Effects of lipoic acid on lipolysis in 3T3-L1 adipocytes

Abstract

Lipoic acid (LA) is a naturally occurring compound with beneficial effects on obesity. The aim of this study was to evaluate its effects on lipolysis in 3T3-L1 adipocytes and the mechanisms involved. Our results revealed that LA induced a dose- and time-dependent lipolytic action, which was reversed by pretreatment with the c-Jun N-terminal kinase inhibitor SP600125, the PKA inhibitor H89, and the AMP-activated protein kinase activator AICAR. In contrast, the PI3K/Akt inhibitor LY294002 and the PDE3B antagonist cilostamide enhanced LA-induced lipolysis. LA treatment for 1 h did not modify total protein content of hormone-sensitive lipase (HSL) but significantly increased the phosphorylation of HSL at Ser(563) and at Ser(660), which was reversed by H89. LA treatment also induced a marked increase in PKA-mediated perilipin phosphorylation. LA did not significantly modify the protein levels of adipose triglyceride lipase or its activator comparative gene identification 58 (CGI-58) and inhibitor G(0)/G(1) switch gene 2 (G0S2). Furthermore, LA caused a significant inhibition of adipose-specific phospholipase A2 (AdPLA) protein and mRNA levels in parallel with a decrease in the amount of prostaglandin E(2) released and an increase in cAMP content. Together, these data suggest that the lipolytic actions of LA are mainly mediated by phosphorylation of HSL through cAMP-mediated activation of protein kinase A probably through the inhibition of AdPLA and prostaglandin E(2).

Fig. 1.

LA stimulates lipolysis in 3T3-L1 adipocytes. Mature 3T3-L1 adipocytes were treated with LA (0–500 μM) for the indicated times (1, 3, 6, or 24 h). A: Lipolysis was assessed by the amount of glycerol released into media in adipocytes treated for 24 h. B: Time-dependent effects of LA (250 and 500 μM) on glycerol release. C: Concentration-dependent effects of LA on FFA release in adipocytes treated for 3 h. Data are expressed as mean ± SE of six independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control (vehicle-treated cells).

Fig. 2.

Signaling pathways involved in the lipolytic effects of LA. A–D: Effects of LA on the phosphorylation of JNK (A), ERK1/2 (B), AMPK (C), and PI3K/AKT (D). Band intensities for each phosphorylated species were normalized to their respective total fractions. E: Effects of LA treatment for 24 h on glycerol release in the presence or absence of the JNK inhibitor SP600125 (SP), the AMPK activator AICAR, the ERK1/2 inhibitor PD98059 (PD), the PKA inhibitor H89, the PI3K/AKT inhibitor LY294002 (LY), and the PDE3B inhibitor Cilostamide. Data are expressed as mean ± SE of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. basal control (vehicle-treated cells). ^#P < 0.05, ^##P <0 .01, and ^###P < 0.001 vs. respective control. ^bP < 0.01 and ^cP < 0.001 vs. basal LA-treated adipocytes.

Fig. 3.

Long-term LA treatment down-regulates total HSL, ATGL, and perilipin transcripts. The effects of LA (250 μM) on total ATGL, HSL, and perilipin protein (A) and mRNA (B) levels were assessed in 3T3-L1 adipocytes after 24 h of treatment. C–E: Effects of the JNK inhibitor SP600125 (SP), the ERK1/2 inhibitor PD98059 (PD), the PKA inhibitor H89, the AMPK activator AICAR, and the PI3K/AKT inhibitor LY294002 (LY) on ATGL (C), HSL (D), and Perilipin mRNA (E) levels in control and LA-treated 3T3-L1 adipocytes. Data are expressed as mean ± SE of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.01 vs. basal control (vehicle-treated cells). ^#P < 0.05 and ^##P < 0.01 vs. respective control. ^aP < 0.05 vs. basal LA-treated adipocytes.

Fig. 4.

LA stimulates PKA-mediated phosporylation of HSL and perilipin. A and B: Representative Western blots for Ser⁵⁶³-phosphorylated HSL (A) and Ser⁶⁶⁰-phosphorylated HSL (B) in differentiated 3T3-L1 adipocytes treated with LA (250 μM) for 1 h in the presence or absence of the JNK inhibitor SP600125 (SP), the AMPK activator AICAR, the ERK1/2 inhibitor PD98059 (PD), and the PKA inhibitor H89. Band intensities were normalized to total HSL. C: Adipocyte lysates were immunoblotted using a phospho-PKA-motif-specific antibody, and the blots were stripped and reprobed with antiperilipin antibodies to detect native perilipins. The density of the protein bands was quantified, and the data (mean ± SE) were expressed as p-PKA substrate/perilipin ratio (n ≥ 3 independent experiments). *P < 0.05 and ***P < 0.001 vs. basal control (vehicle-treated cells). ^#P < 0.05 vs. respective control. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001 vs. basal LA-treated cells.

Fig. 5.

LA does not modify the levels of the ATGL coactivator CGI-58 or the ATGL inhibitor G0S2. A and B: Lysates from 3T3-L1 adipocytes treated with LA (250 μM) for 1 h (A) and 24 h (B) were immunoblotted for ATGL, CGI-58, G0S2, and actin antibody. Band intensities for ATGL, CGI-58, and G0S2 were normalized to actin. Data are expressed as mean ± SE of at least five independent experiments. *P < 0.05 vs. control (vehicle-treated cells).

Fig. 6.

LA reduces AdPLA levels and PGE₂ secretion and increases intracellular cAMP levels in 3T3-L1 adipocytes. A: AdPLA protein levels at 1 and 24 h of treatment with LA (250 μM). B: PGE₂ released to the media in 3T3-L1 adipocytes treated with LA (250 µM) for 24 h. C: Intracellular cAMP levels at 1 and 24 h of treatment with LA (250 μM). D: Effects of PGE₂ (0.5 ng/ml) on the lipolytic action of LA (250 μM) in the presence or absence of the EP3 antagonist L78106 (10 μM). Data are expressed as mean ± SE of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.01 vs. control (vehicle-treated cells). ^#P < 0.05 vs. PGE2-treated cells. ^aP < 0.05 vs. basal LA-treated cells.