The role of SAMM50 in non-alcoholic fatty liver disease: from genetics to mechanisms

Abstract

Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation. SAMM50 encodes Sam50, a mitochondrial outer membrane protein involved in the removal of reactive oxygen species, mitochondrial morphology and regulation of mitophagy. Certain single nucleotide polymorphisms of SAMM50 have been reported to be correlated with NAFLD. However, the contribution of SAMM50 polymorphisms to the occurrence and severity of fatty liver in the Chinese Han cohort has rarely been reported. Here, we investigated the association between SAMM50 polymorphisms (rs738491 and rs2073082) and NAFLD in a Chinese Han cohort, as well as the mechanistic basis of this association. Clinical information and blood samples were collected from 380 NAFLD cases and 380 normal subjects for the detection of genotypes and biochemical parameters. Carriers of the rs738491 T allele or rs2073082 G allele of SAMM50 exhibit increased susceptibility to NAFLD [odds ratio (OR) = 1.39; 95% confidence interval (CI) = 1.14-1.71, P = 0.001; OR = 1.31; 95% CI = 1.05-1.62, P = 0.016, respectively] and are correlated with elevated serum triglyceride, alanine aminotransferase and aspartate aminotransferase levels. The presence of the T allele (TT + CT) of rs738491 (P < 0.01) or G allele (AG + GG) of rs2073082 (P = 0.03) is correlated with the severity of fatty liver in the NAFLD cohort. In vitro studies indicated that SAMM50 gene polymorphisms decrease its expression and SAMM50 deficiency results in increased lipid accumulation as a result of a decrease in fatty acid oxidation. Overexpression of SAMM50 enhances fatty acid oxidation and mitigates intracellular lipid accumulation. Our results confirm the association between the SAMM50 rs738491 and rs2073082 polymorphisms and the risk of fatty liver in a Chinese cohort. The underlying mechanism may be related to decreased fatty acid oxidation caused by SAMM50 deficiency.

Keywords: SAMM50; NAFLD; SNPs; fatty acid oxidation.

Fig. 1

SAMM50 knockdown caused lipid accumulation in human hepatoma cells under fatty acid treatment. (A) Staining of lipid droplets (green) by BODIPY 493/503 in Hep3B cells with PA stimulation. Scale bars = 50 μm. (B) Protein and mRNA levels (n = 3) of SAMM50 in Hep3B cells after PA treatment. (C) The mRNA levels of SAMM50 in patients with or without hepatic steatosis from the GEO database. (D) Representative photos of immunohistochemical staining with SAMM50 on liver tissues with or without steatosis. Scale bars = 50 μm. (E) Hepatic mRNA levels of SAMM50 among genotypes (CC = 8, CT = 5, TT = 4; AA = 8, AG = 5, GG = 6). (F) Hepatic SAMM50 protein levels among genotypes were detected by immunoblotting. (G) Verification of SAMM50 knockdown by immunoblotting. (H,I) Intracellular TG content in SAMM50 knockdown cells with fatty acid stimulation for 24 h. Scale bars = 50 μm. (J) The cell viabilities are compared between SAMM50 knockdown and control groups after fatty acid stimulation (n = 5). Statistical analyses were performed using Student’s t‐test between two groups or one‐way ANOVA followed by a LSD test for more than two groups. Data are expressed as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant

Fig. 2

FAO is impaired as a result of SAMM50 deficiency. (A) The mRNA levels of the fatty acid metabolism‐related genes in both SAMM50 knockdown and control groups (n = 3). Gene expression levels were normalized for Actb. (B) The OCR using palmitate (or BSA) as the only substrate was measured in SAMM50 knockdown and control groups (n = 6). (C) The expression of FAO‐related genes in both SAMM50 knockdown and control groups after PA treatment. (D) Levels of ketone bodies (β‐HB and AcAc) within cells were tested and normalized to protein levels in SAMM50 knockdown cells after PA stimulation (n = 3). (E) The activity of electron transport chain complexes was compared between SAMM50 knockdown and control groups. (F) The levels of mitochondrial proteins involved in β‐oxidation were measured in SAMM50 knockdown group with or without PA treatment. (G,H) The β‐HB levels and the expression of FAO‐related genes between the two groups are shown after GW7647 (10 μm) treatment. Statistical analyses were performed using one‐way ANOVA followed by a LSD test. Data are expressed as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant

Fig. 3

Overexpression of SAMM50 mitigates lipid accumulation and enhances FAO. (A) Overexpression of SAMM50 was verified by immunoblotting. (B) Overexpression of SAMM50 increased the expression of FAO‐related genes after PA stimulation. (C) Overexpression of SAMM50 increased the palmitate‐dependent OCR in Hep3B cells. (D,E) Overexpression of SAMM50 in SAMM50 knockdown cells mitigates lipid accumulation, as indicated by staining of lipid droplets with BODIPY 493/503 and intracellular TG detection. Scale bars = 50 μm. (F) Overexpression of SAMM50 elevated the levels of ketone bodies (β‐HB and AcAc) in SAMM50 knockdown cells after PA challenge for 48 h, n = 3. Statistical analyses were performed using one‐way ANOVA followed by a LSD test. Data are expressed as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant