Reactive oxygen species-induced changes in glucose and lipid metabolism contribute to the accumulation of cholesterol in the liver during aging

Abstract

Aging is a major risk factor for many chronic diseases due to increased vulnerability to external stress and susceptibility to disease. Aging is associated with metabolic liver disease such as nonalcoholic fatty liver. In this study, we investigated changes in lipid metabolism during aging in mice and the mechanisms involved. Lipid accumulation was increased in liver tissues of aged mice, particularly cholesterol. Increased uptake of both cholesterol and glucose was observed in hepatocytes of aged mice as compared with younger mice. The mRNA expression of GLUT2, GK, SREBP2, HMGCR, and HMGCS, genes for cholesterol synthesis, was gradually increased in liver tissues during aging. Reactive oxygen species (ROS) increase with aging and are closely related to various aging-related diseases. When we treated HepG2 cells and primary hepatocytes with the ROS inducer, H₂ O₂ , lipid accumulation increased significantly compared to the case for untreated HepG2 cells. H₂ O₂ treatment significantly increased glucose uptake and acetyl-CoA production, which results in glycolysis and lipid synthesis. Treatment with H₂ O₂ significantly increased the expression of mRNA for genes related to cholesterol synthesis and uptake. These results suggest that ROS play an important role in altering cholesterol metabolism and consequently contribute to the accumulation of cholesterol in the liver during the aging process.

Keywords: aging; cholesterol; lipid metabolism; liver; reactive oxygen species.

Figure 1

Changes in lipid concentration and liver function in mice at different ages (n = 8–10/group). (a) Total cholesterol (T‐CHO). (b) TG. (c) HDL cholesterol. (d) LDL cholesterol in serum. *p < 0.05. (e, f) Primary hepatocytes were isolated from young (2‐month‐old) and old (20‐month‐old) mice, and (e) ATP levels, (f) ADP/ATP ratios, and (g) ROS levels were measured. (h, i) Serum, (h) ALT levels, and (i) AST levels were measured. *p < 0.05 vs. 2 M

Figure 2

Changes in lipid accumulation in liver tissues at different ages of mice (n = 8–10/group). (a) Representative image of Oil Red O staining of liver tissue (Magnification; ×200). (b, c) The Folch method was used to separate lipids from liver tissue of various ages and to measure (b) hepatic TG levels and (c) hepatic cholesterol levels. *p < 0.05 vs. 8 M

Figure 3

Cholesterol uptake and glucose uptake cholesterol synthesis‐related gene expression were increased in aged mice (n = 3/group). (a, b) Primary hepatocytes were isolated from mouse livers at various ages, and cholesterol and glucose uptake were measured. (a) Cholesterol uptake was measured in the absence (CON) or presence of 1.25 μM U18666A, a cholesterol uptake inducer. (b) Glucose uptake was measured in the absence (CON) or presence of 1 μM insulin. (c, d) Total RNA was isolated from mouse livers at various ages, and qRT–PCR was carried out (n = 8–10/group). (c) GLUT2; (d) GK; (e) SREBP2; (f) 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGCR); (g) 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 1 (HMGCS). *p < 0.05

Figure 4

(a–h) Induction of ROS by H₂O₂ treatment increased cholesterol and triglyceride synthesis in HepG2 cells (n = 3/group). (a) HepG2 cells were untreated (CON) or treated with various concentrations of H₂O₂ for 24 hr, and the expression of SREBP2 mRNA was determined by qRT–PCR. (b–d) HepG2 cells were untreated (CON) or treated with 500 µM H₂O₂ for various times. (b) The expression of SREBP2 mRNA was determined by qRT–PCR. (c) SREBP2 protein expression in the cytosol and nucleus was determined using western blotting. (d) Quantitative analysis of (c). (e, f) HepG2 cells were treated with 500 µM H₂O₂ for 48 hr and harvested. (e) Identification of HMGCS mRNA expression using qRT–PCR. (f) Identification of HMGCR mRNA expression using qRT–PCR (g) Intracellular TG level. (h) Intracellular cholesterol level *p < 0.05 vs. CON. (i–l) Induction of ROS by H₂O₂ treatment increased cholesterol synthesis in primary hepatocytes (n = 3/group). (i) Primary hepatocytes were left untreated (CON) or treated with various concentrations of H₂O₂ for 48 hr, and the expression of SREBP2 mRNA was determined by qRT–PCR. (j–l) Primary hepatocytes were treated with 500 μM H₂O₂ for 48 hr and harvested. (j) HMGCS mRNA expression and (k) HMGCR mRNA expression determined by qRT–PCR. (l) Intracellular cholesterol level *p < 0.05 vs. CON

Figure 5

(a–c) mRNA and protein expression of caveolin‐1 and LDLR was increased in H₂O₂‐treated HepG2 cells. HepG2 cells were untreated (CON) or treated with 500 µM H₂O₂ for 48 hr and harvested (n = 3/group). (a) Identification of caveolin‐1 mRNA expression using qRT–PCR. (b) Identification of LDLR mRNA expression using qRT–PCR. (c) Identification of caveolin‐1 and LDLR protein expression using western blotting. (d–f) Increase in glucose uptake in H₂O₂‐treated HepG2 cells. HepG2 cells were untreated (CON) or treated with 500 µM H₂O₂ for 48 hr and harvested (n = 3/group). (d) HepG2 cells were treated without (−) or with of 1 μM insulin, and a glucose uptake assay was performed. (e) Identification of GLUT2 in the cytosol and plasma membrane using western blotting. (f) Quantitative analysis of (e). *p < 0.05 vs. CON. (g, h) Primary hepatocytes were treated with 500 µM H₂O₂ for 48 hr, and glucose uptake and cholesterol uptake were measured (n = 3/group). (g) Glucose uptake was measured in the absence or presence of 1 μM insulin. (h) Cholesterol uptake was measured in the absence or presence of 1.25 μM U18666A, an inducer of cholesterol uptake. *p < 0.05

Figure 6

Changes in glucose metabolism‐related factors by ROS induction. (a–k) HepG2 cells were untreated (CON) or treated with 500 µM H₂O₂ for 48 hr and harvested. (n = 3/group). (a, b, d, e, g, h) Measurement of gene expression by qRT–PCR. (c, f, i) Measurement of protein levels using western blotting. (j) Measurement of intracellular glycogen levels. (k) After GLUT2 knockdown using GLUT2 siRNA, HepG2 cells were untreated (CON) or treated with 500 µM H₂O₂ for 48 hr and harvested; intracellular acetyl‐CoA levels were measured. *p < 0.05 vs. CON. (l–n) Primary hepatocytes were treated with 500 µM H₂O₂ for 48 hr and harvested (n = 3/group). (l) Measurement of mRNA expression of GK by qRT–PCR. (m) Measurement of mRNA expression of PKLR by qRT–PCR. (n) Intracellular acetyl‐CoA levels were measured. *p < 0.05 vs. CON