Elucidation of SIRT-1/PGC-1α-associated mitochondrial dysfunction and autophagy in nonalcoholic fatty liver disease

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) can lead to chronic liver diseases associated with mitochondrial damages. However, the exact mechanisms involved in the etiology of the disease are not clear.

Methods: To gain new insights, the changes affecting sirtuin 1 (SIRT-1) during liver fat accumulation was investigated in a NAFLD mouse model. In addition, the in vitro research investigated the regulation operated by SIRT-1 on mitochondrial structures, biogenesis, functions, and autophagy.

Results: In mice NAFLD, high-fat-diet (HFD) increased body weight gain, upregulated serum total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, blood glucose, insulin levels, and liver malondialdehyde, and decreased liver superoxide dismutase activity. In liver, the levels of SIRT-1 and peroxisome proliferator-activated receptor-gamma coactivator -1α (PGC-1α) decreased. The expression of peroxisome proliferator-activated receptor-α and Beclin-1 proteins was also reduced, while p62/SQSTM1 expression increased. These results demonstrated SIRT-1 impairment in mouse NAFLD. In a well-established NAFLD cell model, exposure of the HepG2 hepatocyte cell line to oleic acid (OA) for 48 h caused viability reduction, apoptosis, lipid accumulation, and reactive oxygen species production. Disturbance of SIRT-1 expression affected mitochondria. Pre-treatment with Tenovin-6, a SIRT-1 inhibitor, aggravated the effect of OA on hepG2, while this effect was reversed by CAY10602, a SIRT-1 activator. Further investigation demonstrated that SIRT-1 activity was involved in mitochondrial biogenesis through PGC-1α and participated to the balance of autophagy regulatory proteins.

Conclusion: In conclusion, in high-fat conditions, SIRT-1 regulates multiple cellular properties by influencing on mitochondrial physiology and lipid autophagy via the PGC-1α pathway. The SIRT-1/PGC-1α pathway could be targeted to develop new NAFLD therapeutic strategies.

Keywords: Mitochondrial autophagy; Mitochondrial dysfunction; Lipid autophagy; Mitochondrial physiology; Nonalcoholic fatty liver disease; Peroxisome proliferator-activated receptor-gamma coactivator -1a; Sirtuin 1.

Fig. 1

Effect of HFD for 8 weeks on liver steatosis, SIRT-1 and PGC-1ɑ mRNA level and protein expression of SIRT-1, PGC-1ɑ, PPAR-ɑ, p62, Beclin-1. a The liver pathology of mice in the two groups. b Lipid deposition in liver tissues. c The mRNA expression level of SIRT-1 and PGC-1ɑ. d The protein expression of SIRT-1, PGC-1a, PPAR-ɑ, p62 and Beclin-1 was analyzed and quantified. *P < 0.05, **P < 0.01, vs. CON. Original magnification: × 200, × 400

Fig. 2

SIRT-1 regulates HepG2 cell viability and apoptosis crosstalk with OA. The cells were pretreated with T6 (2 μM) or CAY (20 μM) 2 h, followed incubated with OA (1.5 mM) for 48 h. a The cell viability was estimated using CCK-8 kit. b, c Flow Cytometry analysis of apoptosis and apoptosis rate quantitative analysis in cells. ^*P < 0.05, ^**P < 0.01, vs. CON group; ^#P < 0.05, ^##P < 0.01, vs. OA group

Fig. 3

SIRT-1 regulates HepG2 intracellular lipid and ROS crosstalk with OA. The cells were pretreated with T6 (2 μM) or CAY (20 μM) 2 h, followed incubated with OA (1.5 mM) for 48 h. a The cells were stained oil red O. b Immunofluorescence detected intracellular ROS. Scale bars are 50 μm and 10 μm respectively

Fig. 4

SIRT-1 involved in regulation of mitochondrial autophagy and lipophages in HepG2 cells. The cells were pretreated with T6 (2 μM) or CAY (20 μM) 2 h, followed incubated with OA (1.5 mM) for 48 h. a Mitochondrial autophagosome was determined. Red: mitochondria; green: LC3; yellow/ orange: merge. Scale bars are 10 μm respectively. b Representative electron micrographs of cells. Mit: mitochondria; SM: swollen and ruptured mitochondria; ER: endoplasmic reticulum; ASS: autophagolysosome; PG: phagophore; va: vacuole; LD: lipid drops; LA: lipid component of autolysosome. Scalar bar = 2.0 μm and 1.0 μm

Fig. 5

Effect of intervene in SIRT-1 and combine with OA on p62, Beclin-1, LC3B, SIRT-1, PGC-1a, MFN1, MFF and COX-IV protein expression in HepG2 cells. HepG2 cells were treated with 1.5 mM OA for 48 h, following T6 (2 μM) or CAY (20 μM) 2 h. a The p62, Beclin-1 and LC3B protein expression was detected by Western blotting. b The same method was used to detect the SIRT-1, PGC-1a, MFN1, MFF and COX-IV protein expression. The bands were normalized using GAPDH. The images were quantified with ImageJ. Data are presented as the mean ± SD; *P < 0.05, **P < 0.01 compared with CON group; ^#P < 0.05, ^##P < 0.01 compared with OA group