title: Short-term regulation of tumor necrosis factor-alpha-induced lipolysis in 3T3-L1 adipocytes is mediated through the inducible nitric oxide synthase/nitric oxide-dependent pathway.
abstract: 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.
authors.affiliation: Department of Physiology, National Yang-Ming University, Number 155, Section 2, Li-Nong Street, Taipei, Taiwan. ccjuan@ym.edu.tw
authors.forename: Chih-Chan
authors.initials: CC
authors.lastname: Lien
authors.forename: Lo-Chun
authors.initials: LC
authors.lastname: Au
authors.forename: Ying-Lan
authors.initials: YL
authors.lastname: Tsai
authors.forename: Low-Tone
authors.initials: LT
authors.lastname: Ho
authors.forename: Chi-Chang
authors.initials: CC
authors.lastname: Juan
mesh_terms: D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D015870:Gene Expression / Q000187:drug effects; D008066:Lipolysis / Q000187:drug effects*; D051379:Mice; D041681:NIH 3T3 Cells; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II