Joanne Mushack

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.

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]

Leptin down-regulates insulin action through phosphorylation of serine-318 in insulin receptor substrate 1

Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine‐318 as a protein kinase C (PKC)‐dependent phosphorylation site in Irs1 (Ser‐318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity‐related insulin resistance, promotes phosphorylation of Ser‐318 in Irs1 by a janus kinase 2, Irs2, and PKC‐dependent pathway. Mutation of Ser‐318 to alanine abrogates the inhibitory effect of leptin on insulin‐induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser‐318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet‐induced obesity Ser‐318 phosphorylation levels were already up‐regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser‐318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser‐318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser‐318 in Irs1 mediates the inhibitory signal of leptin on the insulin‐signaling cascade in obese subjects.—Hennige A. M., Stefan N., Kapp K., Lehmann R., Weigert C., Beck A., Moeschel K., Mushack J., Schleicher E., and Häring H. U. Leptin down‐regulates insulin action through phosphorylation of serine‐318 in insulin receptor substrate 1. FASEB J. 20, E381–E389 (2006)

Protein kinase C (PKC) ϵ enhances the inhibitory effect of TNFα on insulin signaling in HEK293 cells

Recently we have shown that PKC β1 and β2 are able to inhibit the tyrosine kinase activity of the human insulin receptor (HIR). Now we have investigated whether a distinct PKC isoform might be involved in the inhibitory effect of TNFα on insulin signaling in HEK293 cells. TNFα induces a rapid translocation of the PKC isoform ϵ (TNFα 10−9 M, maximal effect within 5–10 min) in rat‐1 fibroblasts, while no effect occurred on other isoforms. Cotransfection of HIR with PKC ϵ did not significantly reduce the insulin stimulated receptor kinase activity; however, when cells were incubated with TNFα for 10 min (10−9 M) a 62±17% (n=5) inhibition of the insulin receptor kinase activity was observed which was significantly (P<0.01) higher than that observed in cells which were not transfected with PKC (32±11.5%, n=5). The data suggest that translocation of PKC ϵ induced by TNFα enables this PKC isoform to interact with insulin signaling and to inhibit the insulin receptor kinase activity.

Subcellular distribution of GLUT 4 in the skeletal muscle of lean type 2 (non-insulin-dependent) diabetic patients in the basal state

SummaryInsulin resistance of the skeletal muscle is a key feature of Type 2 (non-insulin-dependent) diabetes mellitus. To determine whether a decrease of glucose carrier proteins or an altered subcellular distribution of glucose transporters might contribute to the pathogenesis of the insulin resistant state, we measured glucose transporter numbers in membrane fractions of gastrocnemius muscle of 14 Type 2 diabetic patients and 16 non-diabetic control subjects under basal conditions. Cytochalasin-B binding and immunoblotting with antibodies against transporter-subtypes GLUT 1 and GLUT 4 were applied. The cytochalasin-B binding values (pmol binding sites/g muscle) found in a plasma membrane enriched fraction, high and low density membranes of both groups (diabetic patients and non-diabetic control subjects) suggested a reduced number of glucose transporters in the plasma membranes of the diabetic patients compared to the control subjects (diabetic patients: 1.47 ± 1.01, control subjects: 3.61 ± 2.29,p ≤ 0.003). There was no clear difference in cytochalasin-B binding sites in high and low density membranes of both groups (diabetic patients: high density membranes 3.76 ± 1.82, low density membranes: 1.67 ± 0.81; control subjects: high density membranes 5.09 ± 1.68, low density membranes 1.45 ± 0.90). By Western blotting analysis we determined the distribution of the glucose transporter sub-types GLUT 1 and GLUT 4 in the plasma membrane enriched fraction and low density membranes of seven patients of each group. In agreement with the cytochalasin-B binding data and despite a high variance within one group, the results show a clear decrease of GLUT 4 in the plasma membrane enriched fraction of diabetic patients compared to control subjects. In contrast, we found no difference in the distribution of GLUT 1 in diabetic patients and control subjects. In conclusion, despite a high variance of glucose transporter numbers in the skeletal muscle of different individuals fractionation of muscle samples clearly suggests that the number of GLUT 4 is reduced in the plasma membrane fraction of skeletal muscle of lean diabetic patients in the basal state.

Stimulation of phospholipase C activity by insulin is mediated by both isotypes of the human insulin receptor.

