Monika Kellerer

Leptin stimulates glucose transport and glycogen synthesis in C2C12 myotubes : evidence for a PI3-kinase mediated effect

Summary It was recently shown that leptin impairs insulin signalling, i. e. insulin receptor autophosphorylation and insulin-receptor substrate (IRS)-1 phosphorylation in rat-1 fibroblasts, NIH3T3 cells and HepG2 cells. To evaluate whether leptin might impair the effects of insulin in muscle tissue we studied the interaction of insulin and leptin in a muscle cell system, i. e. C2C12 myotubes. Preincubation of C2C12 cells with leptin (1–500 ng/ml) did not significantly affect insulin stimulated glucose transport and glycogen synthesis (1.8 to 2 fold stimulation); however, leptin by itself (1 ng/ml) was able to mimic approximately 80–90 % of the insulin effect on glucose transport and glycogen synthesis. Both glucose transport as well as glycogen synthesis were inhibited by the phosphatidylinositol-3 (PI3)-kinase inhibitor wortmannin and the protein kinase C inhibitor H7 while no effect was observed with the S6-kinase inhibitor rapamycin. We determined whether the effect of leptin occurs through activation of IRS-1 and PI3-kinase. Leptin did not stimulate PI3-kinase activity in IRS-1 immunoprecipitates; however, PI3-kinase activation could be demonstrated in p85α immunoprecipitates (3.04 ± 1.5 fold of basal). In summary the data provide the first evidence for a positive crosstalk between the signalling chain of the insulin receptor and the leptin receptor. Leptin mimics in C2C12 myotubes insulin effects on glucose transport and glycogen synthesis most likely through activation of PI3-kinase. This effect of leptin occurs independently of IRS-1 activation in C2C12 cells. [Diabetologia (1997) 40: 606–609]

Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways

SummaryWe have recently shown that leptin mimicks insulin effects on glucose transport and glycogen synthesis through a phosphatidylinositol-3 (PI) kinase dependent pathway in C2C12 myotubes. The aim of the present study was to identify the signalling path from the leptin receptor to the PI-3 kinase. We stimulated C2C12 myotubes with insulin (100 nmol/1, 5 min) or leptin (0.62 nmol/1,10 min) and determined PI-3 kinase activity in immunoprecipitates with specific non-crossreacting antibodies against insulinreceptor substrate (IRS 1/IRS 2) and against janus kinase (JAK 1 and JAK 2). While insulin-stimulated PI-3 kinase activity is detected in IRS-1 and IRS-2 immunoprecipitates, leptin-stimulated PI-3 kinase activity is found only in IRS-2 immunoprecipitates, suggesting that the leptin signal to PI-3 kinase occurs via IRS-2 and not IRS-1. Leptin-, but not insulin-stimulated PI-3 kinase activity is also detected in immunoprecipitates with antibodies against JAK-2, but not JAK-1. The data suggest that JAK-2 and IRS-2 couple the leptin signalling pathway to the insulin signalling chain. Since we have also detected leptin-stimulated tyrosine phosphorylation of JAK-2 and IRS-2 in C2C12 myotubes it can be assumed that leptin activates JAK-2 which induces tyrosine phosphorylation of IRS-2 leading to activation of PI-3 kinase. As we could not detect the long leptin receptor isoform in C2C12 myotubes we conclude that this signalling pathway is activated by a short leptin receptor isoform.

