B-003 Decrease in Serum apoE-rich HDL-cholesterol May Be Associated With Cholesterol Accumulation in the Liver of Rats Fed A High-cholesterol Diet
Authors: Shinohata R , Takeuchi R , Usui S 
Published: September 2023

Abstract

Background
Free cholesterol accumulation in the liver has been suggested to be one of risk factors for the progression of non-alcoholic fatty liver to non-alcoholic steatohepatitis. Therefore, a non-invasive approach to assess hepatic cholesterol accumulation in the early stage without elevation of liver enzymes (AST and ALT) may be useful in clinical setting. In this study, we show that serum apoE-rich HDL-cholesterol level is a possible indicator for predicting hepatic cholesterol accumulation in rats with diet-induced simple liver steatosis.

Methods
Male Sprague-Dawley rats (8 weeks) were purchased from Charles River Laboratories. Four different diets were obtained from Oriental Yeast; control diet (MF), high-fat diet (HF; 68% MF, 30% palm oil, and 2% cholic acid), high-cholesterol diet (HC; 95.5% MF, 2.5% cholesterol, and 2% cholic acid), and high-fat/high-cholesterol diet (HFC; 81.75% MF, 15% palm oil, 1.25% cholesterol, and 2% cholic acid). The rats were randomly divided into 4 groups and fed as follows. The control and HFC groups received the MF and HFC diets for 8 weeks, respectively. The HF/HC group received the HF diet for the first 4 weeks and the HC diet for the next 4 weeks. The HC/HF group received the HC diet first, then the HF diet. Blood and liver tissue specimens were collected under anesthesia. Total HDL fractions were isolated from whole sera by the polyethylene glycol precipitation method, and applied into a cation-exchange column (HiTrap SPHP, GE healthcare) for determining apoE-rich HDL-cholesterol levels. Correlations between variables were assessed with Spearman’s correlation coefficient (rs).

Results
Total cholesterol content of the liver was significantly higher in the HFC (11.1-fold, P &lt; 0.001), HF/HC (9.1-fold, P &lt; 0.001), and HC/HF (5.8-fold, P &lt; 0.001) groups than the control group. Hepatic free cholesterol was also higher in the HFC (5.5-fold, P &lt; 0.001), HF/HC (3.9-fold, P &lt; 0.001), and HC/HF (3.3-fold, P &lt; 0.001) groups. Hepatic triglycerides were slightly increased in the HC/HF (1.6-fold, P &lt; 0.01) and HFC (1.5-fold, P &lt; 0.05) groups. Serum apoE-rich HDL-cholesterol was significantly decreased in the HF/HC (0.21-fold, P &lt; 0.001), HC/HF (0.25-fold, P &lt; 0.001) and HFC (0.42-fold, P &lt; 0.01) groups. The HFC group had significantly higher serum ALT (3.0-fold, P &lt; 0.05) and AST (2.3-fold, P &lt; 0.01) levels, compared with the control groups. There was a significant increase in hepatic mRNA expression of TNF-α (24.8-fold, P &lt; 0.01) and TGF-β (3.8-fold, P &lt; 0.001) only in the HFC group, indicating liver inflammation and stellate cell activation. To exclude the effects of hepatocyte injury on apoE-rich HDL metabolism, rats with ALT less than 100 U/mL (n = 16) were included in the following analysis. ApoE-rich HDL-cholesterol levels were significantly and inversely correlated with hepatic total cholesterol (rs = −0.535, P &lt; 0.05) and free cholesterol (rs = −0.532, P &lt; 0.05) levels but not with hepatic triglycerides. Furthermore, the ratio of ALT to apoE-rich HDL-cholesterol was more highly correlated with hepatic total cholesterol (rs = 0.626, P &lt; 0.01) and free cholesterol (rs = 0.632, P &lt; 0.01). There was no such correlation for apoE-poor HDL-cholesterol. Conclusion: The present observations suggest that serum apoE-rich HDL-cholesterol levels may be a useful indicator for cholesterol accumulation in the liver of cholesterol-fed rats.