Changes In Expression Of Genes Involved In Liver Fatty Acids Metabolism Of Insulin Resistance And Diabetic Rats

Objective: Insulin resistance is a risk factor of type 2 diabetes mellitus and atherosclerosis. It is a key feature of type 2 diabetes. The serum free fatty acids (FFA) is elevated in insulin resistance and type 2 diabetes status. Free fatty acids in the liver are also elevated and triglycerides are accumulated in liver. Fatty acids enter into liver and can be metabolized by mitochondrialβ-oxidation, peroxisomalβ-oxidation and esterification. Carnitine palmitoyltransferaseⅠ(CPTⅠ) is essential for long-chain acyl-coenzyme A to enter into mitochondria, and is the key enzyme for long-chain fatty acid oxidation. In peroxisome, palmitoyl-CoA oxidase (ACOX1) is involved in the oxidation of very long straight-chain fatty acid oxidation, and pristanoyl-CoA oxidase (ACOX3) is involved in the oxidation of branched-chain fatty acids. Acyl-CoA oxidase 2 (ACOX2) is involved in bile acid synthesis. Diacylglycerol transferases (DGATs) are the key enzymes for triglyceride synthesis.In diabetic state, impaired glucose utilization due to insulin deficiency renders tissues more dependent on lipid oxidation for the energy metabolism, and the lipid oxidation is enhanced. Our previous research had also showed that mitochondrial and peroxisomalβ-oxidation were both enhanced in diabetic rats. Besides, we found mitochondrial and peroxisomalβ-oxidation were both enhanced in insulin resistance state. Kohtaro Asayama found that the enhancement of peroxisomalβ-oxidation in diabetic state was related to the increase of ACOX1 mRNA expression. So, how did the other enzymes involved in peroxisomalβ-oxidation express? Was the enhancement of mitochondrial and peroxisomalβ-oxidation ralated to the expression of biocatalyst genes in insulin resistance state?Peroxisome proliferator activated receptorα(PPARα) was a transcriptional factor,which regulated the transcription of downstream genes when it was activated by ligand. CPTⅠ,ACOX1 and LBP were the downstream genes of PPARαand were regulated by PPARα. Some investigations showed the expression of PPARαin hepatic tissue increased in high-dose STZ induced diabetic rats. How the expression of PPARαin insulin resistance and type 2 diabetic state?Investigations showed that there was TG disposition in hepatic tissue in insulin resistance and type 2 diabetic states and our prior research showed the same phenomenon. Was TG disposition in hepar related to the expression of DGAT biocatalyst gene?In order to study these issues, we developed insulin resistance in male Sprague-Dawley rats by high-fat diet, and further formation of diabetes by peritoneal injection of streptozotocin (STZ).To investigate the changes in lipid metabolism of liver by detecting the expression of the genes involved in fatty acid metabolism in liver of insulin resistance rats and diabetes rats. To investigate the molecular mechanism of hepatic lipid metabolism disorder in insulin resistance and type 2 diabetic states.Methods:1 AnimalsMale SD rats were divided randomly into control group (Con group) and high fat group (HF group). The rats in Con group were fed with standard diet and HF group high-fat diet. lipid in the high-fat diet consisted mainly of lard-based, accounting for 59.8 percent of calory. After feeding for 8 weeks in high-fat diet, the rats were detected for insulin resistance by oral glucose tolerance (OGTT) And then, after overnight fasting, half of the HF rats were injected intraperitoneally with 25 mg/kg of streptozotocin freshly dissolved in ice cold sodium citrate buffer (0.1 M, pH4.5). Diabetes was confirmed three days after the i.p. injection of streptozotocin by the presence of hyperglycemia.The rats whose fasting blood glucose concentration exceeded 11.1 mmol/L were considered diabetes. So, HF group were dvided into two groups: Insulin resistance group (IR group) and diabetic group (DM group).All the rats in three groups were sacrificed 6 weeks after diabetes induction. After fasting overnight, the rats were anesthetized in by 20% urethane (0.12g/100g). 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 glucoseBlood glucose concentration were assayed by Olympus HITACHI 7170A automatic biochemistry analysator.3 Serum and liver total free fatty acids contents were assayed using kit.4 Fatty deposition in liver was detected by oil red o staining.5 Liver triglyceride contents was assayed using kit.6 Assay of mRNA expression Liver total RNA was extracted by Promega SV Total RNA Isolation System. The relative mRNA content was measured by quantitative real time RT-PCR, with 18S rRNA as inner standard.7 Statistical analysisData were expressed as mean±SD. SPSS13.0 soft ware was used. Statistical comparisons were made by Independent-Samples T Test (2-tail). A value of P<0.05 was considered significant.Results:1 Oral glucose tolerance test (OGTT)The AUC of high fat fed rats group (HF) is significantly higher than that of Con group (P<0.01).2 