Angela Cassese

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.

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.

Multiple Members of the Mitogen-activated Protein Kinase Family Are Necessary for PED/PEA-15 Anti-apoptotic Function

293 kidney embryonic cells feature very low levels of the anti-apoptotic protein PED. In these cells, expression of PED to levels comparable with those occurring in normal adult cells inhibits apoptosis induced by growth factor deprivation and by exposure to H2O2 or anisomycin. In PED-expressing 293 cells (293PED), inhibition of apoptosis upon growth factor deprivation was paralleled by decreased phosphorylation of JNK1/2. In 293PED cells, decreased apoptosis induced by anisomycin and H2O2 was also accompanied by block of JNK1/2 and p38 phosphorylations, respectively. Impaired activity of these stress kinases by PED correlated with inhibition of stress-induced Cdc-42, MKK4, and MKK6 activation. At variance with JNK1/2 and p38, PED expression increased basal and growth factor-stimulated Ras-Raf-1 co-precipitation and MAPK phosphorylation and activity. Treatment of 293PED cells with the MEK inhibitor PD98059 blocked ERK1/2 phosphorylations with no effect on inhibition of JNK1/2 and p38 activities. Complete rescue of JNK and p38 functions in 293PED cells by overexpressing JNK1 or p38, respectively, enabled only partial recovery of apoptotic response to growth factor deprivation and anisomycin. However, simultaneous rescue of JNK and p38 activities accompanied by block of ERK1/2 fully restored these responses. Thus, PED controls activity of the ERK, JNK, and p38 subfamilies of MAPKs. PED anti-apoptotic function in the 293 cells requires PED simultaneous activation of ERK1/2 and inhibition of the JNK/p38 signaling systems by PED.

Leptin-Stimulated Endothelial Nitric-Oxide Synthase via an Adenosine 5'-Monophosphate-Activated Protein Kinase/Akt Signaling Pathway Is Attenuated by Interaction with C-Reactive Protein

The AMP-activated protein kinase (AMPK) lies upstream of Akt in the pathway leading to endothelial NO synthase (eNOS) activation. Whether leptin promotes eNOS activation via AMPK-dependent activation of Akt, and which of the two AMPKalpha catalytic subunits is involved, remains unknown. Leptin resistance may be partly attributed to interaction between leptin and C-reactive protein (CRP). We hypothesized that leptin effect on eNOS activation in human aortic endothelial cells might be blunted by direct interaction with human recombinant CRP. Small interfering RNAs (siRNAs) were used to knock down expression of alpha1- or alpha2-AMPK in transient transfection assay to evaluate which is involved in this pathway and whether leptin effect on eNOS activation in human aortic endothelial cells might be blunted by direct interaction with human CRP. siRNA-mediated down-regulation of AMPKalpha1, but not AMPKalpha2, abolished leptin-induced Akt-Ser(473) phosphorylation, eNOS-Ser(1177) phosphorylation, eNOS activation, and cGMP accumulation. By contrast, siRNA-mediated knockdown of Akt1 did not affect AMPKalpha1 phosphorylation, but it abolished leptin-induced phosphorylation of Akt-Ser(473) and eNOS-Ser(1177), suggesting that Akt functions downstream of AMPKalpha1. Preincubation of leptin with human recombinant CRP impaired leptin-induced AMPK activation, eNOS-Ser(1177) phosphorylation, eNOS activity, and intracellular cGMP accumulation. The data are consistent with a model implicating an AMPKalpha1-->Akt-->eNOS pathway leading to NO production in response to leptin supporting the idea that interaction between leptin and CRP may have a role in impairing leptin effect on eNOS activation, suggesting a link between leptin resistance, low-grade inflammation, and endothelial dysfunction.

Prep1 deficiency induces protection from diabetes and increased insulin sensitivity through a p160-mediated mechanism.

ABSTRACT We have examined glucose homeostasis in mice hypomorphic for the homeotic transcription factor gene Prep1. Prep1-hypomorphic (Prep1i/i) mice exhibit an absolute reduction in circulating insulin levels but normal glucose tolerance. In addition, these mice exhibit protection from streptozotocin-induced diabetes and enhanced insulin sensitivity with improved glucose uptake and insulin-dependent glucose disposal by skeletal muscle. This muscle phenotype does not depend on reduced expression of the known Prep1 transcription partner, Pbx1. Instead, in Prep1i/i muscle, we find normal Pbx1 but reduced levels of the recently identified novel Prep1 interactor p160. Consistent with this reduction, we find a muscle-selective increase in mRNA and protein levels of PGC-1α, accompanied by enhanced expression of the GLUT4 transporter, responsible for insulin-stimulated glucose uptake in muscle. Indeed, using L6 skeletal muscle cells, we induced the opposite effects by overexpressing Prep1 or p160, but not Pbx1. In vivo skeletal muscle delivery of p160 cDNA in Prep1i/i mice also reverses the molecular phenotype. Finally, we show that Prep1 controls the stability of the p160 protein. We conclude that Prep1 controls insulin sensitivity through the p160-GLUT4 pathway.

