Naringenin Attenuates Non-Alcoholic Fatty Liver Disease by Enhancing Energy Expenditure and Regulating Autophagy via AMPK

Abstract

Background: The prevalence of non-alcoholic fatty liver disease (NAFLD) keeps growing recently. Purpose: To investigate the effects and mechanisms of naringenin (NAR) on NAFLD. Methods: High-fat diet (HFD)-induced NAFLD rats were orally administered with NAR at 10, 30, and 90 mg/kg for 2 weeks. The serum level of triglyceride (TG), total cholesterol (TC), glutamic-oxaloacetic transaminase (AST), and glutamic-pyruvic transaminase (ALT) was measured. The hepatic histology was detected by H&E and oil red O staining. L02 and Huh-7 cells were induced by sodium oleate to establish a NAFLD cell model. The effects of NAR on lipid accumulation were detected by oil red O staining. The glucose uptake and ATP content of 3T3-L1 adipocytes and C2C12 myotubes were measured. The expression of proteins of the AMPK signaling pathway in 3T3-L1 adipocytes and C2C12 myotubes was assessed by Western blotting. The mitochondrial biogenesis of 3T3-L1 adipocytes and C2C12 myotubes was measured by mitotracker orange staining and Western blotting. The biomarkers of autophagy were detected by Western blotting and immunofluorescence. The binding of NAR to AMPKγ1 was analyzed by molecular docking. Chloroquine and compound C were employed to block autophagic flux and AMPK, respectively. Results: NAR alleviated HFD-induced NAFLD in rats at 10, 30, and 90 mg/kg. NAR attenuated lipid accumulation in L02 and Huh-7 cells at 0.7, 2.2, 6.7, and 20 μM. NAR increased glucose uptake, decreased the ATP content, activated the CaMKKβ/AMPK/ACC pathway, and enhanced the mitochondrial biogenesis in 3T3-L1 adipocytes and C2C12 myotubes. NAR increased autophagy and promoted the initiation of autophagic flux in 3T3-L1 preadipocytes and C2C12 myoblasts, while it inhibited autophagy in NAFLD rats, 3T3-L1 adipocytes, and C2C12 myotubes. Molecular docking showed that NAR binds to AMPKγ1. Compound C blocked effects of NAR on lipid accumulation and autophagy in L02 cells. Conclusion: NAR alleviates NAFLD by increasing energy expenditure and regulating autophagy via activating AMPK directly and indirectly. The direct binding of NAR and AMPKγ1 needs further validation.

Keywords: autophagy; energy expenditure; mitochondrial biogenesis; naringenin; non-alcoholic fatty liver disease.

GRAPHICAL ABSTRACT

FIGURE 1

NAR attenuates HFD-induced NAFLD in rats. Male Sprague-Dawley rats aged 8 weeks were fed with an HFD or ND for 12 weeks. Then they were orally administered with NAR at 10, 30, or 90 mg/kg for 2 weeks n = 5 rats per group. (A) Chemical structure of NAR. (B–E) Serum level of TG, TC, ALT, and AST. (F–H) Representative H&E and oil red O staining of hepatic sections (the objective amplification 10×). Bars represent 50 μM. All values were expressed as the mean ± SD from three or more independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. HFD group. Abbreviation: NAR, naringenin; NAFLD, nonalcoholic fatty liver disease; ND, normal diet; HFD, high-fat diet; TG, triglyceride; TC, total cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; IOD, integrated optical density; H&E, hematoxylin and eosin.

FIGURE 2

NAR alleviates lipid accumulation in L02 and Huh-7 cells, enhances glucose uptake, and decreases the ATP content in 3T3-L1 adipocytes and C2C12 myotubes. Sodium oleate-induced L02. (A,B) and Huh-7 cells (C,D) were treated with NAR (0, 0.7, 2.2, 6.7, and 20 μM) for 24 h. Lipid accumulation was visualized using oil red O staining (the objective amplification 20×). 3T3-L1 adipocytes (E,F) and C2C12 myotubes (G,H) were treated with NAR (0, 2.2, 6.7, and 20 μM), phloretin (100 μM), or metformin (2.5 mM) for 4 h. Glucose uptake was detected by 2-NBDG staining and photographed by fluorescence microscopy (the objective amplification 10×). The ATP content of 3T3-L1 adipocytes (I) and C2C12 myotubes (J) was determined using an ATP calorimetric assay kit. Bars represent 20 μM (A,C), 50 μM (E,G). All values were expressed as the mean ± SD from three or more independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. untreated cells. Abbreviation: NAR, naringenin; Phl, phloretin; Met, metformin; 2-NBDG, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose.

