β-aminoisobutyric acid attenuates LPS-induced inflammation and insulin resistance in adipocytes through AMPK-mediated pathway

Abstract

Background: β-aminoisobutyric acid (BAIBA) is produced in skeletal muscle during exercise and has beneficial effects on obesity-related metabolic disorders such as diabetes and non-alcoholic fatty liver disease. Thus, it is supposed to prevent high fat diet (HFD)-induced inflammation and insulin resistance in adipose tissue though anti-inflammatory effects in obesity. Previous reports have also demonstrated strong anti-inflammatory effects of BAIBA.

Methods: We used BAIBA treated fully differentiated 3T3T-L1 mouse adipocytes to investigate the effects of exogenous BAIBA on inflammation and insulin signaling in adipocytes. Insulin signaling-mediated proteins and inflammation markers were measured by Western blot analysis. Secretion of pro-inflammatory cytokines were measured by ELISA. Lipid accumulation in differentiated 3 T3-L1 cells was stained by Oil red-O. Statistical analysis was performed by ANOVA and student's t test.

Results: BAIBA treatment suppressed adipogenesis assessed by adipogenic markers as well as lipid accumulation after full differentiation. We showed that BAIBA treatment stimulated AMP-activated protein kinase (AMPK) phosphorylation in a dose-dependent manner and lipopolysaccharide (LPS)-induced secretion of pro-inflammatory cytokines such as TNFα and MCP-1 was abrogated in BAIBA-treated 3 T3-L1 cells. Treatment of 3 T3-L1 cells with BAIBA reduced LPS-induced NFκB and IκB phosphorylation. Furthermore, BAIBA treatment ameliorated LPS-induced impairment of insulin signaling measured by IRS-1 and Akt phosphorylation and fatty acid oxidation. Suppression of AMPK by small interfering (si) RNA significantly restored these changes.

Conclusions: We demonstrated anti-inflammatory and anti-insulin resistance effects of BAIBA in differentiated 3 T3-L1 cells treated with LPS through AMPK-dependent signaling. These results provide evidence for the beneficial effects of BAIBA not only in liver and skeletal muscle cells but also in adipose tissue.

Keywords: AMPK; Adipocyte; BAIBA; Inflammation; Insulin resistance; NFκB.

Fig. 1

BAIBA suppresses TG accumulation in 3 T3-L1 cells during differentiation. a Oil-red O staining in differentiated 3 T3-L1 cells in the presence of BAIBA (30 μM) for 0, 6, or 10 days. TG accumulation was quantitated by modified TG assay kit. Quantitative real-time PCR analysis of PPARγ (b), FABP4 (c), adiponectin (d), and fatty acid synthase (e) mRNA expression in differentiated 3 T3-L1 cells treated with BAIBA (0–30 μM) for 10 days. Means ± SEM were obtained from three separated experiments. ^***P < 0.001, ^**P < 0.01, and ^*P < 0.05 when compared to the control

Fig. 2

BAIBA ameliorates LPS-induced inflammation in differentiated 3 T3-L1 cells through AMPK-mediated pathway. a Western blot analysis of AMPK phosphorylation and adiponectin expression in differentiated 3 T3-L1 cells treated with BAIBA (0–30 μM) for 10 days. b Western blot analysis of LPS-induced NFκB and IκB phosphorylation in siRNA for AMPK transfected differentiated 3 T3-L1 cells treated with 10 μg/ml LPS and BAIBA (0–30 μM) for 10 days. Culture media analysis of (c) TNFα and (d) MCP-1 in siRNA for AMPK transfected differentiated 3 T3-L1 cells treated with 10 μg/ml LPS and BAIBA (0–30 μM) for 10 days. Means ± SEM were calculated data obtained from three independent experiments. ^***P < 0.001, ^**P < 0.01, and ^*P < 0.05 when compared to the control.^!!!P < 0.001,^!!P < 0.01, and^!P < 0.05 when compared to the LPS treatment. ^##P < 0.01, and ^#P < 0.05 when compared to the LPS plus BAIBA treatment

Fig. 3

BAIBA attenuates LPS-induced insulin resistance in differentiated 3 T3-L1 cells through AMPK-mediated signaling. a Western blot analysis of phosphorylation of IRS-1 and Akt in siRNA for AMPK transfected differentiated 3 T3-L1 cells treated with 10 μg/ml LPS and BAIBA (0–30 μM) for 10 days. Human Insulin (10 nM) stimulates IRS-1 and Akt phosphorylation for 3 min. b 2-deoxyglucose uptake in scramble or AMPKsiRNA transfected differentiated 3 T3-L1 cells treated with 10 μg/ml LPS and BAIBA (0–30 μM) for 10 days. Human Insulin (10 nM) stimulates glucose uptake for 30 min. Means ± SEM were calculated data obtained from three independent experiments. ^***P < 0.001 when compared to the insulin treatment.^!!!P < 0.001,^!!P < 0.01, and^!P < 0.05 when compared to the insulin plus LPS treatment. ^###P < 0.001, ^##P < 0.01, and ^#P < 0.05 when compared to the insulin, LPS plus BAIBA treatment

Fig. 4

BAIBA stimulates fatty acid oxidation through AMPK signaling. Intracellular acetyl-CoA (a) and ATP levels (b) in scramble or AMPKsiRNA transfected differentiated 3 T3-L1 cells treated with 10 μg/ml LPS and BAIBA (0–30 μM) for 10 days. c LCAD expression levels in intact human primary adipocytes treated with 10 μg/ml LPS and BAIBA (0–30 μM) for 10 days and compound C (10 μM) for 24 h. Western blot analysis of ACC phosphorylation (d) and CPT1 expression (e) in differentiated 3 T3-L1 cells treated with 10 μg/ml LPS and BAIBA (0–30 μM) for 10 days. Means ± SEM were obtained from three separated experiments. ^***P < 0.001 when compared to the insulin treatment.^!!!P < 0.001,^!!P < 0.01, and^!P < 0.05 when compared to the insulin plus LPS treatment. ^###P < 0.001, ^##P < 0.01, and ^#P < 0.05 when compared to the insulin, LPS plus BAIBA treatment

Fig. 5

Schematic diagram of the effects of BAIBA on inflammation and insulin resistance in adipocytes. BAIBA administration stimulates AMPK activation, resulting in suppression of lipogenesis, inflammation, and insulin resistance in differentiated adipocytes