Matilde Caruso

PED/PEA-15 gene controls glucose transport and is overexpressed in type 2 diabetes mellitus.

We have used differential display to identify genes whose expression is altered in type 2 diabetes thus contributing to its pathogenesis. One mRNA is overexpressed in fibroblasts from type 2 diabetics compared with non‐diabetic individuals, as well as in skeletal muscle and adipose tissues, two major sites of insulin resistance in type 2 diabetes. The levels of the protein encoded by this mRNA are also elevated in type 2 diabetic tissues; thus, we named it PED for phosphoprotein enriched in diabetes. PED cloning shows that it encodes a 15 kDa phosphoprotein identical to the protein kinase C (PKC) substrate PEA‐15. The PED gene maps on human chromosome 1q21–22. Transfection of PED/PEA‐15 in differentiating L6 skeletal muscle cells increases the content of Glut1 transporters on the plasma membrane and inhibits insulin‐stimulated glucose transport and cell‐surface recruitment of Glut4, the major insulin‐sensitive glucose transporter. These effects of PED overexpression are reversed by blocking PKC activity. Overexpression of the PED/PEA‐15 gene may contribute to insulin resistance in glucose uptake in type 2 diabetes.

PED/PEA-15: an anti-apoptotic molecule that regulates FAS/TNFR1-induced apoptosis

PED/PEA-15 is a recently cloned 15 kDa protein possessing a death effector domain (DED). In MCF-7 and HeLa cells, a fivefold overexpression of PED/PEA-15 blocked FasL and TNFα apoptotic effects. This effect of PED overexpression was blocked by inhibition of PKC activity. In MCF-7 and HeLa cell lysates, PED/PEA-15 co-precipitated with both FADD and FLICE. PED/PEA-15-FLICE association was inhibited by overexpression of the wild-type but not of a DED-deletion mutant of FADD. Simultaneous overexpression of PED/PEA-15 with FADD and FLICE inhibited FADD-FLICE co-precipitation by threefold. Based on cleavage of the FLICE substrate PARP, this inhibitory effect was paralleled by a threefold decline in FLICE activation in response to TNF-α. TNFα, in turn, reduces PED association with the endogenous FADD and FLICE of the cells. Thus, PED/PEA-15 is an endogenous protein inhibiting FAS and TNFR1-mediated apoptosis. At least in part, this function may involve displacement of FADD-FLICE binding through the death effector domain of PED/PEA-15.

Protein Kinase C (PKC)-α Activation Inhibits PKC-ζ and Mediates the Action of PED/PEA-15 on Glucose Transport in the L6 Skeletal Muscle Cells

Overexpression of the PED/PEA-15 protein in muscle and adipose cells increases glucose transport and impairs further insulin induction. Like glucose transport, protein kinase C (PKC)-alpha and -beta are also constitutively activated and are not further stimulatable by insulin in L6 skeletal muscle cells overexpressing PED (L6(PED)). PKC-zeta features no basal change but completely loses insulin sensitivity in L6(PED). In these cells, blockage of PKC-alpha and -beta additively returns 2-deoxy-D-glucose (2-DG) uptake to the levels of cells expressing only endogenous PED (L6(WT)). Blockage of PKC-alpha and -beta also restores insulin activation of PKC-zeta in L6(PED) cells, with that of PKC-alpha sixfold more effective than PKC-beta. Similar effects on 2-DG uptake and PKC-zeta were also achieved by 50-fold overexpression of PKC-zeta in L6(PED). In L6(WT), fivefold overexpression of PKC-alpha or -beta increases basal 2-DG uptake and impairs further insulin induction with no effect on insulin receptor or insulin receptor substrate phosphorylation. In these cells, overexpression of PKC-alpha blocks insulin induction of PKC-zeta activity. PKC-beta is 10-fold less effective than PKC-alpha in inhibiting PKC-zeta stimulation. Expression of the dominant-negative K(281)-->W PKC-zeta mutant simultaneously inhibits insulin activation of PKC-zeta and 2-DG uptake in the L6(WT) cells. We conclude that activation of classic PKCs, mainly PKC-alpha, inhibits PKC-zeta and may mediate the action of PED on glucose uptake in L6 skeletal muscle cells.

