Leptin Stimulates Catecholamine Synthesis in a PKC-Dependent Manner in Cultured Porcine Adrenal Medullary Chromaffin Cells

Abstract

We have previously shown that murine recombinant leptin directly stimulates catecholamine synthesis through the long form of the leptin receptor (Ob-Rb) expressed in cultured porcine chromaffin cells. Additionally, we found that leptin activates IP3 production after PLC activation. It is well established that activation of PLC elicits IP3 production as well as an increase in diacylglycerol, a compound that stimulates PKC. Therefore, we investigated the involvement of PKC in leptin-induced catecholamine synthesis. Leptin was found to induce significant increases in PKC activity in a dose-dependent manner (1, 10, and 100 nM); chelation of extracellular Ca(2+) by EDTA abolished this PKC stimulatory activity. We also confirmed by Western blot analysis that leptin (at 100 nM) induced significant increases in Ca(2+)-dependent PKC alpha, -beta(I), and -gamma expression. The activity of the rate-limiting enzyme tyrosine hydroxylase (TH) in the biosynthesis of catecholamine is regulated at the transcriptional and posttranscriptional levels. TH enzyme activity and TH mRNA levels induced by 100 nM leptin were significantly inhibited by the PKC inhibitor Ro 32-0432 as well as by EDTA. In addition, increases in TH protein and intracellular catecholamine content stimulated by leptin were completely inhibited by Ro 32-0432. Leptin markedly activated ERKs and, to a lesser extent, JNK; these stimulatory effects on ERKs and JNK were completely inhibited by Ro 32-0432 as well as EDTA. In contrast, leptin did not activate P38 MAPK. Similar to leptin, PMA activated ERK and JNK. Nicardipine and omega-conotoxin GVIA, each at 1 microM, were effective at inhibiting leptin-induced TH enzyme activity, TH mRNA accumulation, PKC activity, and ERK activity. Leptin increased activating protein-1 DNA-binding activity, and this was diminished by Ro 32-0432 as well as EDTA, similar to the reduction of TH mRNA levels. In addition, using supershift analysis, we documented the involvement of c-Fos and, to a lesser extent, c-Jun in leptin-induced activating protein-1 activity. These results indicate that leptin stimulates Ca(2+)-dependent PKC isoform-dependent catecholamine synthesis in porcine chromaffin cells. Previously, we had shown that leptin stimulated cAMP. The present study also showed that H89 (a PKA inhibitor) moderately, but significantly, inhibited leptin-induced ERK and TH mRNA. Consistent with this finding, leptin is shown here to activate novel PKC epsilon, which is assumed to stimulate Raf, upstream of ERKs, via cAMP, supporting the suggestion that Ca(2+)-independent novel PKC may also play some physiological role in regulating catecholamine synthesis.

Leptin Increases Lipoprotein Lipase Secretion by Macrophages: Involvement of Oxidative Stress and Protein Kinase C

“…In accordance with the results of Takekoshi et al (45) in chromaffin cells, we found that leptin enhances the expression of PKC -␣, -␤ I , -␤ II , and -␥ in macrophages. To the best of our knowledge, this is the first report that leptin activates PKC in vascular cells.…”

Leptin Increases Axonal Growth Cone Size in Developing Mouse Cortical Neurons by Convergent Signals Inactivating Glycogen Synthase Kinase-3β

“…5C). Leptin has been reported to trigger PKC activation and elicit PKC-dependent ERK1/2 phosphorylation in peripheral cells (25). Application of the PKC inhibitor GF109203X (1 M) (26) did not significantly affect leptinmediated rise in phospho-ERK1/2 in cortical neurons (Fig.…”

Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2

“…Also in rat adrenal zona glomerulosa cells ERK1/2 activation blocked cell proliferation and stimulated steroidogenesis (Otis et al 2005, Otis & Gallo-Payet 2007. A role of ERK1/2 for functional aspects of cortical ( Wu et al 2002, Chang et al 2008 or medullary cells (Cox & Parsons 1997, Takekoshi et al 2001, Shibuya et al 2002, Yanagihara et al 2005, Shinkai et al 2007) has also been provided by others. In addition to growth and function also migration is under control by ERK1/2 in adrenomedullary cells (Ho et al 2001(Ho et al , 2005.…”