β-Hydroxybutyrate Suppresses Lipid Accumulation in Aged Liver through GPR109A-mediated Signaling
A Kyoung Lee1, Dae Hyun Kim1, EunJin Bang1, Yeon Ja Choi2, Hae Young Chung1,*
1Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 46241, Korea 2Department of Biopharmaceutical Engineering, Division of Chemistry and Biotechnology, Dongguk University, Gyeongju 38066, Korea
Dietary interventions such as prolonged calorie restriction (CR) and intermittent fasting provide health benefits including a reduction in the inflammatory burden and regulation of energy metabolism. During CR, β-hydroxybutyrate (BHB) level is elevated in the serum. BHB is a ligand of GPR109A, which inhibits lipolysis and exerts anti-inflammatory effects on cells. During aging, comorbidities related to dyslipidemia are significantly associated with fatty liver. However, the underlying mechanisms of BHB in hepatic ER stress and dyslipidemia are unclear and remain to be elucidated. Here, we used aged rats that were administered with BHB and compared the modulatory effects of BHB through the GPR109A/AMPK pathway on the hepatic endoplasmic reticulum (ER) stress and lipid accumulation to CR rats. BHB caused suppression of hepatic ER stress and lipid accumulation through GPR109A/AMPK pathway in the aged rats. Aged rats of both treatment groups showed reduced cAMP level and PKA phosphorylation. Furthermore, AMPK-Ser173 phosphorylation via PKA was decreased, whereas AMPK-Thr172 phosphorylation was increased by BHB and CR. Further supporting evidence was provided in HepG2 cells that BHB inhibited ER stress and lipid accumulation induced by palmitate. These results suggest that BHB activates GPR109A and regulates the activation of AMPK. These findings were further confirmed by GPR109A-siRNA transfection in vitro. In addition, BHB treatment elevated the protein levels of AMPK leading to significant inhibition of hepatic steatosis, whereas AMPK-siRNA treatment abolished these effects. Taken together, these findings suggest that BHB could be a effective molecule that mimics CR in ameliorating age-related hepatic lipid accumulation via GPR109A signaling pathway.
Table 2 Changes in body weight, food intake and liver weight intake during the experimental period.
Figure 1. Changes in lipid and β-hydroxybutyrate levels in the serum. A series of plasma profiles from BHB-treated or calorie-restricted aged rats are shown. Aged rats (24 months old) were treated with BHB for 30 days (10 or 100 mg/kg/day P.O.) and the respective parameters were compared with young rats (6 months old). BHB was administered to the aged rats (n = 4 each). (A) Free fatty acid (FFA), (B) triglyceride (TG), and (C) β-hydroxybutyrate levels were measured after 30 days of BHB treatment. One-factor ANOVA was used to determine the significant differences. #p < 0.05, ##p < 0.01 vs. young; *p < 0.05, ***p < 0.001 vs. old. Y, young rats (6 months old).
Figure 2. Effects of BHB on ER stress in the liver of the aged rats. Western blotting was performed to detect the levels of the ER stress markers (p-PERK and p-IRE), and the downstream signal p-JNK. Western blot results from three independent experiments were quantified by densitometry. One-factor ANOVA was used to determine the significant differences. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. young; *p < 0.05, **p < 0.001, ***p < 0.001 vs. old.
Figure 3. Changes in lipid accumulation, lipogenic genes, and β-oxidation genes in the livers of BHB-treated aged rats. (A) Hepatic TG contents, (B) mRNA expression of lipogenic genes, and (C) β-oxidation genes were evaluated by q-PCR. The results were normalized to the expression of a reference gene (GAPDH). One-factor ANOVA was used to determine the significant differences. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. young; *p < 0.05, **p < 0.01, ***p < 0.001 vs. old. (D) Changes in lipid accumulation in calorie-restricted (CR) and BHB-treated aged rats. Aged liver tissues were stained with Oil red O to visualize lipid accumulation. Scale bar: 400 μm.