In NIH-3T3 Fibroblasts, Insulin Receptor Interaction with Specific Protein Kinase C Isoforms Controls Receptor Intracellular Routing*

Insulin increased protein kinase C (PKC) activity by 2-fold in both membrane preparations and insulin receptor (IR) antibody precipitates from NIH-3T3 cells expressing human IRs (3T3hIR). PKC-α, -δ, and -ζ were barely detectable in IR antibody precipitates of unstimulated cells, while increasing by 7-, 3.5-, and 3-fold, respectively, after insulin addition. Preexposure of 3T3hIR cells to staurosporine reduced insulin-induced receptor coprecipitation with PKC-α, -δ, and -ζ by 3-, 4-, and 10-fold, respectively, accompanied by a 1.5-fold decrease in insulin degradation and a similar increase in insulin retroendocytosis. Selective depletion of cellular PKC-α and -δ, by 24 h of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure, reduced insulin degradation by 3-fold and similarly increased insulin retroendocytosis, with no change in PKC-ζ. In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs (3T3Mut), insulin-induced coprecipitation of PKC-α, -δ, and -ζ with the IR was reduced by 10-, 7-, and 3-fold, respectively. Similar to the 3T3hIR cells chronically exposed to TPA, untreated 3T3Mut featured a 3-fold decrease in insulin degradation, with a 3-fold increase in intact insulin retroendocytosis. Thus, in NIH-3T3 cells, insulin elicits receptor interaction with multiple PKC isoforms. Interaction of PKC-α and/or -δ with the IR appears to control its intracellular routing.

DIFFERENTIAL ROLE OF INSULIN RECEPTOR SUBSTRATE (IRS)-1 AND IRS-2 IN L6 SKELETAL MUSCLE CELLS EXPRESSING THE ARG1152 GLN INSULIN RECEPTOR

In L6 muscle cells expressing the Arg1152 → Gln insulin receptor (Mut), basal tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was increased by 35% compared with wild-type cells (WT). Upon exposure to insulin, IRS-1 phosphorylation increased by 12-fold in both the Mut and WT cells. IRS-2 was constitutively phosphorylated in Mut cells and not further phosphorylated by insulin. The maximal phosphorylation of IRS-2 in basal Mut cells was paralleled by a 4-fold increased binding of the kinase regulatory loop binding domain of IRS-2 to the Arg1152 → Gln receptor. Grb2 and phosphatidylinositol 3-kinase association to IRS-1 and IRS-2 reflected the phosphorylation levels of the two IRSs. Mitogen-activated protein kinase activation and [3H]thymidine incorporation closely correlated with IRS-1 phosphorylation in Mut and WT cells, while glycogen synthesis and synthase activity correlated with IRS-2 phosphorylation. The Arg1152 → Gln mutant did not signal Shc phosphorylation or Shc-Grb2 association in intact L6 cells, while binding Shc in a yeast two-hybrid system and phosphorylating Shc in vitro. Thus, IRS-2 appears to mediate insulin regulation of glucose storage in Mut cells, while insulin-stimulated mitogenesis correlates with the activation of the IRS-1/mitogen-activated protein kinase pathway in these cells. IRS-1 and Shc-mediated mitogenesis may be redundant in muscle cells.

In L6 skeletal muscle cells, glucose induces cytosolic translocation of protein kinase C-alpha and trans-activates the insulin receptor kinase.

In L6 skeletal muscle cells expressing human insulin receptors (L6hIR), exposure to 25 mm glucose for 3 min induced a rapid 3-fold increase in GLUT1 and GLUT4 membrane translocation and glucose uptake. The high glucose concentration also activated the insulin receptor kinase toward the endogenous insulin receptor substrates (IRS)-1 and IRS-2. At variance, in L6 cells expressing kinase-deficient insulin receptors, the exposure to 25 mm glucose elicited no effect on glucose disposal. In the L6hIR cells, the acute effect of glucose on insulin receptor kinase was paralleled by a 2-fold decrease in both the membrane and the insulin receptor co-precipitated protein kinase C (PKC) activities and a 3-fold decrease in receptor Ser/Thr phosphorylation. Western blotting of the receptor precipitates with isoform-specific PKC antibodies revealed that the glucose-induced decrease in membrane- and receptor-associated PKC activities was accounted for by dissociation of PKCα but not of PKCβ or -δ. This decrease in PKCα was paralleled by a similarly sized increase in cytosolic PKCα. In intact L6hIR cells, inhibition of PKCα expression by using a specific antisense oligonucleotide caused a 3-fold increase in IRS phosphorylation by the insulin receptor. This effect was independent of insulin and accompanied by a 2.5-fold increase in glucose disposal by the cells. Thus, in the L6 skeletal muscle cells, glucose acutely regulates its own utilization through the insulin signaling system, independent of insulin. Glucose autoregulation appears to involve PKCα dissociation from the insulin receptor and its cytosolic translocation.

