| Objective: Diabetic cardiomyopathy (DCM) is one of main complications of Diabetes mellitus (DM). Disturbances of lipid metabolism may be an important contributor to DCM. As the result of lipid metabolism disorder, the accumulation of lipid is also the etiological factor of other concurrent diseases. What reasons break the balance of lipid revenue and expenditure? It is helpful for understanding of FA metabolism abnormality, prevention and cure of DCM to observe fatty acids metabolism in heart of type-2 diabetic rats.Under normal conditions, fatty acids in the heart have two ways to be consumed: one is to be esterized into triglyceride, DGAT1 is the key enzyme ; the other is to be oxidized in mitochondria and peroxisomes. Muscle-carnitine palmitoyl transferase 1 (M-CPT1) and acyl-CoA oxidase (ACOX) are respectively rate-limiting enzymes of the two pathways. Cardiac FA oxidation is enhanced by upregulation of M-CPT1, ACOX genes with peroxisome proliferator-activated receptorα(PPARα).In this experiment, on the basis of the model of type 2 diabetic rats, we observed the relative expression of ACOX, M-CPT1, PPARα, DGAT1; we alse detected the activity of total fatty acidsβ-oxidation with spectrophotometric method. Triglyceride and total fatty acids in the heart were determined as well.Methods:1 AnimalMale SD rats (body weight 200-250g) were randomly divided into control group (C) Insulin resistance group (IR) and diabetic group (D). IR and D group were feed on high fat food, after D group emerge insulin resistance, the rats were injected intraperitoneally with streptozotocin (STZ, 27mg/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 7.8 mmol/L were considered diabetic. The rats in C and IR received the corresponding volume of citrate buffer. All the rats in three groups were sacrificed 8 weeks after diabetes induction. 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 -80℃for the extraction of total RNA, determination of activity of enzymes and oil red staining.2 Determination of insulin sensibilityIt is estimated by euglycemic clamp and insulin sensibility index (ISI) 3 Assay of blood indexBlood glucose and triglyceride concentration were measured by kit using oxidase method. Insulin were mersured by radio-immunity4 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.5 Assay of mRNA expressionHeart total RNA was extracted by Tizol regent. The relative mRNA content was measured by RT-PCR usingβ-ACTIN as inner standard.6 Detection of the activity of total fatty acidsβ-oxidation with spectrophotometric method.7 Triglyceride and total fatty acids in the heart were determined using kit.8 Oil red staining to estimate TG accumulation.9 Statistical analysisData 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 OGTT experiment, Euglycemic clamp and insulin sensibility index show that insulin resistance occur in IR and D group2 Changes of fasting blood glucose (FBG) at the 72th hour after the STZ injection.At the 72th hour after the STZ injection, FBG in D group (14.49±3.67mmol/L) was significantly higher than that in C group (5.25±0.43 mmol/L,P<0.01). It indicated that type 2 diabetic rats were successfully established.3 Morphologic changes of C , IR and D group rats.In C, cadiocytes were ranked in order, nuclei were uniform in size and ellipse, kytoplasm was well-distributed in staining; In IR, cadiocytes were still ranked in order, nuclei were irregular in size, kytoplasm was uneven in staining; In D, cadiocytes were ranked in disorder, nuclei were karyopyknotic, kytoplasm was autolytic.4 TG staining changes of C , IR and D group rats.In C and IR , red particles were obviously fewer than D. It indicated that TG accumulated in the heart of D group rats.5 Changes of mRNA expression of some target genes of three group rats5.1 The relative expression of ACOX1 mRNA in heart The relative expression of ACOX1 mRNA in C, IR and D were respectively 0.5498±0.07123, 0.6652±0.06484, 0.7417±0.14342. The relative expression of ACOX1 mRNA in D was significantly higher than that in C (P<0.05).5.2 The relative expression of M-CPT1 mRNA in heartThe relative expression of M-CPT1 mRNA in C, IR and D were respectively 0.3730±0.09561, 0.5288±0.05377, 0.5337±0.04740. The relative expression of M-CPT1 mRNA in IR and D were higher than that in C (P<0.01).5.3 The relative expression of DGAT1mRNA in heartThe relative expression of DGAT1mRNA in C, IR and D were respectively 0.3891±0.06180, 0.5494±0.04182, 0.8515±0.08786. The relative expression of DGAT1 mRNA in IR and D were higher than that in C (P<0.01).5.4 The relative expression of PPARαmRNA in heartThe relative expression of PPARαmRNA in C, IR and D were respectively 0.7065±0.09407, 0.8444±0.03091, 0.6635±0.13149. The relative expression of PPARαmRNA in IR were higher than that in C (P<0.05) but D not. It indicated that the relative expression of PPARαwas enhanced at first, then breakdown at 8 weeks diabetic.5.5 The relative expression of ACOX3 mRNA in heartThe relative expression of ACOX3 mRNA in C, IR and D were respectively 0.1380±0.03745, 0.1811±0.01538, 0.2058±0.01704. The relative expression of ACOX3 mRNA in IR and D were higher than that in C (P<0.05 and P<0.01).6 Ability of fatty acidsβ-oxidation in heart(U/mg prot)The ability of fatty acids oxidation in C, IR and D were respectively 0.001712±0.000334, 0.002492±0.000796, 0.003016±0.000714. The ability of fatty acids oxidation in IR and D were higher than that in C (P<0.05 and P<0.01).7 Content of TG in heart measured by kit (nmol/mg organism)The content of TG in heart in C, IR and D were respectively 3.7474±1.6784, 8.3811±3.4601, 18.1837±11.1713. The content of TG in heart in IRand D were higher than C (P <0.01), It indicated that TG accumulated in IR and D.8 Content of FA in heart measured by kit(μmol/g prot)The content of FA in heart in C, IR and D were respectively 158.0271±54.68046, 186.5958±56.95322, 124.8384±56.07559. The content of TG in heart in C, IR and D were not different. It indicated that fatty acids uptaked by cadiocytes were sufficiently transformed.9 Ability of catalase in heart(U/mg prot)The ability of catalase in C, IR and D were respectively 32.11361±2.715636, 10.64068±2.483603, 11.83867±2.54193. The ability of fatty acids oxidation in IR and D were lower than that in C( P <0.01)Conclusion:1 In type 2 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, ACOX3.2 In type 2 diabetic heart, increased triglyceride accumulation is related to higher mRNA expression of DGAT1 which is the key enzyme of triglyceride synthesis.
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