| Research Abstract |
Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at markedly inhibited adipocyte differentiation measured by Oil Red O staining in preadipocyte cells (3T3-L1), while expression of leptin, a marker of adipocyte differentiation, was suppressed by simvastatin for up to 12 days of culture. In mouse stromal cells (ST2), simvastatin at stimulated osteoblastic differentiation in osteogenic medium, while inhibiting adipocyte differentiation determined by Oil Red O staining and leptin mRNA expression in adipogenic medium containing troglitazone. We next elucidate mechanisms underlying the reduction of leptin expression induced by simvastatin, differentiated 3T3-L1 adipocytes were treated with various inhibitors with mevalonate or its metabolite in the presence or absence of simvastatin. Simvastatin time- and dose-dependently suppress
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ed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by simvastatin, while stability of the mRNA was not changed by treatment with simvastatin in transcription-arrested 3T3-L1 cells. Simvastatin inhibition of leptin gene transcription was not abrogated by pretreatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate, a mevalonate metabolite, abolished simvastatin-induced inhibition of leptin expression in 3T3-L1 cells. Suppression of expression was observed upon addition of GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor. Expression was suppressed by treatment with hydroxyfasudil, protein prenylation inhibitors. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells, while PD98059, an inhibitor of the ERK1/2 MAP kinase pathway and SB203580, an inhibitor of the p38MAP kinase pathway, did not affect expression at optimal concentrations. Simvastatin dose-dependently increased intracellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells. H89, an inhibitor of protein kinase A, completely abolished simvastatin-induced suppression of leptin expression. These results suggested that simvastatin reduced geranylgeranylprotein prenylation followed by deactivation of PI3K, leading to cAMP accumulation and subsequent activation of PKA in differentiated 3T3-L1 adipocytes. PKA inhibited leptin gene transcription without new protein synthesis. Furthermore, simvastatin promoted chondrocytic differentiation in ATDC5 cells. These effects of simvastatin cause reduction of adipocyte tissue mass and maintain bone mass, strongly suggesting salutary effects of these agents in prevention and treatment of obesity-related diseases and metabolic bone diseases. Less
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