Francesca Fiory

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.

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.

The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts

The plant hormone abscisic acid (ABA) is released from glucose‐challenged human pancreatic β cells and stimulates insulin secretion. We investigated whether plasma ABA increased during oral and intravenous glucose tolerance tests (OGTTs and IVGTTs) in healthy human subjects. In all subjects undergoing OGTTs (n=8), plasma ABA increased over basal values (in a range from 2‐ to 9‐fold). A positive correlation was found between the ABA area under the curve (AUC) and the glucose AUC. In 4 out of 6 IVGTTs, little or no increase of ABA levels was observed. In the remaining subjects, the ABA increase was similar to that recorded during OGTTs. GLP‐1 stimulated ABA release from an insulinoma cell line and from human islets, by ~10‐ and 2‐fold in low and high glucose, respectively. Human adipose tissue also released ABA in response to high glucose. Nanomolar ABA stimulated glucose uptake, similarly to insulin, in rat L6 myoblasts and in murine 3T3‐L1 cells differentiated to adipocytes, by increasing GLUT‐4 translocation to the plasma membrane. Demonstration that a glucose load in humans is followed by a physiological rise of plasma ABA, which can enhance glucose uptake by adipose tissues and muscle cells, identifies ABA as a new mammalian hormone involved in glucose metabolism.—Bruzzone, S., Ameri, P., Briatore, L., Mannino, E., Basile, G., Andraghetti, G., Grozio, A., Magnone, M., Guida, L., Scarfì, S., Salis, A., Damonte, G., Sturla, L., Nencioni, A., Fenoglio, D., Fiory, F., Miele, C., Beguinot, F., Ruvolo, V., Bormioli, M., Colombo, G., Maggi, D., Murialdo, G., Cordera, R., De Flora, A., Zocchi, E. The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts. FASEB J. 26, 1251‐1260 (2012). www.fasebj.org

Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization

Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-α (PKCα) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms α and δ, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKδ, but not of DGKα expression, was sufficient to prevent the effect of high glucose on PKCα activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCα activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.

Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon.

Protein kinase C (PKC)-α exerts a regulatory function on insulin action. We showed by overlay blot that PKCα directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3ϵ. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCα co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3ϵ, but not with other 14-3-3 isoforms. Overexpression of 14-3-3ϵ in 3T3-hIR cells significantly reduced IRS-1-bound PKCα activity, without altering IRS-1/PKCα co-precipitation. 14-3-3ϵ overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3ϵ by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCα activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCα expression by antisense oligonucleotides reverted the negative effect of 14-3-3ϵ depletion on insulin signaling. Moreover, PKCα inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3ϵ. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCα, and 14-3-3ϵ. The presence of 14-3-3ϵ in the complex is not necessary for IRS-1/PKCα interaction but modulates PKCα activity, thereby regulating insulin signaling and degradation.

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.

Phorbol Esters Induce Intracellular Accumulation of the Anti-apoptotic Protein PED/PEA-15 by Preventing Ubiquitinylation and Proteasomal Degradation

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA)-15 is an anti-apoptotic protein whose expression is increased in several cancer cells and following experimental skin carcinogenesis. Exposure of untransfected C5N keratinocytes and transfected HEK293 cells to phorbol esters (12-O-tetradecanoylphorbol-13-acetate (TPA)) increased PED/PEA-15 cellular content and enhanced its phosphorylation at serine 116 in a time-dependent fashion. Ser-116 → Gly (PEDS116G) but not Ser-104 → Gly (PEDS104G) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-ζ and by the expression of a dominant-negative PKC-ζ mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-ζ increased cellular content and phosphorylation of WT-PED/PEA-15 and PEDS104G but not of PEDS116G. These events were accompanied by the activation of Ca2+-calmodulin kinase (CaMK) II and prevented by the CaMK blocker, KN-93. At variance, the proteasome inhibitor lactacystin mimicked TPA action on PED/PEA-15 intracellular accumulation and reverted the effects of PKC-ζ and CaMK inhibition. Moreover, we show that PED/PEA-15 bound ubiquitin in intact cells. PED/PEA-15 ubiquitinylation was reduced by TPA and PKC-ζ overexpression and increased by KN-93 and PKC-ζ block. Furthermore, in HEK293 cells expressing PEDS116G, TPA failed to prevent ubiquitin-dependent degradation of the protein. Accordingly, in the same cells, TPA-mediated protection from apoptosis was blunted. Taken together, our results indicate that TPA increases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation.

Protein kinase Cα activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase

We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCα, PKCδ and PKCζ activity and that PKCα, not PKCδ and PKCζ, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCα. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCα. We conclude that RET binds to and activates PKCα. PKCα, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

Selective disruption of insulin-like growth factor-1 (IGF-1) signaling via phosphoinositide-dependent kinase-1 prevents the protective effect of IGF-1 on human cancer cell death.

Insulin-like growth factor-1 (IGF-1) signaling system exerts a broad antiapoptotic function and plays a crucial role in resistance to anticancer therapies. Exposure of MCF-7 breast cancer cells to IGF-1 rapidly and transiently induced tyrosine phosphorylation and activation of phosphoinositide-dependent kinase-1 (PDK1). This was paralleled by Akt/protein kinase B and protein kinase C-ζ phosphorylation, at Thr308 and Thr410, respectively. IGF-1 treatment also enhanced PDK1 interaction with IGF-1 receptor (IGF-1R) in intact MCF-7 cells. Pulldown assays revealed that PDK1 bound IGF-1R in vitro and that the region encompassing amino acids 51–359 of PDK1 was necessary for the interaction. Synthetic peptides corresponding to IGF-1R C terminus amino acids 1295–1337 (C43) and to PDK1 amino acids 114–141 reduced in vitro IGF-1R/PDK1 interaction in a concentration-dependent manner. Loading of fluoresceinated-C43 (fluorescein isothiocyanate (FITC)-C43) into MCF-7 cells significantly reduced IGF-1R/PDK1 interaction and phosphorylation of PDK1 substrates. Moreover, FITC-C43 intracellular loading reverted the protective effect of IGF-1 on growth factor deprivation-induced cell death. Finally, the inhibition of IGF-1R/PDK1 interaction and signaling by FITC-C43 was accompanied by 2-fold enhanced killing capacity of cetuximab in human GEO colon adenocarcinoma cells and was sufficient to restore cell death in cetuximab-resistant cell clones. Thus, disruption of PDK1 interaction with IGF-1R reduces IGF-1 survival effects in cancer cells and may enhance cell death by anticancer agents.