FIGURE 3

NAR activates the CaMKKβ/AMPK/ACC pathway in 3T3-L1 adipocytes and C2C12 myotubes. The expressions of CaMKKβ, p-LKB1, LKB1, p-AMPK, AMPK, p-ACC, and ACC of 3T3-L1 adipocytes (A–E) and C2C12 myotubes (F–J) after treatment of NAR were measured by Western blotting. All values were expressed as the mean ± SD from three or more independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. untreated cells.; Abbreviation: NAR, naringenin; CaMKKβ, calmodulin-dependent protein kinase β; LKB1, liver kinase B1; p-LKB1, phosphor-LKB1; AMPK, AMP-activated protein kinase; p-AMPK, phosphor-AMPK; ACC, acetyl-CoA carboxylase 1; p-ACC, phosphor-ACC.

FIGURE 4

NAR promotes mitochondrial biogenesis of 3T3-L1 adipocytes and C2C12 myotubes. The mass of mitochondria in 3T3-L1 adipocytes ((A,B), the objective amplification 63×) and C2C12 myotubes ((H,I), the objective amplification 20×) after treatment of NAR was detected by mitotracker orange staining (red). The expression of SIRT1, PGC-1α, NRF1, and TFAM in 3T3-L1 adipocytes (C–G) and C2C12 myotubes (J–N) was determined by Western blotting. Bars represent 10 μM (A), 20 μM (H). All values were expressed as the mean ± SD from three or more independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. untreated cells.; Abbreviation: NAR, naringenin; SIRT1, sirtuin1; PGC-1α, peroxisome proliferator-activated receptor coactivator 1α; NRF1, nuclear respiratory factor 1; TFAM, mitochondrial transcription factor A.

FIGURE 5

NAR induces autophagy in 3T3-L1 preadipocytes and C2C12 myoblasts. LC3 in 3T3-L1 preadipocytes (A,B) and C2C12 myoblasts (G,H) were detected by immunofluorescence (endogenous LC3 puncta, green), and pictured by confocal microscopy (the objective amplification 63×). The expression of p-ULK1, ULK1, p62, and LC3 in 3T3-L1 preadipocytes (C–F) and C2C12 myoblasts (I–L) was detected by Western blotting. Bars represent 10 μM. All values were expressed as the mean ± SD from three or more independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. untreated cells.; Abbreviation: NAR, naringenin; ULK1, unc-51 like autophagy activating kinase 1; p-ULK1, phosphor-ULK1; p62, ubiquitin-binding protein p62; LC3, microtubule-associated protein 1A/1B-light chain 3.

FIGURE 6

NAR promotes the initiation of autophagy in the autophagic flux of 3T3-L1 preadipocytes and C2C12 myoblasts. LC3 in 3T3-L1 preadipocytes (A,B) and C2C12 myoblasts (F,G) after treatment of NAR or in combination with CQ were detected by immunofluorescence (green), and pictured by confocal microscopy (the objective amplification 63×). The expression of p62 and LC3 in 3T3-L1 preadipocytes (C–E) and C2C12 myoblasts (H–J) was detected by Western blotting. Bars represent 10 μM. All values were expressed as the mean ± SD from three or more independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. untreated cells or as indicated. Abbreviation: NAR, naringenin; CQ, chloroquine.

FIGURE 7

NAR decreases autophagy in livers of HFD-induced NAFLD rats, 3T3-L1 adipocytes, and C2C12 myotubes. The expression of p62 and LC3 in the HFD-induced NAFLD rat livers (A–C), 3T3-L1 adipocytes (D–F), and C2C12 myotubes (G–I) after treatment of NAR was detected by Western blotting. All values were expressed as the mean ± SD from three or more independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. untreated cells.; Abbreviation: NAR, naringenin; NAFLD, nonalcoholic fatty liver disease; HFD, high-fat diet.

FIGURE 8

NAR exerts effects by binding to AMPKγ1. Sodium oleate-induced L02 cells were treated with NAR (6.7 μM) alone or in combination with Comp C (4 μM) for 24 h. (A,B) Lipid accumulation was detected using oil red O staining (the objective amplification 20×). (C,D) Endogenous LC3 in L02 cells were detected with immunofluorescence (green) (the objective amplification 63×). (E) Docking of NAR at the active sites of AMPKγ1 was performed by LeDock software. The red dotted line indicated a hydrogen bond. (F) The schematic diagram of our study. Bars represent 20 μM (A), 10 μM (C). All values were expressed as the mean ± SD from three or more independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. untreated cells or as indicated. Abbreviation: NAR, naringenin; Comp C, Compound C; IOD, integrated optical density; Ala, Alanine; Ser, Serine; Asp, Aspartic acid; ACC, acetyl-CoA carboxylase 1; AMPK, AMP-activated protein kinase; CaMKKβ, calmodulin-dependent protein kinase β; LC3, microtubule-associated protein 1A/1B-light chain 3; NRF1, nuclear respiratory factor 1; PGC-1α, peroxisome proliferator-activated receptor coactivator 1α; p62, ubiquitin-binding protein p62; SIRT1, NAD-dependent protein deacetylase sirtuin-1; TFAM, mitochondrial transcription factor A; ULK1, Unc-51 like autophagy activating kinase 1.