Leptin rapidly activates PPARs in C2C12 muscle cells

Experimental evidence suggests that leptin operates on the tissues, including skeletal muscle, also by modulating gene expression. Using electrophoretic mobility shift assays, we have shown that physiological doses of leptin promptly increase the binding of C2C12 cell nuclear extracts to peroxisome proliferator-activated receptor (PPAR) response elements in oligonucleotide probes and that all three PPAR isoforms participate in DNA-binding complexes. We pre-treated C2C12 cells with AACOCF(3), a specific inhibitor of cytosolic phospholipase A(2) (cPLA(2)), an enzyme that supplies ligands to PPARs, and found that it abrogates leptin-induced PPAR DNA-binding activity. Leptin treatment significantly increased cPLA(2) activity, evaluated as the release of [H-3]arachidonic acid from pre-labelled C2C12 cells, as well as phosphorylation. Further, using MEK1 inhibitor PD-98059 we showed that leptin activates cPLA(2) through ERK induction. These results support a direct effect of leptin on skeletal Muscle cells, and suggest that the hormone may modulate muscle transcription also by precocious activation of PPARs through ERK cPLA(2) pathway. (c) 2005 Elsevier Inc. All rights reserved

Early intracellular events induced by in vivo leptin treatment in mouse skeletal muscle

Experimental evidence suggests that leptin may exert direct effects on peripheral tissues. In this study we investigated some transductional molecules in skeletal muscle, after intraperitoneal leptin injection in wild-type and ob/ob mice. By immunoprecipitation and immunoblotting with anti-phosphotyrosine antibodies, we observed a modified pattern of phosphotyrosine proteins. We then identified an increase in JAK2, IRS1 and IRS2 tyrosine-phosphorylation and in their association with p85, a subunit of PI3K. The increase in PI3K activity in immunoprecipitated p85 did not reach statistical significance, however, both Akt and GSK3 resulted significantly hyper-phosphorylated. Bad, an Akt substrate involved in cell survival, appeared modified in its phosphorylation. ERK1, ERK2 and p38 MAP kinase phosphorylation significantly increased, even if the latter only in wild-type animals. Finally, by EMSA experiments, we documented that leptin increased the DNA binding capacity of Stat3 homodimers and AP-1. Thus, leptin appears to activate, within minutes, some insulin signalling molecules. Stat3 and AP-1 activation by gene expression remodelling could subsequently trigger more leptin-specific effects. Further, leptin might play a still underestimated role in cell survival

Intracellular signal transduction pathways induced by leptin in C2C12 cells

As experimental evidence suggests that leptin may have direct effects on peripheral tissues, we investigated some of the transductional molecules induced by leptin in C2C12 cells. In immunoprecipitation experiments using anti-p85 antibodies (a regulatory subunit of phosphatidylinositol-3-kinase; PI3K), we observed a significant increase in PI3K activity. Immunoblot analyses showed that Akt, GSK3, ERK1, ERK2, and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation significantly increased after leptin treatment. Protein kinase C (PKC)-zeta was also activated by leptin, as documented by an immunocomplex kinase assay and immunoblotting experiments. The treatment of C2C12 cells with Wortmannin before leptin administration inhibited induction of the phosphorylation of ERKs (extracellular signal-regulated kinases) but not that of p38 MAPK, whereas pre-treatment with a PKC-zeta inhibitor partially decreased ERK phosphorylation. Taken together, our in vitro results further support the hypothesis that leptin acts acutely on skeletal muscle tissue through some of the components of insulin signalling, including PKC-zeta. (c) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved

Rapid stimulation of mitogen-activated protein kinase of rat liver by prolactin.

Intraperitoneal prolactin administration to female rats caused a rapid and transient stimulation of hepatic mitogen-activated kinase (MAP kinase) activity measured in vitro as cytosolic phosphotransferase capacity towards two specific substrates. Myelin basic protein kinase activity of MAP kinase immunoprecipitates confirmed the specificity and magnified the prolactin effect. Immunoblot experiments with anti-(MAP kinase) and anti-phosphotyrosine antibodies showed changes in both electrophoretic mobility and phosphotyrosine content of 40 and 44 kDa isoenzymes suggesting that prolactin affects these isoforms. Concomitant with the increase in MAP kinase activity, prolactin induced tyrosine phosphorylation in a number of liver proteins, suggesting a rapid involvement of tyrosine kinases which might be correlated in some way with MAP kinase activation. Protein kinase C activity, which has been implicated in the regulation of MAP kinase and in mediating the prolactin effect, does not seem to participate in MAP kinase activation

Sam68 and ERKs regulate leptin-induced expression of OB-Rb mRNA in C2C12 myotubes

Abstract Acute leptin treatment significantly increases the mRNA of the long isoform of leptin receptor (OB-Rb) in C2C12 myotubes after as little as 30min, without affecting that of the short isoform (OB-Ra). The Sam68 STAR protein has been implicated in leptin signal transduction as an adaptor molecule useful to recruit other signalling proteins. We found that leptin increased Sam68 tyrosine-phosphorylation so decreasing its poly(U)-binding capacity. RT-PCR analysis of the mRNA bound to immunoprecipitated Sam68 showed that Sam68 associated with OB-Rb but not OB-Ra mRNA in control and leptin-treated C2C12 cells. The siRNA-mediated silencing of Sam68 reduced its levels by 89% and abolished the leptin-mediated increase in OB-Rb mRNA. Leptin activates ERKs which in turn might phosphorylate Sam68 modifying its influence on mRNA. We did not observe any changes in Sam68 Ser/Thr phosphorylation but using the specific MEK1 inhibitor PD-98059 showed that leptin-mediated ERK activation is essential for leptin''s effect on OB-Rb mRNA expression. Thus it appears that leptin has a positive short-term effect on the regulation of OB-Rb mRNA in C2C12 cells, involving both Sam68 and ERKs. These results might suggest that leptin signal acutely favours its own sensitivity

The liver response to in vivo heat shock involves the activation of MAP kinases and RAF and the tyrosine phosphorylation of Shc proteins

We have investigated the mechanisms of signal transduction in the response of liver to heat shock in vivo. By immunoblot experiments we have shown that heat shock decreases the electrophoretic mobility of the 40 and 43 kDa mitogen activated protein kinases (MAPKs) and we have found a significant increase of MAPK activity measured as phosphotransferase capacity of both cytosolic extracts and MAPK immunoprecipitates. To elucidate the signalling pathway which accounts for MAPK activation, we focused our attention on its upstream factors, Raf and Ras. We have shown that, heat shock activates Raf-1 kinase and causes an increase in phosphotyrosine content of the 52 kDa Shc protein accompanied by an increment in the amount of coimmunoprecipitated Grb2. These findings provide the first evidence that the Ras-Raf-MAPK pathway is activated in liver during heat shock in vivo