Laquinimod Prevents Adipogenesis and Obesity by Down-Regulating PPAR-γ and C/EBPα through Activating AMPK

Abstract

Background and purpose: obesity is defined as excessive accumulation of adipose tissues and is becoming one of the main global severe public health issues. The present study aims to investigate the anti-adipogenesis of laquinimod and the underlying mechanism.

Methods: a differentiation cocktail was used to differentiate 3T3-L1 cells, and mice were fed with high fat food to establish the obesity animal model. Oil red O staining, glycerol production assay, and the release of triglyceride were used to evaluate the differentiation degree of 3T3-L1 cells. The expression level of sterol regulatory element binding transcription factor 1 (Srebp1), fatty acid binding protein-4 (FABP4), glucose transporter 4 (GLUT4), peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer-binding proteins (C/EBPα), and phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase α (p-AMPKα) was determined by quantitative real time PCRqRT-PCR and western blot analysis. The pathological state of adipose tissues was evaluated by hematoxylin-eosin staining.

Results: the amount and UV absorption of oil red O, glycerol production, release of triglyceride, and the expression of SREBP1, FABP4, and Glut4 in differentiated 3T3-L1 cells were decreased by the administration of laquinimod. PPAR-γ and C/EBPα were down-regulated, and p-AMPKα was up-regulated by laquinimod. The down-regulated PPAR-γ and C/EBPα, as well as the inhibited lipid accumulation functioned by laquinimod, were reversed by the coincubation with the AMPK inhibitor compound C. Decreased body weight, visceral adipocyte tissue weight, and size of adipocytes were observed in in vivo obesity mice after administration with laquinimod.

Conclusion: laquinimod might prevent adipogenesis by down-regulating PPAR-γ and C/EBPα through activating AMPK.

Figure 1

Molecular structure of laquinimod.

Figure 2

Effects of laquinimod during 3T3-L1 adipogenesis. 3T3-L1 cells were differentiated by incubation in cell culture medium by adding a differentiation cocktail (DMI) with laquinimod (5, 10 μM) and stained with Oil Red O on day 8. (A) Representative images of Oil Red O staining; Scale bar, 100 μm; (B) lipid accumulation was assessed by measuring absorbance at 540 nm of Oil Red O (****, p < 0.0001 vs vehicle group; $$, $$$$, P < 0.01, 0.0001 vs DMI treated group).

Figure 3

Effects of laquinimod on triglyceride content and lipolysis during 3T3-L1 adipogenesis. 3T3-L1 cells were diferentiated by incubation in cell culture medium by adding a differentiation cocktail (DMI) with laquinimod (5, 10 μM) for 8 days. (A) Total content of triglyceride; (B) lipolysis is shown as glycerol release (****, p < 0.0001 vs vehicle group; $$, $$$$, P < 0.01, 0.0001 vs DMI treated group).

Figure 4

Effects of laquinimod on the expression of adipogenic and lipogenic genes. 3T3-L1 cells were diferentiated by incubation in cell culture medium by adding a differentiation cocktail (DMI) with laquinimod (5, 10 μM) for 3, 6, 8 days. (A) mRNA of SREBP1 as measured by real time PCR; (B) mRNA of FABP4 as measured by real time PCR; (C) mRNA of Glut4 as measured by real time PCR (****, p < 0.0001 vs vehicle group; $$, $$$$, P < 0.01, 0.0001 vs DMI treated group).

Figure 5

Effects of laquinimod on the expression of adipogenic and lipogenic transcriptional factor. 3T3-L1 cells were differentiated by incubation in cell culture medium by adding a differentiation cocktail (DMI) with laquinimod (5, 10 μM) for 8 days. (A) mRNA of PPAR-γ as measured by real time PCR; (B) mRNA of C/EBPα as measured by real time PCR; (C) protein of PPAR-γ as measured by western blot analysis; (D) protein of C/EBPα as measured by western blot analysis (****, p < 0.0001 vs vehicle group; $$, $$$$, P < 0.01, 0.0001 vs DMI treated group).

Figure 6

Laquinimod suppressed autophagy in 3T3-L1 cells. 3T3-L1 preadipocytes were incubated in adipocyte DM in the presence or absence of laquinimod (5, 10 μM). The expression of the autophagy markers LC3-I and LC3-II was measured by western blot and the LC3-II/LC3-I ratio was quantified by densitometric scanning and graphed (****, p < 0.0001 vs vehicle group; $$, $$$$, P < 0.01, 0.0001 vs DMI treated group).

Figure 7

Inhibitory effects of laquinimod in 3T3-L1 adipogenesis is mediated by AMPK. (A) 3T3-L1 cells were differentiated by incubation in cell culture medium by adding a differentiation cocktail (DMI) with laquinimod (5, 10 μM) for 24 h. Phosphorylation of AMPKα (1, 0.39, 0.68, 0.92) was measured by western blot analysis. (B–D) 3T3-L1 cells were differentiated by incubation in cell culture medium by adding a differentiation cocktail (DMI) with laquinimod (10 μM) in the presence or absence of AMPKα inhibitor compound C (10 μM) for 8 days. mRNA of PPAR-γ; mRNA of C/EBPα; lipid accumulation was assessed by measuring absorbance at 540 nm of Oil Red O (****, p < 0.0001 vs vehicle group; $$, $$$$, P < 0.01, 0.0001 vs DMI treated group).

Figure 8

Laquinimod decreased the weight of visceral adipocyte tissues and the body weight of HFD-induced obese mice. (A) Growth curve; (B) visceral adipocyte tissue weight; (C) histological sections of visceral adipocyte tissue in mice; and (D) quantification of adipocyte size (****, p < 0.0001 vs vehicle group; $$, $$$$, P < 0.01, 0.0001 vs DMI treated group).