PED/PEA-15 Regulates Glucose-Induced Insulin Secretion by Restraining Potassium Channel Expression in Pancreatic β-Cells

The phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (ped/pea-15) gene is overexpressed in human diabetes and causes this abnormality in mice. Transgenic mice with β-cell–specific overexpression of ped/pea-15 (β-tg) exhibited decreased glucose tolerance but were not insulin resistant. However, they showed impaired insulin response to hyperglycemia. Islets from the β-tg also exhibited little response to glucose. mRNAs encoding the Sur1 and Kir6.2 potassium channel subunits and their upstream regulator Foxa2 were specifically reduced in these islets. Overexpression of PED/PEA-15 inhibited the induction of the atypical protein kinase C (PKC)-ζ by glucose in mouse islets and in β-cells of the MIN-6 and INS-1 lines. Rescue of PKC-ζ activity elicited recovery of the expression of the Sur1, Kir6.2, and Foxa2 genes and of glucose-induced insulin secretion in PED/PEA-15–overexpressing β-cells. Islets from ped/pea-15–null mice exhibited a twofold increased activation of PKC-ζ by glucose; increased abundance of the Sur1, Kir6.2, and Foxa2 mRNAs; and enhanced glucose effect on insulin secretion. In conclusion, PED/PEA-15 is an endogenous regulator of glucose-induced insulin secretion, which restrains potassium channel expression in pancreatic β-cells. Overexpression of PED/PEA-15 dysregulates β-cell function and is sufficient to impair glucose tolerance in mice.

Molecular Cloning and Characterization of the Human PED/PEA-15 Gene Promoter Reveal Antagonistic Regulation by Hepatocyte Nuclear Factor 4α and Chicken Ovalbumin Upstream Promoter Transcription Factor II

Overexpression of the ped/pea-15 gene in mice impairs glucose tolerance and leads to diabetes in conjunction with high fat diet treatment. PED/PEA-15 is also overexpressed in type 2 diabetics as well as in euglycemic offspring from these subjects. The cause(s) of this abnormality remains unclear. In the present work we have cloned and localized the promoter region of the human PED/PEA-15 gene within the first 230 bp of the 5®-flanking region. A cis-acting regulatory element located between -320 and -335 bps upstream the PED/PEA-15 gene transcriptional start site (+1) is recognized by both the hepatocyte nuclear factor 4α (HNF-4α) and the chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), two members of the steroid/thyroid superfamily of transcription factors, both of which are involved in the control of lipid and glucose homeostasis. HNF-4α represses PED/PEA-15 expression in HeLa cells, whereas COUP-TFII activates its expression. In hepatocytes, the activation of PED/PEA-15 gene transcription is paralleled by the establishment of a partially dedifferentiated phenotype accompanied by a reduction in mRNA levels encoded by genes normally expressed during liver development. Cotransfection of HeLa cells with a reporter construct containing the PED/PEA-15 response element and various combinations of HNF-4α and COUP-TFII expression vectors indicated that COUP-TFII antagonizes the repression of the PED/PEA-15 gene by HNF-4α. Thus, at least in part, transcription of the PED/PEA-15 gene in vivo is dependent upon the intracellular balance of these positive and negative regulatory factors. Abnormalities in HNF-4α and COUP-TFII balance might have important consequences on glucose tolerance in humans.

Prep1 Controls Insulin Glucoregulatory Function in Liver by Transcriptional Targeting of SHP1 Tyrosine Phosphatase

OBJECTIVE We investigated the function of the Prep1 gene in insulin-dependent glucose homeostasis in liver. RESEARCH DESIGN AND METHODS Prep1 action on insulin glucoregulatory function has been analyzed in liver of Prep1-hypomorphic mice (Prep1i/i), which express 2–3% of Prep1 mRNA. RESULTS Based on euglycemic hyperinsulinemic clamp studies and measurement of glycogen content, livers from Prep1i/i mice feature increased sensitivity to insulin. Tyrosine phosphorylation of both insulin receptor (IR) and insulin receptor substrate (IRS)1/2 was significantly enhanced in Prep1i/i livers accompanied by a specific downregulation of the SYP and SHP1 tyrosine phosphatases. Prep1 overexpression in HepG2 liver cells upregulated SYP and SHP1 and inhibited insulin-induced IR and IRS1/2 phosphorylation and was accompanied by reduced glycogen content. Consistently, overexpression of the Prep1 partner Pbx1, but not of p160MBP, mimicked Prep1 effects on tyrosine phosphorylations, glycogen content, and on SYP and SHP1 expression. In Prep1 overexpressing cells, antisense silencing of SHP1, but not that of SYP, rescued insulin-dependent IR phosphorylation and glycogen accumulation. Both Prep1 and Pbx1 bind SHP1 promoter at a site located between nucleotides −2,113 and −1,778. This fragment features enhancer activity and induces luciferase function by 7-, 6-, and 30-fold, respectively, in response to Prep1, Pbx1, or both. CONCLUSIONS SHP1, a known silencer of insulin signal, is a transcriptional target of Prep1. In liver, transcriptional activation of SHP1 gene by Prep1 attenuates insulin signal transduction and reduces glucose storage.

Targeting of PED/PEA-15 Molecular Interaction with Phospholipase D1 Enhances Insulin Sensitivity in Skeletal Muscle Cells

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-α activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6PED/PEA-15) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (KD = 0.37 ± 0.13 μm), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6PED/PEA-15 cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-α activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by ∼70%. Similar results are obtained by expression of D4 in L6PED/PEA-15. All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.