Glucose dependence of glycogen synthase activity regulation by GSK3 and MEK/ERK inhibitors and angiotensin-(1-7) action on these pathways in cultured human myotubes.

Glycogen synthase (GS) is activated by glucose/glycogen depletion in skeletal muscle cells, but the contributing signaling pathways, including the chief GS regulator GSK3, have not been fully defined. The MEK/ERK pathway is known to regulate GSK3 and respond to glucose. The aim of this study was to elucidate the GSK3 and MEK/ERK pathway contribution to GS activation by glucose deprivation in cultured human myotubes. Moreover, we tested the glucose-dependence of GSK3 and MEK/ERK effects on GS and angiotensin (1-7) actions on these pathways. We show that glucose deprivation activated GS, but did not change phospho-GS (Ser640/1), GSK3ß activity or activity-activating phosphorylation of ERK1/2. We then treated glucose-replete and -depleted cells with SB415286, U0126, LY294 and rapamycin to inhibit GSK3, MEK1/2, PI3K and mTOR, respectively. SB415286 activated GS and decreased the relative phospho-GS (Ser640/1) level, more in glucose-depleted than -replete cells. U0126 activated GS and reduced the phospho-GS (Ser640/1) content significantly in glucose-depleted cells, while GSK3ß activity tended to increase. LY294 inactivated GS in glucose-depleted cells only, without affecting relative phospho-GS (Ser640/1) level. Rapamycin had no effect on GS activation. Angiotensin-(1-7) raised phospho-ERK1/2 but not phospho-GSK3ß (Ser9) content, while it inactivated GS and increased GS phosphorylation on Ser640/1, in glucose-replete cells. In glucose-depleted cells, angiotensin-(1-7) effects on ERK1/2 and GS were reverted, while relative phospho-GSK3ß (Ser9) content decreased. In conclusion, activation of GS by glucose deprivation is not due to GS Ser640/1 dephosphorylation, GSK3ß or ERK1/2 regulation in cultured myotubes. However, glucose depletion enhances GS activation/Ser640/1 dephosphorylation due to both GSK3 and MEK/ERK inhibition. Angiotensin-(1-7) inactivates GS in glucose-replete cells in association with ERK1/2 activation, not with GSK3 regulation, and glucose deprivation reverts both hormone effects. Thus, the ERK1/2 pathway negatively regulates GS activity in myotubes, without involving GSK3 regulation, and as a function of the presence of glucose.

La activación de receptor activado por proliferadores peroxisómicos Beta/ Delta mejora la resistencia a insulina inducida porIL-6 en células hepáticas / No disponible

Introducción: La resistencia a la insulina precede y predice la presencia de diabetes mellitustipo 2, condición que supone un notable incremento del riesgo cardiovascular. La interleucina-6es uno de los mediadores que relacionan la inflamación crónica observada en estados de obesidad con la resistencia a la insulina a través de la activación de STAT3 (signal transducer and activator of transcription 3), con el consiguiente aumento de SOCS3 (suppressor of cytokinesignaling 3) en el hígado. El objetivo de este estudio ha sido evaluar si un agonista del receptor activado por proliferadores peroxisómicos (PPAR) / , GW501516, es capaz de evitar la activación de la vía de senalización IL-6/STAT3/SOCS3 y la resistencia a la insulina en células hepáticas. Material y métodos: Células HepG2 humanas se estimularon con IL-6 (20 ng/ml) en presenciao en ausencia de GW501516 (10 M). También analizamos el hígado de ratones salvajes y con deficiencia PPAR / . Los niveles de ARNm y proteínas se analizaron mediante las técnicas deRT-PCR y Western-Blot, respectivamente. Resultados: GW501516 evitó la fosforilación en Tyr705 y en Ser727 de STAT3 y el aumento deSOCS3 inducidas por la IL-6. Asimismo, el tratamiento con este fármaco evitó la activación por la IL-6 de la ERK1/2, una serina-treonina cinasa implicada en la fosforilación de STAT3 enSer727. Cabe destacar que el hígado de ratones deficientes en PPAR / mostró un aumento (..) (AU)

Introduction: Insulin resistance precedes and predicts the development of type 2 diabetes mellitus, a disease which increases the risk of cardiovascular events. Interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent up regulation of suppressor of cytokine signaling 3 (SOCS3)in liver. The aim of this study was to evaluate whether peroxisome proliferator-activated receptor (PPAR) / agonist GW501516 prevented activation of the IL-6/STAT3/SOCS3 pathway and insulin resistance in hepatic cells. Material and methods: Human HepG2 cells were stimulated for 10 min with IL-6 (20 ng/mL)in the presence or in the absence of 10 M GW501516, then mRNA and protein levels were analyzed by RT-PCR or Western-Blot, respectively. In addition, we also analyzed protein levels from PPAR / null mice and wild-type mice livers. Results: GW501516 prevented IL-6-induced STAT3 phosphorylation on Tyr705 and Ser727 and avoided the increase in SOCS3 caused by this cytokine. In addition, this drug also preventedIL-6-dependent ERK1/2 phosphorylation, a serine-threonine protein kinase involved in STAT3phosphorylation on Ser727. Interestingly, livers from PPAR / null mice showed increased phosphorylations on Tyr 705 and Ser727 of STAT3 as well as phosphorylated ERK1/2 levels. Finally, all (..) (AU)

TNF-α inhibits PPARß/δ activity and SIRT1 expression through NF-κB in human adipocytes.

