosine 960 of the insulin receptor and inhibits insulin signaling. Now we show that in 3T3-L1 adipocytes and in transfected COS-7 cells insulin leads to SOCS-3 tyrosine phosphorylation. This phosphorylation takes place on Tyr(204) and is dependent upon a functional SOCS-3 SH2 domain. Purified insulin receptor directly phosphorylates SOCS-3. However, in intact cells, a mutant of the insulin receptor, IRY960F, unable to bind SOCS-3, was as efficient as the wild type insulin receptor to phosphorylate SOCS-3. Importantly, IRY960F is as potent as the wild type insulin receptor to activate janus-activated kinase (Jak) 1 and Jak2. Furthermore, expression of a dominant negative form of Jak2 inhibits insulin-induced SOCS-3 tyrosine phosphorylation. As transfected Jaks have been shown to cause SOCS-3 phosphorylation, we propose that insulin induces SOCS-3 phosphorylation through Jak activation. Our data indicate that SOCS-3 belongs to a class of tyrosine-phosphorylated insulin signaling molecules, the phosphorylation of which is not dependent upon a direct coupling with the insulin receptor but relies on the Jaks.

3T3 Cells↗