Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis

Abstract

Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30) is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA) biosynthesis. SMP30 also participates in Ca(2+) efflux by activating the calmodulin-dependent Ca(2+)-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO) mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15-24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1) higher plasma levels of triglyceride and aspartate aminotransferase; (2) severe accumulation of hepatic triglyceride and total cholesterol; (3) higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4) decreased mRNA and protein levels of Apolipoprotein B (ApoB) in livers - ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion.

Keywords: AA, l-ascorbic acid; AST, aspartate aminotransferase; ApoB, Apolipoprotein B; Ascorbic acid; DHA, dehydroascorbic acid; DHE, dihydroethidium; DKO, double knockout; EDTA, ethylenediaminetetraacetic acid; FFA, free fatty acid; Grp78, glucose-regulated protein 78 kDa; KO, knockout; MTP, microsomal triglyceride transfer protein; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; Non-alcoholic fatty liver disease; PL, phospholipid; PPARα, peroxisome proliferator-activated receptor-α; Reactive oxygen species; SDS, sodium dodecyl sulfate; SMP30; SMP30, senescence marker protein-30; SOD, superoxide dismutase; SOD1; SREBP, sterol regulatory element binding protein; T-cho, total cholesterol; TBARS, thiobarbituric acid reactive substances; TG, triglyceride; VLDL, very low-density lipoprotein; qPCR, quantitative real-time polymerase chain reaction.

Fig. 1

Establishment of Smp30^Y/−Sod1^−/− male mice and Smp30^−/−Sod1^−/− female mice. (A) Cumulative survival rate in each experimental group of mice. (B) Appearance of Smp30^Y/−Sod1^−/− male mice and Smp30^−/−Sod1^−/− female mice at 14 days of age. (C) Body weight change in each experimental group (n = 10–11) between 7 and 14 days of age. The body weight of mice at 7 days was set at 100%. Open circles, Smp30^Y/+Sod1^+/+ or Smp30^+/+Sod1^+/+ mice; open triangles, Smp30^Y/+Sod1^+/− or Smp30^+/+Sod1^+/− mice; open squares, Smp30^Y/+Sod1^−/− or Smp30^+/+Sod1^−/− mice; filled circles, Smp30^Y/−Sod1^+/+ or Smp30^−/−Sod1^+/+ mice; filled triangles, Smp30^Y/−Sod1^+/− or Smp30^−/−Sod1^+/− mice; filled squares, Smp30^Y/−Sod1^−/− or Smp30^−/−Sod1^−/− mice. Values are given as means ± SEM. (D and E) Ascorbic acid (AA; filled columns) and dehydroascorbic acid (DHA; gray columns) concentration in plasma (D) and livers (E) from each experimental group of mice (n = 7–11) at 14 days. Values are given as means ± SEM (AA plus DHA).

Fig. 2

Increased steatosis in liver sections from Smp30^Y/−Sod1^−/− male mice and Smp30^−/−Sod1^−/− female mice. Representative images of (A) hematoxylin/eosin staining and (B) oil red O staining in liver sections from each experimental group at 14 days of age. PV, portal vein; CV, central vein. Scale bar is 50 μm.

Fig. 3

Increased triglyceride, cholesterol, and thiobarbituric acid reactive substances (TBARS) in livers of Smp30^Y/−Sod1^−/− male mice. (A) Triglyceride, (B) Cholesterol, (C) Phospholipid, (D) Free fatty acid, and (E) TBARS in livers from each experimental group at 14 days of age. Values are given as means ± SEM of five animals.

Fig. 4

Increased generation of superoxide anion radicals in livers from Smp30^Y/−Sod1^−/− male mice. Representative images of dihydroethidium staining in liver sections from each experimental group at 14 days of age. Scale bar is 50 μm.

Fig. 5

Decreased Apolipoprotein B (ApoB) levels in livers of Smp30^Y/−Sod1^−/− male mice. (A) Western blot analysis of ApoB100, ApoB48, microsomal triglyceride transfer protein (MTP), and glucose-regulated protein 78 kDa (Grp78) in liver microsome fractions from each experimental group of mice. (B) Apob gene expression levels in the liver. mRNA for the Apob gene was measured using quantitative real-time polymerase chain reaction and normalized to Rplp2, ribosomal protein, large P2, and the value was expressed as arbitrary unit. The values from Smp30^Y/+Sod1^+/+ male mice were assigned a relative value of 1.0. Values are given as means ± SEM of five animals.

Fig. 6

Decreased sterol regulatory element binding protein (SREBP)-1c, SREBP2, and peroxisome proliferator-activated receptor-α (PPARα) levels in livers from Smp30^Y/−Sod1^−/− male mice. Western blot analysis of (A) precursor (125 kDa) and (B) mature (60 kDa) of SREBP1c, (C) mature SREBP2, and (D) PPARα. Representative images for each experimental group in one blot are shown and the signal intensity for each protein was measured and expressed as an arbitrary unit. The values from Smp30^Y/+Sod1^+/+ mice were assigned a relative value of 1.0. Values are given as means ± SEM of three animals.