The mechanisms linking low-grade chronic inflammation with obesity-induced insulin resistance have only been partially elucidated. PPARß/δ and SIRT1 might play a role in this association. In visceral adipose tissue (VAT) from obese insulin-resistant patients we observed enhanced p65 nuclear translocation and elevated expression of the pro-inflammatory cytokines TNF-α and IL-6 compared to control subjects. Inflammation was accompanied by a reduction in the levels of SIRT1 protein and an increase in PPARß/δ mRNA levels. Stimulation of human mature SGBS adipocytes with TNF-α caused similar changes in PPARß/δ and SIRT1 to those reported in obese patients. Unexpectedly, PPAR DNA-binding activity and the expression of PPARß/δ-target genes was reduced following TNF-α stimulation, suggesting that the activity of this transcription factor was inhibited by cytokine treatment. Interestingly, the PPARß/δ ligand GW501516 prevented the expression of inflammatory markers and the reduction in the expression of PPARß/δ-target genes in adipocytes stimulated with TNF-α. Consistent with a role for NF-κB in the changes caused by TNF-α, treatment with the NF-κB inhibitor parthenolide restored PPAR DNA-binding activity, the expression of PPARß/δ-target genes and the expression of SIRT1 and PPARß/δ. These findings suggest that the reduction in PPARß/δ activity and SIRT1 expression caused by TNF-α stimulation through NF-κB helps perpetuate the inflammatory process in human adipocytes.

Targeting PPARß/δ for the treatment of type 2 diabetes mellitus.

INTRODUCTION: The nuclear receptors Peroxisome Proliferator-Activated Receptors (PPAR)α and PPARγ are therapeutic targets for hypertriglyceridemia and insulin resistance, respectively. Evidence is now emerging that the PPARß/δ isotype is a potential pharmacological target for the treatment of insulin resistance and type 2 diabetes mellitus. AREAS COVERED: In this review, the capacity of PPARß/δ to prevent the development of insulin resistance and type 2 diabetes mellitus is discussed. Special emphasis is placed on preclinical studies and the molecular mechanisms responsible for its actions in the main cell types involved in these pathologies: adipocytes, ß-cells, skeletal muscle cells and hepatocytes. EXPERT OPINION: While several concerns remain for the development of PPARß/δ agonists, these drugs have demonstrated their efficacy in the treatment of insulin resistance and type 2 diabetes mellitus in preclinical studies, as well as in a few short clinical studies in humans. Although this data is promising, additional studies must be performed to confirm the efficacy and safety of these drugs in the treatment of type 2 diabetes mellitus.

Activation of peroxisome proliferator-activated receptor-ß/-δ (PPAR-ß/-δ) ameliorates insulin signaling and reduces SOCS3 levels by inhibiting STAT3 in interleukin-6-stimulated adipocytes.

OBJECTIVE: It has been suggested that interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent upregulation of suppressor of cytokine signaling 3 (SOCS3). We evaluated whether peroxisome proliferator-activated receptor (PPAR)-ß/-δ prevented activation of the IL-6-STAT3-SOCS3 pathway and insulin resistance in adipocytes. RESEARCH DESIGN AND METHODS: Adipocytes and white adipose tissue from wild-type and PPAR-ß/-δ-null mice were used to evaluate the effect of PPAR-ß/-δ on the IL-6-STAT3-SOCS3 pathway. RESULTS: First, we observed that the PPAR-ß/-δ agonist GW501516 prevented both IL-6-dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. In addition, this drug treatment abolished IL-6-induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6-induced STAT3 phosphorylation on Tyr(705) and Ser(727) residues in vitro and in vivo. Moreover, GW501516 prevented IL-6-dependent induction of extracellular signal-related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPAR-ß/-δ-null mice, STAT3 phosphorylation (Tyr(705) and Ser(727)), STAT3 DNA-binding activity, and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPAR-ß/-δ-null mice compared with wild-type mice. CONCLUSIONS: Collectively, our findings indicate that PPAR-ß/-δ activation prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes.

The PPARß/δ activator GW501516 prevents the down-regulation of AMPK caused by a high-fat diet in liver and amplifies the PGC-1α-Lipin 1-PPARα pathway leading to increased fatty acid oxidation.

Metabolic syndrome-associated dyslipidemia is mainly initiated by hepatic overproduction of the plasma lipoproteins carrying triglycerides. Here we examined the effects of the peroxisome proliferator-activated receptors (PPAR)-ß/δ activator GW501516 on high-fat diet (HFD)-induced hypertriglyceridemia and hepatic fatty acid oxidation. Exposure to the HFD caused hypertriglyceridemia that was accompanied by reduced hepatic mRNA levels of PPAR-γ coactivator 1 (PGC-1)-α and lipin 1, and these effects were prevented by GW501516 treatment. GW501516 treatment also increased nuclear lipin 1 protein levels, leading to amplification in the PGC-1α-PPARα signaling system, as demonstrated by the increase in PPARα levels and PPARα-DNA binding activity and the increased expression of PPARα-target genes involved in fatty acid oxidation. These effects of GW501516 were accompanied by an increase in plasma ß-hydroxybutyrate levels, demonstrating enhanced hepatic fatty acid oxidation. Moreover, GW501516 increased the levels of the hepatic endogenous ligand for PPARα, 16:0/18:1-phosphatidilcholine and markedly enhanced the expression of the hepatic Vldl receptor. Interestingly, GW501516 prevented the reduction in AMP-activated protein kinase (AMPK) phosphorylation and the increase in phosphorylated levels of ERK1/2 caused by HFD. In addition, our data indicate that the activation of AMPK after GW501516 treatment in mice fed HFD might be the result of an increase in the AMP to ATP ratio in hepatocytes. These findings indicate that the hypotriglyceridemic effect of GW501516 in HFD-fed mice is accompanied by an increase in phospho-AMPK levels and the amplification of the PGC-1α-lipin 1-PPARα pathway.