Quercetin enhances fatty acid β-oxidation by inducing lipophagy in AML12 hepatocytes

Abstract

Recent evidence demonstrated that chronic intake of quercetin attenuated hepatic fat accumulation in various animal models of obesity and diabetes. However, whether quercetin has the ability to enhance energy metabolism in hepatocytes and its exact mechanisms have yet to be identified. In the present study, we investigated whether quercetin directly enhanced the energy metabolism of cultured hepatocytes by focusing on lipophagy, involving selective autophagic degradation of lipid droplets. As an indicator of mitochondrial respiration, oxygen consumption was measured following 12-h treatment with quercetin or its related flavonoids, isorhamnetin and rutin (10 μM) using an extracellular flux analyzer. Treatment of alpha mouse liver 12 (AML12) hepatocytes with quercetin enhanced mitochondrial respiration, but isorhamnetin and rutin did not. Results of a palmitate-bovine serum albumin fatty acid oxidation assay showed that quercetin significantly increased the oxygen consumption of AML12 hepatocytes, suggesting enhanced fatty acid β-oxidation. However, as expression levels of mitochondrial oxidative phosphorylation proteins were unaltered by quercetin, we explored whether lipophagy contributed to enhanced fatty acid β-oxidation. Increased colocalization of lipid droplets and lysosomes confirmed that quercetin promoted lipophagy in AML12 hepatocytes. Furthermore, pharmacological inhibition of the autophagy-lysosomal pathway abolished the enhancement of fatty acid β-oxidation induced by quercetin in AML12 hepatocytes, suggesting that the enhancement of lipophagy by quercetin contributed to increased fatty acid β-oxidation. Finally, we showed that quercetin could activate AMPK signaling, which regulates autophagy even under nutrient-sufficient conditions. Our findings indicate that quercetin enhanced energy metabolism by a potentially novel mechanism involving promotion of lipophagy to produce the substrate for fatty acid β-oxidation in mitochondria through activation of AMPK signaling. Our results suggest the possibility that nutrient-induced lipophagy might contributes to the reduction of fat in hepatocytes.

Keywords: AMPK; Fatty acid β-oxidation; Hepatocyte; Lipophagy; Quercetin.

Figure 1

Effect of quercetin, isorhamnetin, and rutin on mitochondrial respiration of AML12 hepatocytes. A Cell Mito Stress Test was performed to investigate the effect of (a) quercetin, (b) isorhamnetin, and (c) rutin on mitochondrial respiration. Oligomycin (Oligo), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone with antimycin A (Rot & AA) were sequentially injected onto culture microplates to determine individual parameters of mitochondrial function (non-mitochondrial oxygen consumption, basal respiration, maximum respiration, H⁺ leak, ATP production, and spare respiration capacity). The results show enhanced mitochondrial respiration following quercetin treatment (10 μM) for 12 h. Significance was determined with a two-tailed Student's t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Values represent mean ± SEM (n = 3). C, control; Iso, isorhamnetin; Q, quercetin; R, rutin. (d) Results of Glycolysis Stress Test show enhanced glycolysis following quercetin treatment (10 μM) for 12 h. Glucose (Glu), Oligomycin (Oligo), and 2-deoxy-D-glucose (2-DG) were sequentially injected onto culture microplates to determine individual parameters of glycolysis (glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification).

Figure 2

Effect of quercetin on fatty acid β-oxidation and mitochondrial marker expression of AML12 hepatocytes. (a) Fatty acid oxidation (FAO) assay results indicated enhancement of fatty acid β-oxidation by quercetin treatment (10 μM) for 12 h. FAO was measured by adding palmitate conjugated to BSA into XF base media, combined with Mito stress test (n = 3). (b) Expression of genes related to mitochondrial biogenesis, lipid metabolism, and autophagy (Tfam, Ppara, Pparg, Ppargc1a, Cpt1, Cpt2, Map1lc3b, and Plin2) were measured by qRT-PCR. mRNA expression levels were calculated relative to Gapdh and data are expressed as a fold-increase (n = 6). (c) Expression of mitochondrial oxidative phosphorylation proteins (ATP5A, UQCR2, MTCO1, SDHB, and NDUFB8) was measured by immunoblot (left). Protein expression levels were calculated relative to GAPDH. Right graphs show relative intensity of each band (n = 3). Values represent mean ± SEM. Significance was determined with a two-tailed Student's t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 3

Relationship between quercetin and autophagy/lipophagy in AML12 hepatocytes. (a) Representative images of subcellular location of lipid droplets (BODIPY: green) and lysosomes (lysotracker: red) in AML12 hepatocytes treated with quercetin for 24 h (left: control, right: quercetin). Co-localization of BODIPY and lysotracker was evaluated by ImageJ co-localization analysis (n = 7). Scale bar = 10 μm. (b) Comparison of triglycerides between control and quercetin-treated (50 μM) AML12 hepatocytes (n = 6). (c) Fatty acid oxidation (FAO) assay showed that the enhancement fatty acid β-oxidation by quercetin treatment (10 μM) was cancelled by the autophagy–lysosomal pathway inhibitor 3-Methyladenine (3-MA). FAO was measured by adding palmitate conjugated to BSA into XF base media, combined with Mito stress test (n = 3). Values represent mean ± SEM. Significance was determined with a two-tailed Student's t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 4

Effect of quercetin on autophagy-related signaling pathways in AML12 hepatocytes. The effect of quercetin (50 μM) on phosphorylation levels of AMPK (T172), ERK1/2 (T202 and Y204), Akt (S473), and mTOR (S2443), as well as activation of autophagy (ratio of LC3-II/LC3-I) were measured by immunoblot. Cells were cultured in serum-free DMEM/F-12 for 12 h before quercetin treatment. Phosphorylation levels were calculated relative to total protein (n = 3). Values represent mean ± SEM.