Saturated, but Not Unsaturated, Fatty Acids Induce Apoptosis of Human Coronary Artery Endothelial Cells via Nuclear Factor-κB Activation

High nonesterified fatty acid (NEFA) concentrations, as observed in the metabolic syndrome, trigger apoptosis of human umbilical vein endothelial cells. Since endothelial apoptosis may contribute to atherothrombosis, we studied the apoptotic susceptibility of human coronary artery endothelial cells (HCAECs) toward selected NEFAs and the underlying mechanisms. HCAECs were treated with single or combined NEFAs. Apoptosis was quantified by flow cytometry, nuclear factor κB (NFκB) activation by electrophoretic mobility shift assay, and secreted cytokines by enzyme-linked immunosorbent assay. Treatment of HCAECs with saturated NEFAs (palmitate and stearate) increased apoptosis up to fivefold (P < 0.05; n = 4). Unsaturated NEFAs (palmitoleate, oleate, and linoleate) did not promote apoptosis but prevented stearate-induced apoptosis (P < 0.05; n = 4). Saturated NEFA-induced apoptosis neither depended on ceramide formation nor on oxidative NEFA catabolism. However, NEFA activation via acyl-CoA formation was essential. Stearate activated NFκB and linoleate impaired stearate-induced NFκB activation. Pharmacological inhibition of NFκB and inhibitor of κB kinase (IKK) also blocked stearate-induced apoptosis. Finally, the saturated NEFA effect on NFκB was not attributable to NEFA-induced cytokine production. In conclusion, NEFAs display differential effects on HCAEC survival; saturated NEFAs (palmitate and stearate) are proapoptotic, and unsaturated NEFAs (palmitoleate, oleate, and linoleate) are antilipoapoptotic. Mechanistically, promotion of HCAEC apoptosis by saturated NEFA requires acyl-CoA formation, IKK, and NFκB activation.

Protein kinase C isoforms α, δ and θ require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells)

Summary Protein kinase C (PKC) isoforms are potentially important as modulators of the insulin signalling chain and could be involved in the pathogenesis of cellular insulin resistance. We have previously shown that phorbol ester stimulated PKC β1 and β2 as well as tumor necrosis factor-α (TNFα) stimulated PKC ɛ inhibit human insulin receptor (HIR) signalling. There is increasing evidence that the insulin receptor substrate-1 (IRS-1) is involved in inhibitory signals in insulin receptor function. The aim of the present study was to elucidate the role of IRS-1 in the inhibitory effects of protein kinase C on human insulin receptor function. HIR, PKC isoforms (α, β1, β2, γ, δ, ɛ, η, θ and ζ) and IRS-1 were coexpressed in human embryonic kidney (HEK) 293 cells. PKCs were activated by preincubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (CTPA) (10––7 mol/l) following insulin stimulation. While PKCs α, δ and θ were not inhibitory in HEK 293 cells which were transfected only with HIR and PKC, additional transfection of IRS-1 induced a strong inhibitory effect of these PKC isoforms being maximal for PKC θ (99 ± 1.8 % inhibition of insulin stimulated receptor autophosphorylation, n = 7, p < 0.001). No effect was seen with PKC γ, ɛ, ζ and η while the earlier observed insulin receptor kinase inhibition of PKC β2 was further augmented (91 ± 13 %, n = 7, p < 0.001 instead of 45 % without IRS-1). The strong inhibitory effect of PKC θ is accompanied by a molecular weight shift of IRS-1 (183 kDa vs 180 kDa) in the sodium dodecyl sulphate polyacrylamide gel. This can be reversed by alkaline phosphatase treatment of IRS-1 suggesting that this molecular weight shift is due to an increased phosphorylation of IRS-1 on serine or threonine residues. In summary, these data show that IRS-1 is involved in the inhibitory effect of the PKC isoforms α, β2, δ and θ and it is likely that this involves serine/threonine phosphorylation of IRS-1. [Diabetologia (1998) 41: 833–838]

Prevention by Protein Kinase C Inhibitors of Glucose-Induced Insulin-Receptor Tyrosine Kinase Resistance in Rat Fat Cells