Figure 4. Effects of BHB on palmitate-induced ER stress. HepG2 cells incubated with BHB (200 and 400 µM) for 3 h followed by treatment with palmitate (500 µM) for 24 h. Western blotting was performed to detect the levels of the ER stress markers (p-PERK and p-IRE). and the downstream signal p-JNK. Western blot results from three independent experiments were quantified by densitometry. One-factor ANOVA was used to determine the significant differences. ##p < 0.01 vs. normal; *p < 0.05, **p < 0.01 vs. palmitate (500 µM).
Figure 5. Changes in lipid accumulation in the BHB-treated HepG2 cells. (A) Cellular TG content and (B) Oil red O staining of palmitate-induced HepG2 cells are shown. HepG2 cells were pre-treated with BHB (200 and 400 µM) for 3 h followed by treatment with palmitate (500 µM) for 24 h. Scale bar: 100 µm. mRNA expression of (C) lipogenic genes and (D) β-oxidation genes were evaluated by q-PCR. The results were normalized to the expression of a reference gene (GAPDH). One-factor ANOVA was used to determine the significant differences. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. normal; *p < 0.05, **p < 0.01 vs. palmitate (500 µM).
Figure 6. Changes in AMPK phosphorylation through the GPR109A signaling pathway in BHB-treated rat liver. (A) cAMP level in the liver homogenate. (B) Western blotting was performed to detect the levels of p-PKA, p-AMPK (Ser), p-AMPK (Thr), and AMPK in rat liver. Western blot results from three independent experiments were quantified by densitometry. One-factor ANOVA was used to determine the significant differences. #p < 0.05, ##p < 0.01 vs. young; *p < 0.05, **p < 0.01 vs. old.
Figure 7. Effects of BHB on AMPK-Thr172 phosphorylation through the GPR109A signaling pathway in HepG2 cells. (A) Intracellular cAMP was treated with 400 µM BHB for 1 h in HepG2 cells. The results of Student’s t-test are shown. *p < 0.005 vs. normal. (B) HepG2 cells were incubated with BHB (200 and 400 µM) for 6 h. Western blotting was performed to detect the levels of p-PKA, p-AMPK (Ser), p-AMPK (Thr), and AMPK in HepG2 cells. Western blot results from three independent experiments were quantified by densitometry. One-factor ANOVA was used to determine the significant differences. *p < 0.05, **p < 0.01 vs. normal. (C) The possible mechanisms of the protective effects of BHB on hepatic lipid accumulation during aging are shown. Activation of GPR109A inhibited adenylyl cyclase and subsequent reduction in PKA activity by BHB. PKA dephosphorylation reduced AMPK-Ser173 and increased AMPK-Thr172 phosphorylation by BHB. Increased AMPK activity inhibited ER stress and lipid accumulation.
Figure 8. BHB suppressed ER stress and lipid accumulation through GPR109A/PKA/AMPK in the cells. (A) The cells were treated with the BHB (400 µM) for 4 h. The cells pre-incubated with GPR109A-siRNA (20 nM) for 44 h were subjected to western blotting analysis using GAPDH as control. GPR109A, p-PKA, PKA, p-AMPK (Ser), p-AMPK (Thr), and AMPK levels were assessed. (B) The cAMP level measured after stimulation with BHB in the absence (-) or presence (+) of GPR109A-siRNA in the liver. The results of Student’s t-test are shown. #p < 0.05 vs. normal; *p < 0.05 vs. GPR109A-siRNA treated cells. (C) HepG2 cells were transfected and pre-incubated with PKI (10 µM) for 1 h. The cells were analyzed by Western blotting using PKA, p-PERK, PERK, p-IRE, IRE, and GAPDH antibodies. (D) The cells were treated with the BHB (400 µM) for 4 h. The cells pre-incubated with AMPK-siRNA (20 nM) for 44 h were subjected to western blotting analysis using GAPDH as control. AMPK and ER stress markers (p-PERK, PERK, p-IRE, and IRE) were assessed using cytosolic proteins from HepG2 cells. (E) The cells incubated with BHB (400 µM) for 4 h. The cells pre-transfected with AMPK-siRNA (20 nM) for 44 h were subjected to qPCR analysis using actin as a control. The mRNA expression of the lipogenic genes (SREBP-1c, PPAR, FAS, and SCD) was assessed. The results were normalized with respect to the actin level. #p < 0.05 vs. Normal; *p < 0.05 vs. AMPK-siRNA group.
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