| Objective: Hepatic cholesterol metabolism is an important process in the maintenance of total body cholesterol homeostasis. Was the concentration of hepatic cholesterol perturbed in type 2 diabetic state? How was the concentration of hepatic cholesterol in insulin resistance as a risk related to type 2 diabetes?Transport of cholesterol plays a major part in the liver. It has been shown that SR-BI is responsible primarily for reverse cholesterol transport in the liver, Cholesterol esters from HDL are taken up into the liver through selective uptake by the HDL receptor, scavenger receptor class BI (SR-BI),and SR-BI mRNA expressions affects the concentration of reverse cholesterol. Hepatocytes using acetyl-CoA for cholesterol synthesis. HMG-CoA reductase is the rate-limiting enzyme of synthesis pathway. The amount of the expression of rate-limiting enzyme reflects the amount of cholesterol synthesis. Cholesterol 7-hydroxylase (CYP7A1) catabolized cholesterol to bile acids is rate-limiting enzyme, CYP7A1 mRNA expressions reflects the amount of cholesterol by bile acid synthesis. A second major catabolic route is the direct efflux of cholesterol from the liver into the bile via the ATP-binding cassette transporter G5 (ABCG5) and ATP-binding cassette transporter G5 (ABCG8). Liver X receptors are ligand-activated transcription factors that belong to the nuclear receptor superfamily, and target genes of LXR is CYP7A1, ABCG5, ABCG8. Is LXR activated in type 2 diabetes and insulin state? Is HMGR, SR-BI, ABCG5, ABCG8, CYP7A1 expression at the mRNA level related to the content of liver cholesterol?There are two LXR subtypes, LXRαand LXRβ, they are expressed in the liver. How to change of LXRαand LXRβexpression at the mRNA level in a state of insulin resistance and diabetes? Study of changes for expression of genges involved in hepatic cholesterol metabolism on insulin resistance and type 2 diabetic rats.Methods:1 Animals and materialsMale SD rats (body weight 200-250 g, n=40) were randomly divided into control diet rats (n=12) and high diet rats (n=28). Control diet rats were treated with normal chow (10.3% of calories as fat) for 14 weeks as control group (Con). High diet rats were treated with high fatty chow (59.8% of calories as fat) for 8 weeks. Plasma insulin concentrations in response to control diet rats were increased (P < 0.05), the result showed that the model of insulin resistance is setted up. High diet rats were randomly divided into insulin resistance rats (IR, n=12) and diabetes rats (DM, n=16). Diabetes rats injected with streptozotocin (2%STZ, 25 mg/kg intravenously). After 72 h, blood glucose concentration of diabetes rats were increased compaired with insulin resistance rats and control diet rats. All the rats in three groups were sacrificed 6 weeks after diabetes induction. After fasting overnight, the rats were killed. Blood were collected for blood index assay. Liver tissues were removed and immediately submerged in liquid Nitrogen, then stored at -80℃for the extraction of total RNA.2 Assay of blood glucose and serum cholesterolBlood glucose concentration and serum cholesterol were observed by Olympus HITACHI 7170A automatic biochemistry analysator.3 The level of serum high-density lipoprotein cholesterol were observed by Olympus HITACHI 7170A automatic biochemistry analysator.4 The level of cholesterol in liver were observed using Kit.5 Observation of mRNA expressionBrain total RNA was purchased by Promega SV Total RNA Isolation System. Using reverse transcriptase, cDNA were used as template for quantitative real time PCR , with 18S rRNA as inner standard.6 Statistical analysisData were expressed as mean±S.D., SPSS 13.0 soft ware was used. Statistical comparisons were made by Independent-Samples T Test (2-tail). A value of P < 0.05 was considered significant. The value of control group was expressed as one. The fold change was relatived to control rats by insulin resistance rats and diabetic rats.Results:1 The result of oral glucose tolerance test (OGTT) showed that the model of insulin resistance is setted up. The result of changes of fasting blood glucose (FBG) proved that the model of type 2 diabetes is setted up.2 The level of cholesterol in liver (mg/g tissue) The level of HDL in Con, IR and DM groups were respectively 225.96±28.21, 346.48±71.02 and 212.03±20.73. Compared with Con groups, the level of cholesterol in liver of IR groups significantly increased (P<0.01) and DM groups had no significant difference (P > 0.05).The results