Hyperglycemia causes insulin-receptor kinase (IRK) resistance in fat cells. We characterized the mechanism of IRK inhibition and studied whether it is the consequence of a glucose-induced stimulation of protein kinase C (PKC). Fat cells were incubated for 1 or 12 h in culture medium containing either a low- (5-mM) or high- (25-mM) glucose concentration. IRK was isolated, insulin binding was determined, and autophosphorylation was studied in vitro with [γ-32P]ATP or was determined by Western blotting with anti-phosphotyrosine antibodies. Substrate phosphorylation was investigated with the artificial substrate poly(Glu80-Tyr20). Partially purified insulin receptor from rat fat cells, which were cultured under high-glucose conditions for 1 or 12 h, showed no alteration of insulin binding but a reduced insulin effecton autophosphorylation (30 ± 7% of control) and poly(Glu80-Tyr20) phosphorylation (55.5 ± 9% of control). Lineweaver-Burk plots of the enzyme kinetics revealed, beside a reduced Vmax, and increased KM (from 30 μM to 80 μM) for ATP of IRK from high-glucose–treated cells. Because a similar inhibition pattern was earlier found for IRK from fat cells afteracute phorbol ester stimulation, we investigated whether activation of PKC might be the cause of the reduced IRK activity. We isolated PKC from the cytosol and the membrane fraction of high- and low-glucose fat cells and determined the diacylglycerol- and phospholipid-stimulated PKC activity toward the substrate histone. There was no significant change of cytosolic PKC; however, membrane-associated PKC activity was increased in high-glucose–treated cells. To evaluate whether the activation of PKC causes the inhibitors (H 7, staurosrine, and polymyxin B) and tested whether the effect of hyperglycemia was stillpresent when fat cells were pretreated with phorbolester (tetradecanoylphorbol acetate) for 24 h. Indeed, H 7, staurosporine, and polymyxin B blocked the inhibitory effect of hyperglycemia on IRK. Furthermore, in cells treated for 24 h with tetradecanoylphorhol acetate, no inhibitory effect of hyperglycemia was observed. This and the effect of the PKC inhibitors are consistent with a causal relationship between IRK inhibition and PKC activation

Troglitazone Prevents Glucose-Induced Insulin Resistance of Insulin Receptor in Rat-1 Fibroblasts

Troglitazone (CS045), a compound belonging to the thiazolidine diones, is being tested as a new oral antidiabetic agent. Evidence exists from animal studies and clinical trials with non-insulin-dependent diabetes mellitus patients that Troglitazone might reduce insulin resistance. The molecular mechanism of this effect is not understood. In this study, we investigated whether Troglitazone might interfere with the mechanism of glucose-induced insulin resistance. Several studies indicate that hyperglycemia reduces the kinase activity of the insulin receptor in different cell types. This effect is paralleled by translocation of several protein kinase C (PKC) isoforms, and it can be prevented by PKC inhibitors, which suggests that glucose-induced receptor desensitization is mediated by activation of PKC. We studied the effect of hyperglycemia on the insulin receptor kinase activity and its modulation by Troglitazone in rat-1 fibroblasts that stably overexpress the human insulin receptor. Before stimulation with insulin (10−7 M), cells were acutely exposed to hyperglycemic conditions in the absence or presence of Troglitazone (0.01–2 μ/ml). The insulin receptor was solubilized from a plasma membrane fraction or whole cell lysates, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted against antiphosphotyrosine and anti-insulin receptor β-subunit (CT 104) antibodies. Acute hyperglycemia (25 mM glucose) induced a significant inhibition of the insulin receptor kinase (IRK) activity within 30 min (inhibition to 30 ± 12.5% of maximal insulin-stimulated β-subunit phosphorylation, n = 9, P < 0.01).The glucose-induced inhibition of the insulin receptor kinase could be antagonized by a preincubation of the cells with Troglitazone before addition of 25 mM glucose (72 ± 13.5% of maximal insulin-stimulated 3-subunit phosphorylation after 20–30 min of preincubation at a concentration of 2 μg/ml Troglitazone, n = 9, P < 0.01). In addition, Troglitazone is also able to reverse the inhibition of the insulin receptor kinase caused by a prior glucose incubation. In parallel with the decrease of the IRK activity, the phosphorylation of the insulin receptor substrate-1 (IRS-1) was inhibited (inhibition to 45 ± 11.8% of maximal insulin-stimulated IRS-1 phosphorylation, n = 4, P ± 0.01), and this inhibition also could be reduced by Troglitazone (84 ± 15.7% of maximal insulin-stimulated IRS-1 phosphorylation in the presence of 2 μg/ml Troglitazone, n = 4, P ± 0.01). Taken together, the data suggest that Troglitazone is able to prevent and reverse hyperglycemia-induced insulin resistance of the insulin receptor in rat-1 fibroblasts. This mechanism might be relevant for the in vivo activity of this compound.

