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Short-Term Regulation of Tumor Necrosis Factor--Induced Lipolysis in 3T3-L1 Adipocytes Is Mediated through the Inducible Nitric Oxide Synthase/Nitric Oxide-Dependent Pathway

Department of Physiology (C.-C.L., C.-C.J.), National Yang-Ming University, Taipei 11221, Taiwan; Department of Medical Research and Education (L.-C.A., L.-T.H., C.-C.J.), Taipei Veterans General Hospital, Taipei 11217, Taiwan; Department of Athletic Training and Health (Y.-L.T.), National Taiwan Sport University, Taoyuan County 33301, Taiwan; and Department of Education and Research (C.-C.J.), Taipei City Hospital, Taipei 10341, Taiwan

Address all correspondence and requests for reprints to: Chi-Chang Juan, Ph.D., Department of Physiology, National Yang-Ming University, Number 155, Section 2, Li-Nong Street, Taipei, Taiwan. E-mail: ccjuan{at}ym.edu.tw.

TNF- 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- increases lipolysis in adipocytes. However, the short-term (<4 h) effects of TNF- on lipolysis have not been well investigated. The aim of this study was to investigate the short-term regulatory mechanism of TNF--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--induced lipolysis. Transient transfection with iNOS short hairpin RNA was performed to confirm the involvement of iNOS in TNF--induced lipolysis. Phosphorylation of hormone-sensitive lipase (HSL) was measured by immunoprecipitation and Western blotting. Results showed that short-term TNF- 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--induced lipolysis and HSL phosphorylation. Suppression of TNF--induced iNOS expression using short hairpin RNA significantly reduced TNF--induced lipolysis. In conclusion, short-term TNF- 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.