| Objective: Under physiological conditions, heart acquires most of its energy from metabolism of glucose and fatty acid (FA). During insulin resistance and diabetes, characterized by inadequate glucose utilization, cardiac FA consumption supersedes glucose oxidation. The heart relies almost exclusively on fatty acids to generate energy. So what will happen to FAβoxidation related genes? During diabetes, lipids deposition were ovserved in heart. Whether or not the lipids are associated with the rate-limiting enzymes in triglyceride synthesis? In this experiment, cardiac FA oxidation and triglyceride synthesis related genes were ovserved at transcriptional level, in order to investigate the effect of FA metabolism in the development of myocardial lesions during insulin resistance and diabetes.Fatty acids are oxidized in mitochondria and peroxisomes. Muscle-carnitine palmitoyltransferase 1(m-CPT1) is the key enzyme in mitochondria oxidation in heart. Peroxisome is in charge of oxidation of very long chain fatty acids and branched fatty acids. In peroxisomal pathways ofβ-oxidation, enzymes of catalytic dehydrogenation are acyl-CoA oxidase 1(ACOX1) and acyl-CoA oxidase 3(ACOX3); enzymes of catalytic imbitition and redehydrogenation are L-bifunctional protein(LBP) and D-bifunctional protein(DBP); enzymes of catalytic thioclastic reaction are 3-Ketoacyl-CoA thiolaseA/B(THLA/B) and sterol carrier protein x(SCPx). Peroxisome proliferator-activated receptorα(PPARα) plays a key role in the selection of energy substrate, activated PPARαcan promote the expression of its target genes to enhance the level ofβ-oxidation. In insulin resistance rats and type 2 diabetes rats'heart, are there some connection with the enhancing level ofβ-oxidation and the expressions of genes above, has not been reported.Normally, the amount of TG's synthesis and storage in heart are very little. Diacylglycerol transferase 1(DGAT1) and Diacylglycerol transferase 2(DGAT2) are key enzymes in TG synthesis in the heart, the enzymes can catalyze FA transformed to TG. In insulin resistance rats and type 2 diabetes rats'heart,are there some connection with the abnormal accumulation of TG and the expressions of DGATs, has not been reported.In order to study these issues, we developed insulin resistance in male Sprague-Dawley rats by high-fat diet; and further formation of type 2 diabetes by peritoneal injection of streptozotocin(STZ). To investigate the changes in lipid metabolism of heart by detecting the expression of the genes at mRNA level involved in fatty acid metabolism in heart of insulin resistance rats and type 2 diabetes rats. These genes conclude PPARα,m-CPT1,ACOX1,LBP,THLA,THLB,ACOX3,DBP,SCPx,DGAT1 and DGAT2.Methods:1 AnimalsAccording to the method of Xiaoling W etc.:Male 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. The rats whose fasting blood glucose concentration exceeded 11.1 mmol/L were considered diabetes. Con rats and the other half HF rats received the corresponding volume of citrate buffer. Blood were collected for blood index assay. Cardiac tissues were removed and immediately submerged in liquid Nitrogen, then stored at -80℃for the extraction of total RNA.2 Assay of blood index Blood glucose concentration were detected by Olympus HITACHI 7170A automatic biochemistry analysator.3 Serum and heart total free fatty acids were determined using kit.4 Assay of mRNA expressionHeart 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.5 Statistical analysisData were expressed as mean±SD. SPSS13.0 soft ware was used. Statistical comparisons were made by T test. A value of P<0.05 was considered significant.Results:1 Oral glucose tolerance test(OGTT)The AUC of HF group is significantly higher than that of Con group(P<0.01).2 Changes of fasting blood glucose (FBG) at the 72th hour after the STZ injection.At the 72th hour after the STZ injection, FBG in DM group (12.7±1.9 mmol/L) was significantly higher than that in Con group (5.1±0.6 mmol/L,P<0.01). 