Maeda Toyonobu, Horiuchi Noboru
Section of Biochemistry, Department of Oral Function and Molecular Biology, Ohu University School of Dentistry, Koriyama 963-8611, Japan.
J Biochem. 2009 Jun;145(6):771-81. doi: 10.1093/jb/mvp035. Epub 2009 Mar 2.
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.
辛伐他汀可抑制3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶，该酶催化HMG-CoA转化为甲羟戊酸，这是胆固醇合成中的限速步骤。我们证明，1微摩尔的辛伐他汀可显著抑制前脂肪细胞（3T3-L1）中通过油红O染色测定的脂肪细胞分化，而1微摩尔的辛伐他汀在长达12天的培养过程中可抑制脂肪细胞分化标志物瘦素的表达。接下来，为了阐明辛伐他汀诱导瘦素表达降低的潜在机制，在有或无辛伐他汀的情况下，用甲羟戊酸或其代谢物的各种抑制剂处理分化的3T3-L1脂肪细胞。辛伐他汀可时间和剂量依赖性地抑制瘦素mRNA表达。与瘦素mRNA相关的不均一核RNA被10微摩尔的辛伐他汀抑制，而在转录停滞的3T3-L1细胞中，辛伐他汀处理并未改变mRNA的稳定性。用蛋白质合成抑制剂环己酰亚胺预处理并未消除辛伐他汀对瘦素基因转录的抑制作用。添加甲羟戊酸或香叶基香叶基焦磷酸（GGPP，一种甲羟戊酸代谢物）可消除辛伐他汀诱导的3T3-L1细胞中瘦素表达的抑制。添加香叶基香叶基转移酶I抑制剂GGTI-298后观察到表达受到抑制，但添加法尼基转移酶抑制剂FTI-277后未观察到。用蛋白质异戊二烯化抑制剂羟基法舒地尔处理可抑制表达。用磷脂酰肌醇3-激酶（PI3K）抑制剂LY294002和渥曼青霉素处理可降低3T3-L1细胞中的瘦素表达。辛伐他汀可剂量依赖性地增加3T3-L1细胞内的环磷酸腺苷（cAMP）浓度，在10微摩尔时获得最大刺激。添加GGPP可消除辛伐他汀诱导的cAMP积累和蛋白激酶A（PKA）活性的刺激。PKA抑制剂H89可完全消除辛伐他汀诱导的瘦素表达抑制。这些结果表明，辛伐他汀可降低香叶基香叶基蛋白异戊二烯化，随后使PI3K失活，导致cAMP积累，并随后激活分化的3T3-L1脂肪细胞中的PKA。最后，PKA可抑制瘦素基因转录，而无需新的蛋白质合成。
