Insulin inhibits isoproterenol-stimulated lipolysis in 3T3-L1 adipocytes via the AKT/PKA/HSL pathway.

We treated differentiated adipocytes with 100 nM insulin and 1 μM isoproterenoland examined the effects on lipolysis(in triplicate). (a)Western blot analyses ofAKT, PDE3B, PKA, HSL and ATGL protein levels in the insulin, isoproterenol and control groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (b)The phosphorylated protein/total protein ratios of AKT, PKA and HSL were calculated, and the control ratio was normalized to 1. (c)Western blot analyses of PDE3B protein levels in the insulin, isoproterenol and control groups. Differentiated 3T3-L1 adipocytes were treated with different doses of insulin (1, 10,100 nM) and 1 μM isoproterenol for 24 h(in triplicate). (d) Cellular triglycerides were stained with Oil red O. Bar, 50 μm. (e) The amount of lipids was quantified by the Oil red O staining technique. (f)The concentration of glycerol in the medium was detected in different groups(g, i)Western blot analyses of AKT, PDE3B, PKA, HSL and ATGL protein levels in different groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (h) The phosphorylated protein/total protein ratios were calculated, and the control ratio was normalized to 1.(j)cAMP levels in different groups. (k) The expression of PDE3Bwas determined in different groups by Western blot.