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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 B/akt binds and phosphorylates PED/PEA-15, stabilizing its antiapoptotic action

ABSTRACT The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser116. In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser116 PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser116. In addition, a mutant of PED/PEA-15 featuring the substitution of Ser116→Gly (PEDS116→G) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also induced phosphorylation of PED/PEA-15 at Ser116. Based on pull-down and coprecipitation assays, PED/PEA-15 specifically bound Akt, independently of Akt activity. Serum activation of Akt as well as BAD phosphorylation by Akt showed no difference in 293 cells transfected with PED/PEA-15 and in untransfected cells (which express no endogenous PED/PEA-15). However, the antiapoptotic action of PED/PEA-15 was almost twofold reduced in PEDS116→G compared to that in PED/PEA-15WT cells. PED/PEA-15 stability closely paralleled Akt activation by serum in 293 cells. In these cells, the nonphosphorylatable PEDS116→G mutant exhibited a degradation rate threefold greater than that observed with wild-type PED/PEA-15. In the U373MG glioma cells, blocking Akt also reduced PED/PEA-15 levels and induced sensitivity to tumor necrosis factor-related apoptosis-inducing ligand apoptosis. Thus, phosphorylation by Akt regulates the antiapoptotic function of PED/PEA-15 at least in part by controlling the stability of PED/PEA-15. In part, Akt survival signaling may be mediated by PED/PEA-15.

In Skeletal Muscle Advanced Glycation End Products (AGEs) Inhibit Insulin Action and Induce the Formation of Multimolecular Complexes Including the Receptor for AGEs

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Cα (PKCα). Here we show that HGA-induced PKCα activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCα in HGA-treated L6 cells. A direct interaction of PKCα with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCα co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCα activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCα. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCα.

The cannabinoid CB1 receptor antagonist rimonabant stimulates 2-deoxyglucose uptake in skeletal muscle cells by regulating the expression of phosphatidylinositol-3-kinase.

The endocannabinoid system regulates food intake, energy, and glucose metabolism at both central and peripheral levels. We have investigated the mechanism by which it may control glucose uptake in skeletal muscle cells. Detectable levels of the cannabinoid receptor type 1 (CB1) were revealed in L6 cells. Exposure of differentiated L6 myotubes to the CB1 antagonist rimonabant (SR141716) selectively increased 2-deoxyglucose uptake (2-DG) in a time- and dose-dependent manner. A similar effect was induced by genetic silencing of CB1 by small interfering RNA. Protein expression profiling revealed that both the regulatory p85 and the catalytic p110 subunits of the phosphatidylinositol-3-kinase (PI3K) were increased by SR141716. No significant change in the cellular content of other known molecules regulating PI3K was observed. However, phosphoinositide-dependent kinase-1, Akt/protein kinase B, and protein kinase Cζ activities were rapidly induced after SR141716 treatment of L6 cells in a PI3K-dependent manner. The stimulatory effect of SR141716 on PI3K expression and activity was largely prevented by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of the cAMP-dependent protein kinase. Moreover, SR141716-stimulated 2-DG uptake was blunted by the coincubation either with H-89 or with the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), both in L6 cells and in mouse primary myocytes. Thus, modulation of CB1 regulates glucose uptake at the level of the PI3K signaling system in skeletal muscle cells. Interfering with CB1 signaling may therefore ameliorate glucoregulatory functions in peripheral tissues.

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.

Age-Related Impairment in Insulin Release: The Essential Role of β2-Adrenergic Receptor

In this study, we investigated the significance of β2-adrenergic receptor (β2AR) in age-related impaired insulin secretion and glucose homeostasis. We characterized the metabolic phenotype of β2AR-null C57Bl/6N mice (β2AR−/−) by performing in vivo and ex vivo experiments. In vitro assays in cultured INS-1E β-cells were carried out in order to clarify the mechanism by which β2AR deficiency affects glucose metabolism. Adult β2AR−/− mice featured glucose intolerance, and pancreatic islets isolated from these animals displayed impaired glucose-induced insulin release, accompanied by reduced expression of peroxisome proliferator–activated receptor (PPAR)γ, pancreatic duodenal homeobox-1 (PDX-1), and GLUT2. Adenovirus-mediated gene transfer of human β2AR rescued these defects. Consistent effects were evoked in vitro both upon β2AR knockdown and pharmacologic treatment. Interestingly, with aging, wild-type (β2AR+/+) littermates developed impaired insulin secretion and glucose tolerance. Moreover, islets from 20-month-old β2AR+/+ mice exhibited reduced density of β2AR compared with those from younger animals, paralleled by decreased levels of PPARγ, PDX-1, and GLUT2. Overexpression of β2AR in aged mice rescued glucose intolerance and insulin release both in vivo and ex vivo, restoring PPARγ/PDX-1/GLUT2 levels. Our data indicate that reduced β2AR expression contributes to the age-related decline of glucose tolerance in mice.

