Lien Chih-Chan, Au Lo-Chun, Tsai Ying-Lan, Ho Low-Tone, Juan Chi-Chang
Department of Physiology, National Yang-Ming University, Number 155, Section 2, Li-Nong Street, Taipei, Taiwan.
Endocrinology. 2009 Nov;150(11):4892-900. doi: 10.1210/en.2009-0403. Epub 2009 Oct 9.
TNF-alpha has several effects on adipocytes that may be related to the development of type 2 diabetes in obese subjects. Many studies demonstrated that long-term treatment with TNF-alpha increases lipolysis in adipocytes. However, the short-term (<4 h) effects of TNF-alpha on lipolysis have not been well investigated. The aim of this study was to investigate the short-term regulatory mechanism of TNF-alpha-induced lipolysis in 3T3-L1 adipocytes. Well-differentiated 3T3-L1 adipocytes were used. Lipolysis was determined by measuring glycerol release. Expression of inducible nitric oxide (iNOS) and nitric oxide (NO) production were measured, respectively, by Western blots and the Griess reagent. A selective iNOS inhibitor (s-ethylisothiourea . HBr), an adenylyl cyclase inhibitor (SQ22536), and a guanylyl cyclase inhibitor (LY83583) were used to investigate the involvement of iNOS, cAMP, and cGMP in TNF-alpha-induced lipolysis. Transient transfection with iNOS short hairpin RNA was performed to confirm the involvement of iNOS in TNF-alpha-induced lipolysis. Phosphorylation of hormone-sensitive lipase (HSL) was measured by immunoprecipitation and Western blotting. Results showed that short-term TNF-alpha treatment significantly increased lipolysis, iNOS expression, and NO production in a time- and dose-dependent manner. Furthermore, treatment with the NO donor S-nitroso-N-acetylpenicillamine also stimulated lipolysis and HSL phosphorylation in 3T3-L1 adipocytes. Moreover, pretreatment with inhibitors of iNOS and guanylate cyclase, but not an adenylate cyclase inhibitor, abolished TNF-alpha-induced lipolysis and HSL phosphorylation. Suppression of TNF-alpha-induced iNOS expression using short hairpin RNA significantly reduced TNF-alpha-induced lipolysis. In conclusion, short-term TNF-alpha treatment induces lipolysis in 3T3-L1 adipocytes by increasing iNOS expression and NO production, which activates the guanylyl cyclase/cGMP-dependent pathway and induces phosphorylation of HSL.
肿瘤坏死因子-α（TNF-α）对脂肪细胞有多种作用，这些作用可能与肥胖受试者2型糖尿病的发生发展有关。许多研究表明，长期用TNF-α治疗可增加脂肪细胞的脂解作用。然而，TNF-α对脂解的短期（<4小时）作用尚未得到充分研究。本研究的目的是探讨TNF-α诱导3T3-L1脂肪细胞脂解的短期调节机制。使用分化良好的3T3-L1脂肪细胞。通过测量甘油释放来测定脂解作用。分别通过蛋白质免疫印迹法和格里斯试剂测定诱导型一氧化氮合酶（iNOS）的表达和一氧化氮（NO）的产生。使用选择性iNOS抑制剂（s-乙基异硫脲·HBr）、腺苷酸环化酶抑制剂（SQ22536）和鸟苷酸环化酶抑制剂（LY83583）来研究iNOS、环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）在TNF-α诱导的脂解中的作用。进行iNOS短发夹RNA的瞬时转染以证实iNOS参与TNF-α诱导的脂解。通过免疫沉淀和蛋白质免疫印迹法测量激素敏感性脂肪酶（HSL）的磷酸化。结果表明，短期TNF-α处理以时间和剂量依赖性方式显著增加脂解、iNOS表达和NO产生。此外，用NO供体S-亚硝基-N-乙酰青霉胺处理也刺激了3T3-L1脂肪细胞的脂解和HSL磷酸化。此外，用iNOS和鸟苷酸环化酶抑制剂预处理，但不是腺苷酸环化酶抑制剂，消除了TNF-α诱导的脂解和HSL磷酸化。使用短发夹RNA抑制TNF-α诱导的iNOS表达显著降低了TNF-α诱导的脂解。总之，短期TNF-α处理通过增加iNOS表达和NO产生诱导3T3-L1脂肪细胞的脂解，这激活了鸟苷酸环化酶/cGMP依赖性途径并诱导HSL磷酸化。
