Changes In Expression Of Genes Involved In Cholesterol Metabolism In Liver Of STZ-induced Diabetes Rat

Objective: The major function of HDL is to transfer cholesterol from peripheral tissues to the liver for biliary excretion. There is a major role for hepatocyte ATP binding cassette transporter A1 (ABCA1) in generating a critical pool of HDL precursor particles that enhance further HDL generation. HDL precursor particles are released to blood and recruit cholesterol from peripheral tissues. With the effect of lecithin cholesterol acyl transferase (LCAT), HDL precursor particles change into cholesterol esters and become mature HDL particles. The scavenger receptor class B type I (SR-BI) is the liver cell-surface HDL receptor and mediates selective HDL cholesterol uptake. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid biosynthesis. During diabetes course, with high blood cholesterol, more cholesterol was englobed to liver and enzyme activity of CYP7A1 become higher. Then whether the increasing of cholesterol to liver is related to the expression changes of ABCA1, SR-BI and whether the high activity of CYP7A1 is related to alter of the expression of CYP7A1 are not reported.Peroxisome proliferator activated receptorα(PPARα) is the main nuclear receptor which regulate fatty acid metabolism. Liver X receptorα(LXRα) is the main nuclear receptor which regulate cholesterol metabolism. There is an interaction between fatty acid metabolism and cholesterol metabolism. In diabetic state fat mobilization upgrade and fatty acid increase, then hepatic PPARαis activating. Whether the relative expression of LXRαand its target genes were changed accompany with PPARαactivating are not reported by present.The Fibrate group of drugs, used extensively as lipid-lowering agents, are pharmacological activators of PPARα. It can lower blood triglyceride by the way of activating some fatty acid catabolism genes, decomposing fatty acid, and it also has little effect on decreasing blood total cholesterol. In diabetic state if it can activate PPARαand affect some cholesterol metabolism gene expression to decrease the level of blood cholesterol and ameliorate the morphosis and function of hepatocyte are not reported by present.In present experiment, diabetes mellitus (DM) was induced by intraperitoneal injection with streptozocin (STZ). In the condition of injured liver structure and function, such as the decreasing of serum aspartate aminotransferase (AST)/ alanine aminotransferase (ALT) in DM rat, we observe the total cholesterol level in liver, detecte the expression of liver PPARα, ACOX1, LXRα, ABCA1, SR-BI and CYP7A1 genes by RT-PCR. We also observe the effect of Fenofibrate-the activating ligand of PPARαon the above indexes, in order to investigate the molecule mechanism of lipid metabolism in DM rat liver, deepening the recognition of lipid metabolic disorder in DM.Methods:1 Animals and materialsMale Wistar rat weighed 200-250g were divided randomly into: control group (CON), diabetes group (DM), diabetes+Fenofibrate (DM+F), control+Fenofibrate (CON+F). DM was induced by intraperitoneal injection with 2% STZ. Four weeks after DM was successfully induced, DM+F group and CON+F group were perfused with 100mg/kg/day of Fenofibrate for two weeks. CON group and DM group were perfused with 100mg/kg/day of isotonic Na chloride for two weeks. After abrosia for one night, blood was collected by carotid artery bloodletting, and the serum was used for the detection of serum glucose, lipids, bile acid, ALT, AST. The liver was used for histological section and extraction of RNA and lipids.2 Observation of the pathological changes by light microscopeThe liver tissue was routinely fixed by citromint, and prepared for light microscope observing.3 The extraction of lipids in rat hepatocyte Refer to reported methods .4 Detection of blood glucose, lipids, bile acid, ALT, AST, liver cholesterolWhat above-mentioned were detected by Olympus Au2700 automatic biochemistry analysator.5 Detection of the relative expression of related genes in the liverTotal RNA was extracted with Trizol method. The relative expression of PPARα, ACOX1, LXRα, ABCA1, SR-BI and CYP7A1 were evaluated by RT-PCR, with GAPDH as inner standard.Results:1 All results in control and diabetes group1.1 The changes of blood AST/ALT and liver morphous: DM group blood AST/ALT were singnificantly decreased (1.776±0.279 vs 2.261±0.445, p<0.01); hepatic cells structure confused, hepatic sinusoid broaden. The results showed that DM group hepatic structure and function were injured.1.2 The changes of blood lipids: In diabetic state, blood total cholesterol (1.870±0.180mmol/L vs 1.359±0.197mmol/L, p<0.01), blood HDL-C (1.308±0.188mmol/L vs 1.011±0.209mmol/L, p<0.01), blood bile acid (59.626±16.349μmol/L vs 9.334±2.011μmol/L, p<0.01), blood triglyceride (1.346±0.393mmol/L vs 0.651±0.130mmol/L, p<0.01) were singnificantly increased. The results showed that in diabetic state blood cholesterol