In Skeletal Muscle, Glucose Storage and Oxidation Are Differentially Impaired by the IR1152 Mutant Receptor

L6 myotubes expressing the constitutively active Arg1152→Gln insulin receptor (L61152) featured a 31% increased glucose consumption as compared with L6 cells expressing wild-type receptors (L6WT). However, insulin treatment decreased glucose consumption of the mutant cells by 20% while increasing that of the L6WT by 30%. In the L6WT, insulin elicited a significant increase in glucose transport and GLUT1 and GLUT4 plasma membrane expression, while in the L61152, all of these functions were constitutively activated and not further stimulated by insulin. Similarly, glycogen content and glycogen synthase activity were increased by 80 and 125%, respectively, in the L61152 versus the L6WT and unaffected by insulin (while a 2-fold increase was measured in insulin-exposed L6WT). Glucose oxidation and pyruvate dehydrogenase activity were also 25% higher in the mutant compared with the L6WT. However, in the L61152, both functions decreased by 35% in response to insulin (while increasing by 60 and 80%, respectively, in the L6WT). Similarly as in the L61152, in vivo, forearm glucose uptake in IR1152 patients was 2-fold higher than in control subjects. This difference was not accounted for by higher plasma glucose levels. We conclude that, in skeletal muscle, glucose storage and oxidation are differentially impaired by the expression of IR1152, suggesting that their regulation by insulin involves divergent signaling pathways. Muscle expression of IR1152 may contribute to impairing glucose tolerance in IR1152 individuals.

The IR1152 mutant insulin receptor selectively impairs insulin action in skeletal muscle but not in liver.

In patients harboring the IR1152 mutant insulin receptor, hepatic glucose production was normally suppressed by insulin. Hepatocytes without the insulin receptor gene and expressing IR1152 (Hep(MUT)) also showed normal insulin suppression of glucose production and full insulin response of glycogen synthase. In contrast, expression of the IR1152 mutant in skeletal muscle maximally increased glucose uptake and storage, preventing further insulin stimulation. IRS-1 phosphorylation was normally stimulated by insulin in both intact Hep(MUT) and L6 skeletal muscle cells expressing the IR1152 mutant (L6(MUT)). At variance, IRS-2 phosphorylation exhibited high basal levels with no further insulin-dependent increase in L6(MUT) but almost normal phosphorylation, both basal and insulin-stimulated, in the Hep(MUT) cells. In vitro, IR1152 mutant preparations from both the L6(MUT) and the Hep(MUT) cells exhibited increased basal and no insulin-stimulated phosphorylation of IRS-2 immobilized from either muscle or liver cells. IR1152 internalization in liver and muscle cells closely paralleled the ability of this mutant to phosphorylate IRS-2 in vivo in these cells. Block of receptor internalization (wild-type and mutant) in the liver and muscle cells also inhibited IRS-2, but not IRS-1, phosphorylation. Thus, the mechanisms controlling insulin receptor internalization differ in liver and skeletal muscle cells and may enable IR1152 to control glucose metabolism selectively in liver. In both cell types, receptor internalization seems necessary for IRS-2 but not IRS-1 phosphorylation.

Vanadate regulates the insulin mitogenic effect by modulating SHP-2 association with insulin receptor substrate 1 in JAr human choriocarcinoma cells

Maternal hyperglycemia alters placental glucose metabolism and induces placental hypercellularity. In this study we investigated ,in JAr cells ,the effect of a protein tyrosine phosphatase inhibitor ,vanadate, on the insulin receptor substrate 1 (IRS1)-mitogen-activated protein kinase (MAPK) pathway and on cell proliferation in the presence of normal or high glucose concentration. When JAr cells were cultured in the presence of 25 mmol/l glucose ,treatment with vanadate completely prevented SHP-2 association with IRS1. However ,vanadate treatment reverted the effect of high glucose on basal and insulin-stimulated insulin receptor and IRS1 phosphorylation. Similar effects were observed on MAPK activation. These events determined a related modification in cell proliferation. Indeed ,after high glucose and vanadate treatment ,thymidine incorporation levels were comparable to those observed in the presence of normal glucose concentration and in the absence of vanadate. Therefore ,in JAr cells ,vanadate exerts an inhibitory effect on cell proliferation. This action is related to a modulation of the SHP-2 association with IRS1 that in turn might regulate the phosphorylation state of the main substrates involved in mitogenesic signaling of the insulin receptor.

Expression of Concern. The IR 1152 Mutant Insulin Receptor Selectively Impairs Insulin Action in Skeletal Muscle but Not in Liver. Diabetes 2000;49:1194–1202. DOI: https://doi.org/10.2337/diabetes.49.7.1194. PMID: 10909978

On the basis of the recommendation of the American Diabetes Association’s Panel on Ethical Scientific Programs (ESP), the American Diabetes Association, the publisher of Diabetes, is issuing this expression of concern to alert readers to questions about the reliability of the data in the above-cited article. After readers of the journal contacted Diabetes about potentially duplicated images in the article, the ESP reviewed the following issues: