Simvastatin suppresses leptin expression in 3T3-L1 adipocytes via activation of the cyclic AMP-PKA pathway induced by inhibition of protein prenylation.
Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyses conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at 1 microM 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 1 muM simvastatin for up to 12 days of culture. Next, to 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 suppressed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by 10 muM 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 pre-treatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate (GGPP), 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, a protein prenylation inhibitor. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells. Simvastatin dose-dependently increased intra-cellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells, with maximal stimulation obtained at 10 muM. Addition of GGPP abolished simvastatin-induced stimulation of cAMP accumulation and protein kinase A (PKA) activity. H89, an inhibitor of PKA, 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. Finally, PKA inhibited leptin gene transcription without new protein synthesis.
Maeda
Toyonobu
T
Section of Biochemistry, Department of Oral Function and Molecular Biology, Ohu University School of Dentistry, Koriyama 963-8611, Japan.
Horiuchi
Noboru
N
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D020738:Leptin / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000031:analogs & derivatives / Q000494:pharmacology; D008798:Mevalonic Acid / Q000494:pharmacology; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D017368:Protein Prenylation / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D019821:Simvastatin / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D001549:Benzamides; D002867:Chromones; C096856:FTI 277; C102521:GGTI 298; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007546:Isoquinolines; D020738:Leptin; D009025:Morpholines; D011106:Polyisoprenyl Phosphates; D011500:Protein Synthesis Inhibitors; D013449:Sulfonamides; C431085:hydroxyfasudil; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003513:Cycloheximide; D008715:Methionine; D019821:Simvastatin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C002963:geranylgeranyl pyrophosphate; D008798:Mevalonic Acid; D000077191:Wortmannin