Sidt2 regulates hepatocellular lipid metabolism through autophagy

Abstract

SID1 transmembrane family member 2 (Sidt2) is an integral lysosomal membrane protein. To investigate its explicit function, we generated a global Sidt2 knockout mouse model (Sidt2^-/-). Compared with the littermate controls, Sidt2^-/- mice exhibited a remarkable accumulation of lipid droplets in liver. First, it was observed that food consumption, hepatocyte fatty acid uptake and de novo lipogenesis, hepatocyte lipolysis, and TG secretion in the form of very low density lipoprotein were comparable between Sidt2^-/- and WT mice. However, the hepatic β-oxidation of fatty acids decreased significantly as revealed by a low level of serum β-hydroxybutyrate in the Sidt2^-/- mice along with normal mRNA expression of genes involved in fatty acid oxidation. In addition, the classical autophagy pathway marker proteins, p62 and LC3-II, increased in liver, along with compromised autophagic flux in primary hepatocytes, indicating a block of autophagosome maturation due to Sidt2 deficiency, which was also supported by electron microscopy image analysis both in livers and in primary hepatocytes from Sidt2^-/- mice. It was concluded that Sidt2 plays an important role in mouse hepatic lipid homeostasis by regulating autophagy at the terminal stage.

Keywords: SID1 transmembrane family member 2; lipid droplets; liver metabolism; triglycerides.

Fig. 1.

Sidt2 knockout causes lipid accumulation in the liver. A: Gross view and weights of livers in chow and HF diet groups (n = 5). B: Body weights of WT and Sidt2^−/− mice maintained on a HF diet for 12 weeks. C: H&E staining of liver from mice fed with chow diet (scale bar, 50 µm). The graph shows the quantification of hepatocyte size in more than 100 cells from three different mice in each group. D: Oil Red O staining (scale bar, 100 µm). E: Electron micrographs of liver from chow diet-fed mice without intake of food for 20 h. Scale bars, 5 µm (left) and 2 µm (right). The graphs show quantifications of LD number and size (n = 3). F: Oil Red O staining of primary hepatocytes from Sidt2^−/− and WT mice treated with 0.2 mM OA for 24 h. Microscopic images (left, 400×) and quantification of Oil Red O-stained area (right) (n = 46–54 cells from three independent experiments). G: Immunoblot analysis of ADFP levels in livers from mice fed with chow diet (n = 4). H: Hepatic TG concentration of mice fed chow or HF diets (n = 6). I: Serum ALT and AST levels (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001. TBW, total body weight.

Fig. 2.

Sidt2 knockout does not influence lipogenesis and TG secretion. A: Total food consumption of WT or Sidt2^−/− mice under a chow diet measured for 4 weeks beginning at 8 weeks of age (n = 8). B: Quantitative real-time PCR analysis of genes involved in lipid transport and lipogenesis in the livers of 14-week-old WT or Sidt2^−/− mice fed a chow diet (n = 3). C: Hepatic TG production rate (n = 6). D: Serum NEFA level (n = 6). E: The weight of adipose tissues from WT or Sidt2^−/− mice under a chow diet (n = 6). TBW, total body weight.

Fig. 3.

Sidt2 knockout reduces hepatic β-oxidation of fatty acids. A: Serum β-hydroxybutyrate levels of mice fed a chow diet (n = 6). B: Quantitative real-time PCR analysis of genes involved in lipolysis and fatty acid oxidation in livers of 14-week-old WT or Sidt2^−/− mice fed a chow diet (n = 3). **P < 0.01.

Fig. 4.

Lipid metabolism in 6-week-old mice fed with chow diet. A: Liver TG levels (n = 5). B: Immunoblot and densitometry analysis of ADFP levels in murine livers (n = 4). C: Serum β-hydroxybutyrate levels (n = 5). D: Serum NEFA levels of 6-week-old mice. (n = 5). *P < 0.05.

Fig. 5.

Sidt2 knockout blocks hepatic autophagy in vivo. A–C: Immunoblot analysis of LC3-II and p62 levels in livers from 14-week-old mice fed with chow diet (n = 4). D–F: Immunoblot analysis of LC3-II and p62 levels in livers from mice fed with HF diet (n = 4). EM of liver tissues from WT (G) and Sidt2^−/− (H–J) mice. Abundant vacuolar structures were observed in Sidt2^−/− livers. White arrows indicate autophagosomes and multi-lamellar membrane structures. Black arrows indicate autolysosomes. The asterisks indicate LDs. Scale bars: 2 μm (G); 1 μm (H–J). K: Quantification of autophagic vacuoles (n = 3). *P < 0.05, **P < 0.01.

Fig. 6.

Sidt2 knockout blocks autophagy in vitro. A: Immunoblot analysis of p62 levels of primary hepatocytes from Sidt2^−/− and WT mice in condition of normal medium. B: Immunoblot analysis of LC3-II levels in primary hepatocytes from Sidt2^−/− and WT mice. Cells were either kept in normal medium or starved in Earle’s balanced salt solution medium for 2 h. C: Primary hepatocytes were treated for 2 h with or without 30 μM chloroquine, then the protein levels of LC3-II were detected by Western blot (n = 4). D–I: Electron micrographs of primary hepatocytes cultured in normal medium from WT (D) and Sidt2^−/− mice (E–I). F–I: Higher magnification images of the boxed area in E showing autophagosomes marked by white arrows and autolysosomes marked by black arrows. Scale bars: 1 μm (D, E); 0.5 μm (F); 0.2 μm (G–I). J: Graph shows quantification of number of autophagic vacuoles (AVs) in hepatocytes (n = 14 cells). *P < 0.05, **P < 0.01, and ***P < 0.001. CQ, chloroquine.

Fig. 7.

Autophagy-related gene expression does not significantly change in livers of Sidt2^−/− mice. A: Quantitative real-time PCR analysis of genes involved in autophagy and lysosomal proteases in livers of 14-week-old WT or Sidt2^−/− mice fed a chow diet (n = 3). B: Immunoblot analysis of Beclin1, Atg5, and Atg7 levels in livers from 14-week-old mice fed with chow diet (n = 4). C: Phospho-S6K1, S6K1, phospho-4E-BP1, and 4E-BP1 of primary hepatocytes in RM from Sidt2^−/− and WT mice (n = 4). D–F: Immunoblot analysis of TFEB (D), Lamp2 (E), cathepsin B and cathepsin D (in their immature and mature forms) (F) in mouse livers. G: ASMase activity in liver lysates from Sidt2^−/− and WT mice fed a chow diet (n = 3–4). *P < 0.05. Ctsb, cathepsin B; Ctsd, cathepsin D.