Changes In Gene Expression Of Enzymes Involved In Fatty Acid Oxidation In Heart Of STZ-induced Diabetic Rats

Objective: Diabetic cardiomyopathy (DCM) is one of main complications of Diabetes mellitus (DM). Disturbances of fatty acid (FA) metabolism may be an important contributor to DCM. It is helpful for understanding of FA metabolism abnormality, prevention and cure of DCM to study the changes in expression of genes involved in FA oxidation in diabetic heart.Under normal conditions, cardiac FAβ-oxidation occurs in both mitochondria and peroxisome. Muscle-carnitine palmitoyltransferaseⅠ(M-CPTⅠ) and acyl-CoA oxidase (ACOX) are respectively rate-limiting enzymes of the two pathways. In addition to ACOX, L-bifunctional protein (LBP) and D-bifunctional protein (DBP) are also invoved in peroxisomal FAβ-oxidation. Cardiac FA oxidation is enhanced by upregulation of M-CPTⅠ, ACOX genes with peroxisome proliferator-activated receptorα(PPARα) .It was shown that the relative expression of PPARα, M-CPTⅠand ACOX mRNA were increased in the heart of STZ-induced diabetic mice. But it is unclear how about the relative expression of DBP, LBP mRNA is. It was also reported that PPARαexpression was decreased in latter stage heart of STZ-induced diabetic rats. It is unknown whether the relative expression of ACOX, M-CPTⅠ, DBP and LBP mRNA involved in FA oxidation is changed with the progression of DM.Fenofibrate, used for lipid-lowering clinically, regulates lipid metabolism by activating PPARα. It is unclear whether the fenofibrate influences the expression of genes involved in cardiac FA oxidation or not in both normal and diabetic rats. In this experiment, we observed the relative expression of ACOX, M-CPTⅠ, DBP and LBP mRNA in the heart of STZ-induced diabetic rats. In addition, we treated normal and diabetic rats with PPARαagonist fenofibrate to observe the changes in above enzyme mRNA expression.Methods:1 AnimalMale Wistar rats (body weight 200-250g) were randomly divided into control group (C) and diabetic group (D). After fasting overnight, the rats in D were injected intraperitoneally with streptozotocin (STZ, 65mg/kg), dissolved in 0.1M citrate buffer (pH4.5). At the 72th hour after the injection of STZ, the rats whose fasting blood glucose concentration exceeded 16.7mmol/L were considered diabetic. The rats in C received the corresponding volume of citrate buffer. Some of control and diabetic rats were respectively sacrificed 6 and 12 weeks after diabetes induction, as control group of 6 weeks (C6), control group of 12 weeks (C12), diabetic group of 6 weeks (D6) and diabetic group of 12 weeks (D12). Starting 4 weeks after diabetes induction, the other control and diabetic rats were administered orally with fenofibrate (100mg/kg/day) for 2 weeks, as control group with fenofibrate (C+F) and diabetic group of 6 weeks with fenofibrate (D6+F), and then were sacrificed. After fasting overnight, the rats were intubated in carotid artery under 10% chloral hydrate anesthesia (0.37g/kg). Blood were collected for blood index assay. Heart tissues were removed and rinsed with normal saline. A part of tissue was used for morphologic analyses, the other part was immediately submerged in liquid Nitrogen, then stored at -70℃for the extraction of total RNA.2 Assay of blood indexBlood glucose and triglyceride concentration were measured by Auto-biochemical-analyst using oxidase method.3 Detection of cadiocyte morphologyLeft ventricular structures were isolated and fixed in paraform. Paraffin-embedded sections were stained with hematoxylin and eosin (H&E). The change of cadiocyte morphology was observed by light microscope.4 Assay of mRNA expressionHeart total RNA was extracted by Tizol regent. The relative mRNA content was measured by RT-PCR using GAPDH as inner standard.5 Statistical analysis Data were expressed as mean±SEM. SPSS13.0 soft ware was used. Statistical comparisons were made by one-way ANOVA. A value of P<0.05 was considered significant.Results:1 Changes of fasting blood glucose (FBG) at the 72th hour after the STZ injection.At the 72th hour after the STZ injection, FBG in diabetic group (25.0415±2.35903mmol/L) was significantly higher than that in control group (6.9056±1.08383mmol/L,p<0.01). It indicated that STZ-induced diabetic rats were successfully established.2 Changes of FBG of normal and diabetic rats at different stagesFBG in both D6 (27.9576±4.64537mmol/L) and D12 (27.7820±4.23858mmol/L) was significantly higher than that in C (5.5256±2.22392mmol/L , p<0.01). It indicated that STZ-induced diabetic rats were in the condition of high blood glucose.3 Morphologic changes of normal and diabetic rats at different stagesIn C, cadiocytes were ranked in order, nuclei were uniform in size and ellipse, kytoplasm was well-distributed in staining; In D6, cadiocytes were still ranked in order, nuclei were irregular in size, kytoplasm was uneven in staining; In D12, cadiocytes were ranked in disorder, nuclei were karyopyknotic, kytoplasm was autolytic. 4 Changes of mRNA expression of some target genes of normal and diabetic rats at different stages4.1 The relative expression of ACOX1 mRNA in heart The relative expression of ACOX1 mRNA in C, D6 and D12 were respectively 0.8733±0.10765, 0.7076±0.08473, 1.2333±0.22422. The relative expression of ACOX1 mRNA in D12 was significantly higher than that in C (p<0.01). It indicated that PPARαwas activated, which indirectly reflected peroxisome straight chain fatty acid oxidation was enhanced in D12.4.2 The relative expression of ACOX2 mRNA in heart The relative expression of ACOX2 mRNA in C, D6 and D12 were respectively 0.2308±0.07548, 0.2525±0.06791, 0.3582±0.03890. The relative expression of ACOX2 mRNA in D12 was significantly higher than that in C (p<0.01). It indicated that PPARαwas activated, which indirectly reflected peroxisome branched chain fatty acid oxidation was enhanced in D12.4.3 The relative expression of M-CPTⅠmRNA in heart The relative expression of M-CPTⅠmRNA in C, D6 and D12 were respectively 0.3356±0.05994, 0.3779±0.06062, 0.4582±0.06817. The relative expression of M-CPTⅠmRNA in D12 was higher than that in C (p<0.01). It indicated that PPARαwas activated, which indirectly reflected mitochondria fatty acid oxidation was enhanced in D12.4.4 The relative expression of DBP mRNA in heart The relative expression of DBP mRNA in C, D6 and D12 were respectively 0.6760±0.12554, 0.6798±0.12798, 1.2939±0.24959. The relative expression of DBP mRNA in D12 was significantly higher than that in C (p<0.01). The result of DBP was coincident with that of ACOX1 or M-CPTⅠ. It also indirectly reflected peroxisome fatty acid oxidation was enhanced in D12.4.5 The relative expression of LBP mRNA in heartThe relative expression of LBP mRNA in C, D6 and D12 were respectively 0.2232±0.05619, 0.2397±0.06629, 0.3129±0.02448. The relative expression of LBP mRNA in D12 was higher than that in C (p<0.01). The result of LBP was coincident with that of ACOX1 or M-CPTⅠ. It also indirectly reflected mitochondria fatty acid oxidation was enhanced in D12.5 Effect of fenofibrate on serum triglyceride (TG) of diabetic ratsSerum triglyceride in D6 (1.4000±0.46279) was significantly higher than that in C (0.6471±0.18172, p<0.01). Serum triglyceride in D6+F (0.7111±0.31167) was significantly lower than that in D6 (p<0.01). It indicated that fenofibrate could reduce serum triglyceride of diabetic rats.6 Effect of fenofibrate on mRNA expression of some target genes in heart6.1 Effect of fenofibrate on the relative expression of ACOX1 mRNA in heart The relative expression of ACOX1 mRNA in C+F (1.5508±0.21766) was significantly higher than that in C (0.8733±0.10765, p<0.01). There was no difference in mRNA expression of ACOX1 between D6+F (0.9002±0.20649) and D6 (0.7076±0.08473, P>0.05). It indicated that PPARαwas activated in C+F, but unactivated in D6+F.6.2 Effect of fenofibrate on the relative expression of ACOX2 mRNA in heartThe relative expression of ACOX2 mRNA in C+F (0.3954±0.13927) was significantly higher than that in C (0.2308±0.07548, p<0.01). There was no difference in mRNA expression of ACOX1 between D6+F (0.3012±0.06550) and D6 (0.2525±0.06791, P>0.05). It indicated that PPARαwas activated in C+F, but unactivated in D6+F.6.3 Effect of fenofibrate on the relative expression of M-CPTⅠmRNA in heartThe relative expression of M-CPTⅠmRNA in C+F (0.4591±0.07063) was higher than that in C (0.3356±0.05994, p<0.01). There was no difference in mRNA expression of M-CPTⅠbetween D6+F (0.3963±0.06215) and D6 (0.3779±0.06062, P>0.05). It also indicated that PPARαwas activated in C+F, but unactivated in D6+F.6.4 Effect of fenofibrate on the relative expression of DBP mRNA in heartThe relative expression of DBP mRNA in C+F (1.2245±0.07843) was significantly higher than that in C (0.6760±0.12554, p<0.01). There was no difference in mRNA expression of DBP between D6+F (0.8563±0.18792) and D6 (0.6798±0.12798, P>0.05). It indicated that fenofibrate increased DBP mRNA expression in C+F, but had no effect on that in D6+F.6.5 Effect of fenofibrate on the relative expression of LBP mRNA in heartThe relative expression of LBP mRNA in C+F (0.3606±0.03506) was higher than that in C (0.2232±0.05619, p<0.01). There was no difference in mRNA expression of LBP between D6+F (0.2791±0.07178) and D6 (0.2397±0.06629, P>0.05). It also indicated that fenofibrate increased LBP mRNA expression in C+F, but had no effect on that in D6+F. Conclusion:1 In diabetic heart, increased fatty acid oxidation is related to enhanced fatty acid oxidation not only in mitochondria, but also in peroxisome, because of higher mRNA expression of peroxisomal ACOX1, ACOX2, DBP and LBP.2 In diabetic heart, the abnormality of fatty acid metabolism might be related to changes in PPARα―ACOX, PPARα―M-CPTⅠregulation pathway.