The human insulin receptor exists in two isoforms, HIR-A and HIR-B. We studied whether both insulin receptor isotypes are able to mediate an insulin signal to phospholipase C. Plasma membranes were prepared from rat-1 fibroblasts transfected either with HIR-A or HIR-B and insulin stimulated PIP-hydrolysis was determined. We found that insulin stimulates PIP-hydrolysis in a similar dose dependent manner and to a similar extent in plasma membranes expressing HIR-A and HIR-B. These data suggest that both receptor isoforms are equally able to activate phospholipase-C.

TPA inhibits insulin stimulated PIP hydrolysis in fat cell membranes: Evidence for modulation of insulin dependent phospholipase C by proteinkinase C

Proteinkinase-C (PKC) stimulating phorbolesters induce in vitro insulin resistance of isolated adipocytes. This effect might be explained by an inhibition of insulin signal transduction at the level of the insulin receptor kinase. There is now some evidence that a phospholipase C is a potential candidate as a signal transducer at the postreceptor level. In order to determine whether phorbol esters might inhibit insulin signalling also at the level of a phospholipase C, we studied the insulin dependent [3H] phosphatidylinositol 4-monophosphate (PIP) hydrolysis of fat cell membranes. PIP hydrolysis was measured after in vitro stimulation with and without insulin. Insulin stimulated PIP hydrolysis in a dose dependent way. When plasma membranes from phorbolester (TPA) treated fat cells were used, this insulin stimulated phospholipase C activity was suppressed, provided, membranes have been prepared in a buffer containing serine phosphatase inhibitors. These data suggest that fat cell membranes contain an insulin dependent phospholipase C which is inhibited by TPA most likely via serine phosphorylation through proteinkinase C.

The phorbol ester TPA induces a translocation of the insulin sensitive glucose carrier (GLUT4) in fat cells

Insulin activates the glucose transport in isolated fat cells through a translocation of the insulin sensitive glucose carrier subtype (GLUT4) and by activation of glucose carriers in the plasma membrane. Protein kinase C stimulating phorbol esters are able to mimick partially the insulin effect on glucose transport. In order to determine whether this phorbol ester effect occurs through a translocation of the insulin sensitive glucose carrier (GLUT4) we used a monoclonal antibody against GLUT4 to determine its distribution in subcellular fractions of rat adipocytes. We found that the phorbol ester TPA is able to increase the amount of GLUT4 in the plasma membrane fraction about two-fold.

A 973 valine to methionine mutation of the human insulin receptor: Interaction with insulin-receptor substrate-1 and Shc in HEK 293 cells

Summary A population-based study in the Netherlands has recently demonstrated that a mutation of the human insulin receptor (HIR-973 valine to methionine) is associated with hyperglycaemia and an increased prevalence of non-insulin-dependent diabetes mellitus (NIDDM). The aim of the present study was to assess whether this mutation leads to a functional alteration of the insulin receptor. We prepared the HIR-973 mutant by in vitro mutagenesis. This mutant was transiently overexpressed in HEK 293 cells either alone or together with insulin-receptor substrate-1 (IRS-1) or Shc. Insulin stimulated autophosphorylation, phosphorylation of the substrates IRS-1 and Shc as well as activation of phosphatidylinositol-3 (PI3)-kinase were studied. Autophosphorylation of HIR-973 and its susceptibility to hyperglycaemia induced inhibition was not different from HIR-wt. Human insulin receptor with a juxtamembrane deletion HIR-ΔJM which is known to impair HIR/IRS-1 interaction was used as control. While the HIR-ΔJM induces a reduced IRS-1 phosphorylation HIR-973 showed even a slightly increased ability to phosphorylate IRS-1 (n = 7, 115 % of control, p < 0.01). Shc phosphorylation was only mediated by HIR-wt and HIR-973 but not by HIR-ΔJM. Again a tendency to higher phosphorylation of Shc was seen with HIR-973 (n = 7, 109 % of control, NS). When PI3-kinase activity was measured in IRS-1 precipitates similar activity was found for HIR-wt and HIR-973 whereas PI3-kinase stimulation was reduced with HIR-ΔJM. In summary, the data suggest that HIR-973 does not impair the first steps of the insulin signalling cascade. It is therefore unlikely that this mutation may cause cellular insulin resistance. The close vicinity of this mutation to insulin receptor domains which are involved in IRS-1 and Shc binding may, however, alter the interaction of the insulin receptor with these substrates. This could explain the slightly increased insulin effect on tyrosine phosphorylation of these docking proteins. These characteristics of HIR-973 might have a compensatory function of impaired signal transduction further downstream of the signalling chain in this specific subgroup of NIDDM patients. [Diabetologia (1997) 40: 1135–1140]