Lack of SIRT1 (Mammalian Sirtuin 1) activity leads to liver steatosis in the SIRT1+/- mice: a role of lipid mobilization and inflammation

Abstract

Mammalian sirtuin 1 (SIRT1) may control fatty acid homeostasis in liver. However, this possibility and underlying mechanism remain to be established. In this study, we addressed the issues by examining the metabolic phenotypes of SIRT1 heterozygous knockout (SIRT1(+/-)) mice. The study was conducted in the mice on three different diets including a low-fat diet (5% fat wt/wt), mediate-fat diet (11% fat wt/wt), and high-fat diet (HFD, 36% fat wt/wt). On low-fat diet, the mice did not exhibit any abnormality. On mediate-fat diet, the mice exhibited a significant increase in hepatic steatosis with elevated liver/body ratio, liver size, liver lipid (triglyceride, glycerol, and cholesterol) content, and liver inflammation. The hepatic steatosis was deteriorated in the mice by HFD. In the liver, lipogenesis was increased, fat export was reduced, and beta-oxidation was not significantly changed. Body weight and fat content were increased in response to the dietary fat. Fat was mainly increased in sc adipose tissue and liver. Inflammation was also elevated in epididymal fat. Whole body energy expenditure and substrate utilization were reduced. Food intake, locomotor activity, and fat absorption were not changed. These data suggest that a reduction in the SIRT1 activity increases the risk of fatty liver in response to dietary fat. The liver steatosis may be a result of increased lipogenesis and reduced liver fat export. The inflammation may contribute to the pathogenesis of hepatic steatosis as well. A reduction in lipid mobilization may contribute to the hepatic steatosis and low energy expenditure.

Figure 1

Increased fat content and reduced energy expenditure on MFD in SIRT1^+/− mice. A, Time course of body weight gain. Body weight (BW) was determined every 3–5 wk after 12 wk of age. B, Fat mass. C, Lean mass. The fat and lean mass were determined using NMR. D, Food intake. Food intake was monitored daily for 3 d. Average daily food intake (g) was converted into kilocalories and normalized with body weight (kg) and time (hours). E, TAG in feces of MFD-fed mice. F, Energy expenditure in SIRT1^+/− mice. Energy expenditure was examined in the mice at 13 wk of age using the metabolic chamber. The unit is kilocalories per kilogram of lean body mass. G, Carbohydrate (CHO) and fatty acid oxidation at night time normalized with the lean body mass. H, Substrate preference. It was expressed by RER, which is a volume ratio of oxygen consumed vs. CO₂ exhaled. I, Western blot of SIRT1 protein in liver tissue. Values are the means ± se (n = 7). *, P ≤ 0.05.

Figure 2

Hepatic steatosis in SIRT1^+/− mice on MFD. Liver was examined in mice of 28 wk of age on MFD. A, Liver. Tissue was stained with H&E. Pictures were taken using a microscopy with ×10 or ×40 object lenses, respectively. B, TAG, glycerol, and cholesterol content in the liver of mice. C, ALT and IL-6 levels in the serum. D, Inflammatory gene mRNA in liver. E, Gluconeogenic and lipogenic gene expression in liver. F, Western blot of PPARγ and SREBP-1 protein in nucleus of liver. G, β-Oxidation and mitochondrial genes. H, Fatty acid oxidation in liver tissue. Fold change is used to express mRNA expression. Values are the means ± se (n = 7). *, P ≤ 0.05; **, P ≤ 0.001.

Figure 3

Reduced energy expenditure in SIRT1^+/− mice on HFD. The mice were fed on HFD at 12 wk of age, and examined for energy expenditure after 3 wk on HFD. A, Time course of body weight gain. Body weight was weighed every 2–4 wk from 12 wk of age. B, Fat mass. C, Lean mass. D, Food intake. Food intake was monitored daily for 3 d. Average daily food intake (g) was converted into kilocalories and normalized with lean body mass (kg) and time (hours). E, Energy expenditure in SIRT1^+/− mice. Energy expenditure was examined using the metabolic chamber and normalized with lean body mass. F, Fatty acid utilization normalized with lean body mass. G, Carbohydrate utilization normalized with lean body mass. H, RER is a volume ratio of oxygen consumed vs. CO₂ exhaled. In this figure, values are the means ± se (n = 7). *, P ≤ 0.05.

Figure 4

Hepatic steatosis was accelerated by HFD in SIRT1^+/− mice. The liver was examined in the SIRT1^+/− mice on HFD for 16 wk (28 wk of age). A, Increased liver weight and size in the SIRT1^+/− Tg mice. B, TAG, glycerol, and cholesterol content in liver. C, H&E staining of liver. Pictures were taken under a microscopy with object lenses of ×10 or ×40, respectively. D, Gluconeogenic and lipogenic gene mRNA in liver. E, Inflammatory gene mRNA in liver. F, mRNA in liver. Values are the means ± se (n = 7). *, P ≤ 0.05; **, P ≤ 0.001.

Figure 5

Energy expenditure genes in brown fat, muscle, and liver. In this figure, mRNA expression was determined for fatty acid oxidation-related genes in different tissues of new born mice except for panel D. A, BAT. B, Muscle. C, Liver. D, BAT of adult mice. E, Lipogenic genes in liver. F, Inflammatory genes. Values are the means ± se (n = 6–8). ERRα, Estrogen-related receptor-α.

Figure 6

Inflammation in adipose tissue of SIRT1^+/− mice on MFD. Epididymal fat was collected from mice at 28 wk of age and used in the analysis. A, H&E staining of epididymal fat. B, Lipogenic genes in epididymal fat. C, mRNA for adipokines and Pref-1. D, mRNA for inflammatory genes. E, mRNA for endothelial and angiogenic genes. F, Oil-Red O staining in differentiated MEFs. Values are the means ± se (n = 7). *, P ≤ 0.05. Apelin, an angiogenic factor produce by adipocytes.

Figure 7

TAG export in liver. Plasma lipids and liver gene expression were determined in mice on HFD. A, FFA level in plasma. B, Total TAG, glycerol, and cholesterol in plasma. C, Expression of genes in VLDL production pathway. Apob, Apolipoprotein B; Apobec1, apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1; Apoe, apolipoprotein E; Dgat1, diacylglycerol acyltransferase 1; Mttp, microsomal TAG transfer protein. Results are means ± sem (n = 7). *, P < 0.05.