2 Changes of blood glucoseCompared with Con group (5.0±0.4mmol/L), the blood glucose contents of DM (12.7±1.9mmol/L) and IR (5.5±0.3mmol/L) groups increased (P<0.01). It indicated that type 2 diabetic rats were successfully established.3 Changes of total Serum and heart free fatty acids.Compared with Con group (0.73±0.25 mmol/L), the total serum FFA contents of DM (1.57±0.16mmol/L) and IR (1.41±0.31mmol/L) groups increased (P<0.01). It indicated that metabolism of FA in type 2 diabetic rats were in choas.Compared with Con group (0.29±0.05 mmol/L), the total heart FFA contents of DM (0.49±0.11mmol/L) and IR (0.49±0.17mmol/L) groups increased (P<0.01). It indicated that metabolism of FA in type 2 diabetic rats'heart were in choas.4 The expression of heart genes compared with 18s RNA.4.1 The relative expression of PPARαmRNA in heart.The relative expression of PPARαmRNA in Con, IR and DM groups were respectively 1.19±0.29, 1.32±0.40, 1.00±0.17. The relative expression of PPARαmRNA in DM and IR group were no different than that in Con group (P>0.05).4.2 The relative expression of m-CPT1 mRNA in heart.The relative expression of m-CPT1 mRNA in Con, IR and DM groups were respectively 6.9±1.3, 7.6±1.9, 8.5±1.2. The relative expression of m-CPT1 mRNA in DM groups were higher than that in Con group(P<0.01)but DM not(P>0.05). It indicated that the oxidation ability of long chain fatty acids of DM group heart was enhanced but IR not.4.3 The relative expression of ACOX1 mRNA in heart.The relative expression of ACOX1 mRNA in Con, IR and DM groups were respectively 4.6±0.3, 5.6±1.2, 5.0±0.9. The relative expression of ACOX1 mRNA in IR group were higher than that in Con group(P<0.01)but DM not.4.4 The relative expression of LBP mRNA in heart.The relative expression of LBP mRNA in Con, IR and DM groups were respectively 0.098±0.029, 0.119±0.037, 0.148±0.022. The relative expression of LBP mRNA in DM group were higher than that in Con group(P<0.01)but IR not(P >0.05).4.5 The relative expression of THLA mRNA in heart.The relative expression of THLA mRNA in Con, IR and DM groups were respectively 2.3±0.9, 3.6±1.4, 3.7±1.7. The relative expression of THLA mRNA in DM and IR groups were higher than that in Con group(P<0.05).4.6 The relative expression of THLB mRNA in heart.The relative expression of THLB mRNA in Con, IR and DM groups were respectively 3.2±1.7, 3.1±1.6, 2.6±0.3. The relative expression of THLB mRNA in DM and IR groups were no different than that in Con group (P>0.05). 4.7 The relative expression of ACOX3 mRNA in heart.The relative expression of ACOX3 mRNA in Con, IR and DM groups were respectively 0.235±0.028, 0.420±0.106, 0.332±0.057. The relative expression of ACOX3 mRNA in DM and IR groups were higher than that in Con group(P<0.01).4.8 The relative expression of DBP mRNA in heart.The relative expression of DBP mRNA in Con, IR and DM groups were respectively 0.82±0.09, 1.90±0.68, 2.11±0.64. The relative expression of DBP mRNA in DM and IR groups were higher than that in Con group(P<0.01).4.9 The relative expression of SCPx mRNA in heart.The relative expression of SCPx mRNA in Con, IR and DM groups were respectively 0.23±0.09, 0.26±0.05, 0.32±0.08. The relative expression of SCPx mRNA in DM and IR groups were no different than that in Con group (P>0.05).The result from 4.3 to 4.9 indicated that: in type 2 diabetes rats'heart, the enhancing level ofβ-oxidation is connected with the pathway of ACOX3-LBP/DBP-THLA/SCPx; in insulin resistance rats'heart, the enhancing level ofβ-oxidation is connected with the pathway of ACOX1/ACOX3- DBP-THLA.4.10 The relative expression of DGAT1 mRNA in heart. The relative expression of DGAT1 mRNA in Con, IR and DM groups were respectively 0.382±0.070, 0.695±0.163, 0.481±0.002. The relative expression of DGAT1 mRNA in DM and IR groups were higher than that in Con group(P<0.01).4.11 The relative expression of DGAT2 mRNA in heart. The relative expression of DGAT2 mRNA in Con, IR and DM groups were respectively 0.65±0.13, 1.01±0.18, 1.12±0.22. The relative expression of DGAT2 mRNA in DM and IR groups were higher than that in Con group(P<0.01).The result from 4.10 to 4.11 indicated that the accumulation of TG in DM and IR groups were relatively with the expression of DGAT1 and DGAT2 in heart.Conclusion:1 In type 2 diabetes rats, the enhanced level ofβ-oxidation is not relative with the mRNA expression of PPARα; the enhanced level ofβ-oxidation of mitochondria is relative with the mRNA expression of m-CPT1; the enhanced level ofβ-oxidation of mitochondria is relative with the mRNA expression of ACOX3,DBP,LBP,THLA and SCPx.2 In insulin resistance rats, the enhanced level ofβ-oxidation is not relative with the mRNA expression of PPARα; the enhanced level ofβ-oxidation of mitochondria is not relative with the mRNA expression of m-CPT1; the enhanced level ofβ-oxidation of mitochondria is relative with the mRNA expression of ACOX1,ACOX3,DBP and THLA mRNA.3 In insulin resistance rats and type 2 diabetes rats, the abnormal accumulation of TG were relatively with the expression of DGAT1 and DGAT2 in heart.
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