and triglyceride metabolism abnormality.1.3 Cholesterol content in hepatic cells: DM group compared to CON group, (32.974±5.526mmol/mg vs 31.977±4.782mmol/mg, p>0.05) the changes of cholesterol content in liver were no statistical significance.1.4 The expression of liver genes 1.4.1 The relative expression of PPARαand ACOX1 mRNA . DM group compared to CON group, the relative expression of PPARα(0.921±0.123 vs 0.621±0.132, p<0.01) and ACOX1 (0.801±0.128 vs 0.587±0.081, p<0.01) were singnificantly increased. The results showed that in diabetic state PPARαwas activated and the expression of PPARαmRNA was increased.1.4.2 The relative expression of LXRα, ABCA1, SR-BI and CYP7A1 mRNA. DM group compared to CON group, the relative expression of LXRα(1.216±0.128 vs 0.702±0.097, p<0.01), ABCA1 (0.725±0.127 vs 0.177±0.042, p<0.01), SR-BI (0.672±0.105 vs 0.220±0.040, p<0.01) and CYP7A1 (1.137±0.212 vs 0.697±0.141, p<0.01) were singnificantly increased. The results showed that the expression of LXRαwas increased with the increasing of PPARαin liver of STZ-induced rats; HDL precursor particles formation, HDL-C intaking and bile acid synthesis were all increased in liver of STZ-induced rats.2 All results of Fenofibrate to DM group and CON group2.1 The changes of blood AST/ALT and liver morphous: DM+F group compared to DM group (1.772±0.193 vs 1.776±0.279, p>0.05) and CON+F group compared to CON group (2.687±0.512 vs 2.261±0.445, p>0.05) the changes of blood AST/ALT were no statistical significance; Hepatic cells structure had no singnificant change between CON+F group and CON group, DM+F group and DM group . The results showed that Fenofibrate didn't alter the structure and function of liver. 2.2 The changes of blood lipids: DM+F group compared to DM group, the changes of blood total cholesterol (1.903±0.312mmol/L vs 1.870±0.180mmol/L, p>0.05), blood HDL-C (1.332±0.249mmol/L vs 1.308±0.188mmol/L, p>0.05), blood bile acid (56.671±29.694μmol/L vs 59.626±16.349μmol/L, p>0.05) were no statistical significance; Blood triglyceride (0.743±0.269mmol/L vs 1.346±0.393mmol/L, p<0.01) were singnificantly decreased. CON+F group compared to CON group, blood total cholesterol (0.979±0.156mmol/L vs 1.359±0.197mmol/L, p<0.01) and blood HDL-C (0.566±0.111mmol/L vs 1.011±0.209mmol/L, p<0.01) were singnificantly decreased; blood bile acid (30.880±9.226μmol/L vs 9.334±2.011μmol/L, p<0.01) was singnificantly increased; the changes of blood triglyceride (0.757±0.108mmol/L vs 0.651±0.130mmol/L, p>0.05) were no statistical significance. The results showed that Fenofibrate can decrease normal rats blood total cholesterol and diabetic rats blood triglyceride but not STZ-induced rats blood cholesterol. Fenofibrate has different regulation between normal and STZ-induced rats.2.3 Cholesterol content in hepatic cells: DM+F group compared to DM group (26.167±2.937mmol/mg vs 32.974±5.526mmol/mg, p<0.01), CON+F group compared to CON group (24.417±1.429mmol/mg vs 31.977±4.782mmol/mg, p<0.01), cholesterol content in hepatic cells were all decreased. The results showed that Fenofibrate can lower cholesterol level of liver. 2.4 The expression of liver genes2.4.1 The relative expression of PPARαand ACOX1 mRNA: DM+F group compared to DM group (0.784±0.138 vs 0.921±0.123, p>0.05), CON+F group compared to CON group (0.651±0.133 vs 0.621±0.132, p>0.05), the changes of relative expression of PPARαmRNA were no statistical significance. DM+F group compared to DM group (1.253±0.125 vs 0.801±0.128, p<0.01) and CON+F group compared to CON group (1.273±0.194 vs 0.587±0.081, p<0.01) the relative expression of ACOX1 mRNA were increased. The results showed that Fenofibrate activated PPARαof normal and STZ-induced rats liver.2.4.2 The relative expression of LXRα, ABCA1, SR-BI and CYP7A1 mRNA. DM+F group compared to DM group, the changes of LXRα(1.090±0.087 vs 1.216±0.128, p>0.05), ABCA1 (0.809±0.050 vs 0.725±0.127, p>0.05) and SR-BI (0.554±0.081 vs 0.672±0.105, p>0.05) were no statistical significance; CYP7A1 (1.399±0.149 vs 1.137±0.212, p<0.01) was increased. CON+F group compared to CON group, the relative expression of LXRα, (0.944±0.189 vs 0.702±0.097, p<0.01), ABCA1 (0.807±0.131 vs 0.177±0.042, p<0.01), SR-BI (0.540±0.140 vs 0.220±0.040,p<0.01) and CYP7A1(1.038±0.116 vs0.697±0.141, p<0.01) were all increasd. The results showed that there are some different effects of Fenofibrate on the expression of genes involved in cholesterol metabolism in liver between normal and STZ-induced rats. Conclusion:1 LXRαis activated with the activation of PPARαin liver of STZ-induced rats; disorder of fatty acid metabolism is accompanied with the abnormal expression of genes involved in cholesterol metabolism.2 There are some different effects of Fenofibrate on the expression of genes involved in cholesterol metabolism in liver between normal and STZ-induced rats. Under this experiment Fenofibrate can decreases liver cholesterol level, but not blood cholesterol level in STZ-induced rats.