showed that abnormal cholesterol metabolism in liver tissue in insulin state.3 Observation of mRNA expression in liver3.1 The relative expression of LXRαmRNAThe relative expression of LXRαmRNA in Con, IR and DM groups were 59.00±9.67, 73.61±18.28 and 75.10±15.12, respectively. Compared with Con groups, IR groups had no significant difference (P < 0.05) and DM groups significantly increased (P<0.01).The value of control group was expressed as one. Relatived to Con groups, IR groups was 1.27 and DM groups was 1.29.The results showed that the expression of LXRαmRNA significantly increased in insulin resistance and type 2 diabetic state.3.2 The relative expression of LXRβmRNAThe relative expression of LXRβmRNA in Con, IR and DM groups were 28.46±7.98, 30.53±4.84 and 32.69±8.17, respectively. Compared with Con groups, IR groups and DM groups had no significant difference (P > 0.05).The value of control group was expressed as one. Relatived to Con groups, IR groups was 1.15 and DM groups was 1.18.The results showed that the liver LXRβmRNA relative expression levels of the same, and less than the relative expression levels of LXRα. 3.3 The relative expression of SR-BI mRNAThe relative expression of SR-BI mRNA in Con, IR and DM groups were 9.60±2.70, 17.79±4.45 and 18.58±4.05, respectively. Compared with Con groups, IR groups and DM sigificantly increased (P<0.01).The value of control group was expressed as one. Relatived to Con groups, IR groups was 2.17 fold change and DM groups was 2.20 fold change. Hepatic SR-BI is a high-density lipoprotein-specific receptors. Results suggest that the relative expression of SR-BI increased more intake of the HDL-mediated transport of excess cholesterol from peripheral tissues to the liver for excretion or recycling.3.4 The relative expression of HMGR mRNAThe relative expression of HMGR mRNA in Con, IR and DM groups were respectively 13.58±2.42, 25.28±6.32 and 32.75±7.23. Compared with Con groups, IR groups and DM groups significantly increased (P < 0.01). The value of control group was expressed as one. Relatived to Con groups, IR groups was 1.97 fold change and DM groups was 2.46.HMGR is the rate-limited enzyme in the pathway to synthesize cholesteror. The results showed that the amount of the mRNA relative expression of HMGR is a significant increase in hepatic cholesterol synthesis.3.5 The relative expression of CYP7A1 mRNAThe relative expression of CYP7A1 mRNA in Con, IR and DM groups were 42.69±10.80, 51.98±4.22 and 69.65±20.69, respectively. Compared with Con groups, IR groups non-significantly increased (P < 0.05) and DM groups significantly increased (P < 0.01).The value of control group was expressed as one. Relatived to Con groups, IR groups was 1.26 and DM groups was 1.62.CYP7A1 is the rate-limited enzyme in the classical pathway of cholesterol synthesis of bile acids. The results showed that a significant increase of cholesterol into bile acids.3.6 The relative expression of ABCG5 mRNAThe relative expression of ABCG5 mRNA in Con, IR and DM groups were 6.90±1.53, 8.59±1.81 and 12.52±3.09, respectively. Compared with Con groups, IR groups significantly increased (P < 0.05) and DM groups significantly increased (P < 0.01).The value of control group was expressed as one. Relatived to Con groups, IR groups was 1.37 and DM groups was 1.97.The role of ABCG5 is the direct efflux of cholesterol from the liver into the bile. The results show that an significant increase of cholesterol into bile.3.7 The relative expression of ABCG8 mRNAThe relative expression of ABCG8 mRNA in Con, IR and DM groups were respectively 1.16±0.30, 1.54±0.31 and 3.58±0.38. Compared with Con groups, IR groups and DM groups significantly increased (P < 0.01).The value of control group was expressed as one. Relatived to Con groups, IR groups was 1.46 and DM groups was 3.41.The role of ABCG8 is the direct efflux of cholesterol from the liver into the bile. The results show that an significant increase of cholesterol into bile.Conclusion:1 In type 2 diabetic state, no changes in the hepatic cholesterol concentration.2 In insulin resistance state, the elevated of hepatic cholesterol concentration was related to the mRNA expressions of HMGR, SR-BI higher than ABCG5, ABCG8, and relative to the enhancement of the mRNA expression level of LXRα.3 Compared with Con groups, the relative expression levels of LXRαis increased in insulin resistance and type 2 diabetic state. LXR is activated in the liver .
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