Synergistic effects of cAMP-dependent protein kinase A and AMP-activated protein kinase on lipolysis in kinsenoside-treated C3H10T1/2 adipocytes.

We previously showed that 3-O-β-D-glucopyranosyl-(3R)-hydroxybutanolide (kinsenoside), a major compound of Anoectochilus formosanus, increased lipolysis through an AMP-activated protein kinase (AMPK)-dependent pathway.To extend our previous finding, we investigated the in vivo and in vitro effects of kinsenoside on lipolysis and the involvement of cyclic AMP (cAMP)-dependent protein kinase A (PKA) and AMPK in kinsenoside-mediated lipolysis.Mice were fed a high-fat diet for six weeks to induce lipid deposition and then treated with 50 and 100 mg/kg kinsenoside for two weeks. The coordination of PKA and AMPK activation in lipolysis in C3H10T1/2 adipocytes was evaluated in vitro by using PKA and AMPK's corresponding inhibitors, oil-red O staining, a glycerol production assay, and Western blot analysis.Kinsenoside reduced body weight, fat pad mass, and hepatic lipid accumulation in obese mice, and concurrently increased the induction and activation of hormone-sensitive lipase (HSL), perilipin, adipose triglyceride lipase (ATGL), and carnitine palmitoyltransferase I (CPT1). Kinsenoside concentration-dependently increased PKA activation by increasing the phosphorylation of Ser/Thr-PKA substrates in vitro. These increases were accompanied by a reduction in fat accumulation. Using H89 and Rp-8-Br-cAMPs to inhibit PKA reduced the release of glycerol but did not alter the activation of peroxisome proliferator-activated receptor alpha or the expression of CPT1 or ATGL. By contrast, compound C, an AMPK inhibitor, inhibited CPT1 and ATGL expression in kinsenoside-treated C3H10T1/2 adipocytes. In addition, H89 caused the reactivation of AMPK downstream targets by increasing the levels of the active form of pAMPK-Thr172, suggesting that PKA negatively modulates AMPK activity.Kinsenoside increased HSL activation through PKA-mediated phosphorylation at Ser660/563 and concomitantly increased perilipin activation in lipolysis. These lipolytic effects of kinsenoside were validated using 6-Bnz-cAMPs, a PKA agonist. In this study, we demonstrated that in addition to AMPK, PKA also plays a crucial role in kinsenoside-mediated lipolysis.