HDAC inhibitor Trichostatin A suppresses adipogenesis in 3T3-L1 preadipocytes

Abstract

Background and purpose: Obesity is becoming a major global health issue and is mainly induced by the accumulation of adipose tissues mediated by adipogenesis, which is reported to be regulated by peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα). Trichostatin A (TSA) is a novel histone deacetylase inhibitor (HDACI) that was recently reported to exert multiple pharmacological functions. The present study will investigate the inhibitory effect of TSA on adipogenesis, as well as the underlying mechanism.

Methods: The adipogenesis of 3T3-L1 cells was induced by stimulation with a differentiation cocktail (DMI) medium for 8 days. MTT assay was used to measure the cell viability and Oil Red O staining was used to evaluate the adipogenesis of 3T3-L1 cells. The total level of triglyceride and released glycerol were detected to evaluate the lipolysis during 3T3-L1 adipogenesis. The expression levels of Leptin, fatty acid-binding protein 4 (FABP4), and sterol regulatory element-binding protein (SREBP1C) were determined by qRT-PCR. qRT-PCR assay was utilized to detect the expression levels of PPARγ and C/EBPα in 3T3-L1 cells. A high-fat diet (HFD) was used to construct an obese mice model, followed by the treatment with TSA. HE staining was conducted to evaluate the pathological state of adipose tissues. Body weights and the weights of adipose tissues were recorded to evaluate the anti-obesity property of TSA.

Results: Firstly, the promoted lipid accumulation induced by DMI incubation was significantly reversed by the treatment with TSA in a dose-dependent manner. The elevated expression levels of Leptin, FABP4, SREBP1C, PPARγ, and C/EBPα induced by the stimulation with DMI incubation were dramatically inhibited by the introduction of TSA, accompanied by the upregulation of phosphorylated AMP-activated protein kinase (p-AMPK). Secondly, the inhibitory effect of TSA against the expression level of PPARγ and lipid accumulation was greatly abolished by an AMPK inhibitor. Lastly, the increased body weights and visceral adipocyte tissue weight, as well as the enlarged size of adipocytes induced by HFD were pronouncedly reversed by the administration of TSA.

Conclusion: TSA inhibited adipogenesis in 3T3-L1 preadipocytes by activating the AMPK pathway.

Keywords: AMPK; PPARγ; Trichostatin A; adipogenesis; obesity.

Figure 1

The effects of TSA on cell viability of 3T3-L1 cells. Cells were stimulated with 0.1, 0.25, 0.5, 1, 2.5, and 5 μM TSA for 4 days and 8 days. (A) Cell viability was determined using an MTT on day 4; (B) Cell viability was determined with MTT on day 8 (n= 5-6, *, **, ***, P<0.05, 0.01, 0.001 vs. vehicle group).

Figure 2

TSA inhibits adipogenesis of 3T3-L1 cells. Cells were incubated with a differentiation cocktail (DMI) medium with TSA (0.5 and 1 μM). (A) Cells were stained with Oil Red O on day 8; (B) Lipid accumulation was examined by measuring absorbance at 540 nm of Oil Red O staining. Scale bar, 100 μm (n=5-6, ####, P<0.0001 vs. vehicle group; &&, &&&, P<0.01, 0.001 vs. DMI treatment group).

Figure 3

TSA promotes lipolysis during 3T3-L1 adipogenesis. Cells were incubated with differentiation cocktail (DMI) medium with TSA (0.5 and 1 μM). (A) Total level of triglyceride; (B) Lipolysis is shown as glycerol release (n=5-6, ##, ####, P<0.01, 0.0001, VS. vehicle group; &&, &&&, P<0.01, 0.001 vs. DMI treatment group).

Figure 4

TSA suppresses the expression of Leptin, FABP4, and SREBP1C during 3T3-L1 adipogenesis. Cells were incubated with a differentiation cocktail (DMI) medium with TSA (0.5 and 1 μM). (A) mRNA of Leptin; (B) mRNA of FABP4; (C) mRNA of SREBP1C (n=5-6, ####, P<0.0001 vs. vehicle group; &&, &&&, P<0.01, 0.001 vs. DMI treatment group).

Figure 5

TSA suppresses the expression of adipogenic and lipogenic transcriptional factors in 3T3-L1 cells. Cells were incubated with a differentiation cocktail (DMI) medium with TSA (0.5 and 1 μM). (A) mRNA of PPAR-γ; (B) mRNA of C/EBPα (n=5-6, ####, P<0.0001 vs. vehicle group; &&, &&&, P<0.01, 0.001 vs. DMI treatment group).

Figure 6

TSA activates the AMPK pathway. (A) Cells were incubated with a differentiation cocktail (DMI) medium with TSA (1 μM). Phosphorylated and total levels of AMPKα were measured; (B) Cells were incubated with differentiation cocktail (DMI) medium with TSA (1 μM) or Compound C (10 μM) for 8 days. mRNA of PPAR-γ and C/EBPα; (C) Lipid accumulation was examined by measuring absorbance at 540 nm of Oil Red O (n=5-6, ####, P<0.0001 vs. vehicle group; &&&, P< 0.001 vs. DMI treatment group; $$$, P<0.001 vs. DMI+TSA group).

Figure 7

TSA reduces the weight of visceral adipocyte tissue and the bodyweight of HFD-induced obese mice (10 weeks, start with 6-8 weeks old B6 male mice). (A) Bodyweight; (B) Histological sections of visceral adipocyte tissue; (C) Quantification of adipocyte size; (D) Visceral adipocyte tissue weight (n=5-6, ###, P<0.001 vs. vehicle group; &, &&, P<0.05, 0.01 vs. HFD group).