Overexpression of the ped/pea-15 Gene Causes Diabetes by Impairing Glucose-Stimulated Insulin Secretion in Addition to Insulin Action

ABSTRACT Overexpression of the ped/pea-15 gene is a common feature of type 2 diabetes. In the present work, we show that transgenic mice ubiquitously overexpressing ped/pea-15 exhibited mildly elevated random-fed blood glucose levels and decreased glucose tolerance. Treatment with a 60% fat diet led ped/pea-15 transgenic mice to develop diabetes. Consistent with insulin resistance in these mice, insulin administration reduced glucose levels by only 35% after 45 min, compared to 70% in control mice. In vivo, insulin-stimulated glucose uptake was decreased by almost 50% in fat and muscle tissues of the ped/pea-15 transgenic mice, accompanied by protein kinase Cα activation and block of insulin induction of protein kinase Cζ. These changes persisted in isolated adipocytes from the transgenic mice and were rescued by the protein kinase C inhibitor bisindolylmaleimide. In addition to insulin resistance, ped/pea-15 transgenic mice showed a 70% reduction in insulin response to glucose loading. Stable overexpression of ped/pea-15 in the glucose-responsive MIN6 beta-cell line also caused protein kinase Cα activation and a marked decline in glucose-stimulated insulin secretion. Antisense block of endogenous ped/pea-15 increased glucose sensitivity by 2.5-fold in these cells. Thus, in vivo, overexpression of ped/pea-15 may lead to diabetes by impairing insulin secretion in addition to insulin action.

The insulin receptor substrate (IRS)-1 recruits phosphatidylinositol 3-kinase to Ret: evidence for a competition between Shc and IRS-1 for the binding to Ret.

Tyrosine 1062 of Ret, which represents an intracytoplasmic docking site for multiple signaling molecules, is essential for Ret-mediated activation of phosphatidylinositol 3-Kinase (PI3-K). PI3-K, in turn, has been implicated in inducing cell survival and neoplastic transformation mediated by Ret. We have examined the mechanisms by which Ret stimulates PI3-K. Here we show that the Insulin Receptor Substrate-1 (IRS-1) is tyrosine phosphorylated and associated with the p85 regulatory subunit of PI3-K in response to Ret activation. IRS-1 coimmunoprecipitates with Ret and co-expression of IRS-1 results in the potentiation of Ret-mediated activation of Akt(PKB), a bona fide effector of PI3-K. The association with the PTB domain of IRS-1 depends on the phosphorylation of tyrosine 1062 of Ret. The deletion of asparagine 1059 (delN1059) and the substitution of leucine 1061 (L1061P), two Ret mutations identified in families affected by congenital megacolon (Hirschsprungs disease), impair the binding of IRS-1 to Ret as well as Ret-mediated Akt(PKB) stimulation. Finally, we show that Shc, which was previously identified as another ligand of Y1062 of Ret, competes with IRS-1 for the binding to Ret pY1062. All together, these findings suggest that IRS-1 is a component of the signaling pathway which leads to Ret-mediated PI3-K activation, a pathway which can be targeted by Hirschsprung-associated Ret mutations. The alternative binding of Shc and IRS-1 to Ret pY1062 can be a system to modulate the activation of different intracellular signaling pathways and to elicit different biological responses following Ret activation.

Frontiers: PED/PEA-15, a multifunctional protein controlling cell survival and glucose metabolism

PED/PEA-15 is a 15-kDa ubiquitously expressed protein implicated in a number of fundamental cellular functions, including apoptosis, proliferation, and glucose metabolism. PED/PEA-15 lacks enzymatic function and serves mainly as a molecular adaptor. PED/PEA-15 is an endogenous substrate for protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CAM kinase II), and Akt. In particular, PKC phosphorylates PED/PEA-15 at Ser(104) and CAM kinase II or Akt at Ser(116), modifying its stability. Evidence obtained over the past 10 years has indicated that PED/PEA-15 regulates cell survival by interfering with both intrinsic and extrinsic apoptotic pathways. In addition, it may also control cell proliferation by interfering with ERK1/2-mediated pathways. Indeed, PED/PEA-15 has been identified as an ERK1/2 interactor, which modifies its subcellular localization and targeting to a specific subset of substrates. Increased PED/PEA-15 levels may affect tumorigenesis and cancer progression as well as sensitivity to anticancer agents. Moreover, PED/PEA-15 affects astrocyte motility and increases susceptibility to skin carcinogenesis in vivo. PED/PEA-15 expression is regulated at the transcriptional and the posttranslational levels. Increased PED/PEA-15 expression has been identified in individuals with type 2 diabetes early during the natural history of the disease. Evidence generated over the past 10 years indicated that this defect contributes to altering glucose tolerance by impairing insulin action and insulin secretion and might play a role in the development of diabetes-associated neurological disorders. Strategies are being devised to target key signaling events in PED/PEA-15 action aimed at improving glucose tolerance and at facilitating cancer cell death.