Medical Antihyperglycaemic Treatment of Type 2 Diabetes Mellitus * Update of the evidence-based guideline of the German Diabetes Association

Correspondence Prof. Stephan Matthaei Spokesperson for the Panel of Experts Quakenbr ü ck Diabetes Centre, DDG clinical diabetes centre Department of Diabetology, Metabolism and Endocrinology at the Christliches Krankenhaus Academic Teaching Hospital of the Hannover Medical University Danziger Stra ß e10 49610 Quakenbr ü ck Tel.: + + 49 / 5431 / 15 2830 Fax: + + 49 / 5431 / 15 2831 S.Matthaei@ckq-gmbh.de

Acute Hyperglycemia Provides an Insulin-Independent Inducer for GLUT4 Translocation in C2C12 Myotubes and Rat Skeletal Muscle

GLUT4 translocation and activation of glucose uptake in skeletal muscle can be induced by both physiological (i.e., insulin, nerve stimulation, or exercise) and pharmacological (i.e., phorbol ester) means. Recently, we demonstrated that high glucose levels may mimic the effects of phorbol esters on protein kinase C (PKC) and insulin receptor function (J Biol Chem 269:3381–3386, 1994). In this study, we tested whether the previously described effects of phorbol esters on translocation of GLUT4 in myotubes in culture and also in rat skeletal muscle might be mimicked by glucose. We found that stimulation of C2C12 myotubes with both insulin (10–7) mol/l, 5 min) and glucose (25 mmol/l, 10 min) induces a comparable increase of the GLUT4 content in the plasma membrane. To test whether this effect occurs in intact rat skeletal muscle as well, two different model systems were used. As an in vitro model, isolated rat hindlimbs were perfused for 80 min with medium containing 6 mmol/l glucose ± insulin (1.6 × 10–9 mmol/l, 40 min) or 25 mmol/l glucose. As an in vivo model, acute hyperglycemia (> 11 mmol/l glucose, 20 min) was induced in Wistar rats by intraperitoneal injection of glucose under simultaneous suppression of the endogenous insulin release by injection of somatostatin. In both models, subcellular fractions were prepared from hindlimb skeletal muscle, and plasma membranes were characterized by the enrichment of the marker enzyme α1 Na+ -K+ -ATPase. Acute hyperglaycemia in vivo (n = 5) and in vitro (n = 6) induced an increases of GLUT4 content in the α1 Na+ -K+ -ATPase–enriched fraction (in vivo, 2.45 ± 0.47-fold increase to basal [mean ±SE]; in vitro, 1.71 ± 0.14-fold increase to basal), which was Quantitatively similar to that obtained after insulin treatment (in vivi, 2.35 ± 0.62-fold increase to basal; in vitro, 1.91 ± 0.21-fold increase to basal). Glucose-in-duced GLUT4 translocation in myotubes was prevented by prior addition of the PKC inhibitor 1-(5-isoquinolinyl-by prior addition of the PKC inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine; in rat skeletal muscle,GLUT4 translocation was paralleled by a translocation of PKC β, while no effect on PKC α, δ, ∊, and ζ was observed. These results suggest that glucose-induced GLUT4 translocation might represenet an insulin-independent autoregulatory mechanism of the skeletal muscle to rapidly increase glucose uptake in acute hyperglycemia. Activation of PKC β might be involved in this mechanisum in skeletal muscle. GLUT4 translocation was paralleled by a translocation of PKC β, while no effect on PKC α, δ, μ, and ¶ was observed. These results suggest that glucose-induced GLUT4 translocation might represent an insulin-independent autoregulatory mechanism of the skeletal muscle to rapidly increase glucose uptake in acute hyperglycemia. Activation of PKC P might be involved in this mechanism in skeletal muscle.

Altered pattern of insulin receptor isotypes in skeletal muscle membranes of type 2 (non-insulin-dependent) diabetic subjects

SummaryThe human insulin receptor exists in two isoforms (HIR-A α-subunit 719 amino acids and HIR-B α-subunit 731 amino acids) which are generated by alternative splicing of a small exon and display distinct patterns of tissue-specific expression. Using the polymerase chain reaction we have recently shown that skeletal muscle of non-diabetic individuals contains predominantly mRNA encoding HIR-A while in skeletal muscle derived from subjects with Type 2 (non-insulin-dependent) diabetes mellitus similar amounts of each mRNA are expressed. We used a polyclonal antibody which discriminates between HIR-A and HIR-B to assess the isoform expression at the protein level. The antibody showed clearly distinct displacement of insulin binding in skeletal muscle membranes of non-diabetic subjects compared to Type 2 diabetic subjects (displacement of specific 125I-insulin binding: 13 non-diabetic subjects 70.0%±14.34, 12 Type 2 diabetic subjects 32.6%±17.45). A control antibody which does not discriminate between both isoforms showed similar displacement of 125I-insulin in membranes of non-diabetic and Type 2 diabetic subjects. These data suggest that the altered expression of receptor isotype mRNA in the skeletal muscle of Type 2 diabetic subjects leads to an altered receptor isoform pattern in the plasma membrane. While skeletal muscle membranes of non-diabetic subjects contain predominantly HIR-A, membranes of Type 2 diabetic subjects show an increased level of HIR-B in addition to HIR-A.

Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms β2 and θ

Aims/hypothesis. Inhibition of the signalling function of the human insulin receptor (HIR) is one of the principle mechanisms which induce cellular insulin resistance. It is speculated that serine residues in the insulin receptor β-subunit are involved in receptor inhibition either as inhibitory phosphorylation sites or as part of receptor domains which bind inhibitory proteins or tyrosine phosphatases. As reported earlier we prepared 16 serine to alanine point mutations of the HIR and found that serine to alanine mutants HIR-994 and HIR-1023/25 showed increased tyrosine autophosphorylation when expressed in human embryonic kidney (HEK) 293 cells. In this study we examined whether these mutant receptors have a different susceptibility to inhibition by serine kinases or an altered tyrosine kinase activity.¶Methods. Tyrosine kinase assay and transfection studies.¶Results. In an in vitro kinase assay using IRS-1 as a substrate we could detect a higher intrinsic tyrosine kinase activity of both receptor constructs. Additionally, a higher capacity to phosphorylate the adapter protein Shc in intact cells was seen. To test the inhibition by serine kinases, the receptor constructs were expressed in HEK 293 cells together with IRS-1 and protein kinase C isoforms β2 and θ. Phorbol ester stimulation of these cells reduced wild-type receptor autophosphorylation to 58 % or 55 % of the insulin simulated state, respectively. This inhibitory effect was not observed with HIR-994 and HIR-1023/25, although all other tested HIR mutants showed similar inhibition induced by protein kinase C.¶Conclusion/interpretation. The data suggest that the HIR-domain which contains the serine residues 994 and 1023/25 is important for the inhibitory effect of protein kinase C isoforms β2 and θ on insulin receptor autophosphorylation. [Diabetologia (2000) 43: 443–449]