Changes of fasting blood glucose (FBG) of rats before being sacrificedCompared with Con group (4.966±0.433mmol/L), the FBG of DM (12.717±1.862mmol/L) and IR (5.511±0.345mmol/L) groups increased (P<0.01).3 Changes of total Serum free fatty acids contents. Compared with Con group (0.581±0.114 mmol/L), the total FFA contents of DM(1.55 8±0.084 mmol/L)and IR (1.602±0.112 mmol/L)groups increased (P<0.01).4 Changes of total liver free fatty acids contents. Compared with Con group(81.365±10.609μmol/gprot), the total FFA contents of DM(156.080±19.268μmol/gprot)and IR(213.922±40.809μmol/gprot)groups increased (P <0.01).5 Detected fatty deposition in liver by oil red o staining Little fat drops could be found in Ctrl. group, but more fat drops were found in IR and DM groups. It showed that there were severe fat deposition in IR and DM rats'livers.6 Changes of liver triglyceride contents.Compared with Con group (11.30±1.89μmol/gtissue), the total triglyceride contents of DM (51.47±7.77μmol/gtissue) and IR (34.94±4.93μmol/gtissue) groups increased (P < 0.01).7 The expression of liver genes7.1 The relative expression of PPARαmRNA in liver The relative expression of PPARαmRNA in Con, IR and DM groups were respectively 1.758±0.231, 1.885±0.262, 2.340±0.458. The relative expression of PPARαmRNA in DM group was significantly higher than that in Con group (P <0.01).7.2 The relative expression of CPTⅠmRNA in liverThe relative expression of CPTⅠmRNA in Con, IR and DM groups were respectively 4.338±0.923, 3.087±0.446, 2.964±0.078. The relative expression of CPT1 mRNA in IR and DM groups were lower than that in Con group (P <0.01).7.3 The relative expression of ACOX1 mRNA in liverThe relative expression of ACOX1 mRNA in Con, IR and DM groups were respectively 12.061±1.154, 13.074±0.640, 14.335±0.561. The relative expression of ACOX1 mRNA in IR and DM groups were higher than that in Con group (P <0.05, P <0.01).7.4 The relative expression of ACOX2 mRNA in liverThe relative expression of ACOX2 mRNA in Con, IR and DM groups were respectively 0.099±0.018, 0.072±0.015, 0.098±0.016. The relative expression of ACOX2 mRNA in IR groups were lower than that in Con group (P <0.01).7.5 The relative expression of ACOX3 mRNA in liverThe relative expression of ACOX3 mRNA in Con, IR and DM groups were respectively 4.315±0.312, 5.041±0.392, 4.564±0.528. The relative expression of ACOX3 mRNA in IR group was higher than that in Con groups (P <0.01).7.6 The relative expression of LBP mRNA in liverThe relative expression of LBP mRNA in Con, IR and DM groups were respectively 2.316±0.604, 3.332±0.340, 4.349±0.544. The relative expression of LBP mRNA in IR and DM groups were higher than that in Con ( P<0.01).7.7 The relative expression of DBP mRNA in liverThe relative expression of DBP mRNA in Con, IR and DM groups were respectively 6.588±0.317, 8.084±0.225, 8.369±0.762. The relative expression of DBP mRNA in IR and DM groups were higher than that in Con (P < 0.01).7.8 The relative expression of THLA mRNA in liverThe relative expression of THIA mRNA in Con, IR and DM groups were respectively 0.0294±0.0032, 0.0371±0.0063, 0.0391±0.0014. The relative expression of THIA mRNA in IR and DM groups were higher than that in Con (P <0.01).7.9 The relative expression of THLB mRNA in liverThe relative expression of THIB mRNA in Con, IR and DM groups were respectively 0.388±0.048, 0.646±0.125, 1.956±0.236. The relative expression of THIB mRNA in IR and DM groups were higher than that in Con(P <0.01).7.10 The relative expression of SCPx mRNA in liverThe relative expression of SCPx mRNA in Con, IR and DM groups were respectively 12.136±1.980, 18.762±3.446 12.661±0.976. The changes of relative expression of SCPx mRNA in IR groups were higher than that in Con(P < 0.05).7.11 The relative expression of DGAT1 mRNA in liverThe relative expression of DGAT1 mRNA in Con, IR and DM groups were respectively 0.485±0.051, 0.659±0.031, 0.702±0.053. The relative expression of DGAT1 mRNA in IR and DM groups were higher than that in Con( P <0.01). 7.12 The relative expression of DGAT2 mRNA in liver The relative expression of DGAT2 mRNA in Con, IR and DM groups were respectively 4.267±0.687, 6.030±0.952, 4.886±0.447. The relative expression of DGAT2 mRNA in IR and DM groups were higher than that in Con(P <0.01, P <0.05).Conclusion:1 In insulin resistance state, the enhancement of hepatic peroxisomal fatty acidβ-oxidation was related to the increase expressions of ACOX1, LBP, THLA/B, ACOX3, DBP, and SCPx. The enhancement of hepatic mitochondrial fatty acidβ-oxidation was not related to the expression of CPTⅠ.2 In type 2 diabetic state, the enhancement of hepatic peroxisomal fatty acidβ-oxidation was related to the increase expressions of ACOX1, LBP, DBP and THLA/B. The enhancement of hepatic mitochondrial fatty acidβ-oxidation was not related to the expression of CPTⅠ.3 In insulin resistance and type 2 diabetic state, increased triglyceride accumulation in liver was related to higher mRNA expression of DGAT 1 and DGAT 2.