Causes Of Fatty Acid And Fat Accumulation In Skeletal Muscle Of Diabetes Rats

Objective: Fatty acid and triglyceride accumulation in skeletal muscle cell is an important cause of the decreased insulin sensitivity in skeletal muscle, whidh leds to insulin resistance in type 2 diabetes mellitus (T2DM).Fatty acid is one major energy substrate in skeletal muscle. Fatty acidβ-oxidation occurs both in mitochondria and peroxisome. Theβ-oxidation pathway consists of four reactions (oxidation, hydration, dehydrogenation, thiolysis). Short- , medium- and long- chain fatty acids are oxidized though mitochondrialβ-oxidation. Muscle-carnitine acetyl transferaseⅠ(M-CPTⅠ) is the rate-limiting enzyme of mitochondrialβ-oxidation. Peroxisomalβ-oxidation is involved in very long chain fatty acids. There are two two pathways ofβ-oxidation in peroxisomal. In the first pathway, straight-chain fatty acids are catalyzed by fatty acyl-CoA oxidase 1(ACOX1), L-bifunctional protein (LBP), and thiolase. In the second pathway, 2-methyl-branched fatty acids are catalyzed by ACOX3, D-bifunctional protein (DBP), and sterol carrier protein (SCP) x. Peroxisome proliferator-activated receptorα(PPARα) can affect fatty acids oxidation in skeletal muscle by regulating downstream genes expression (CPTⅠ, ACOX, et al). Fatty acids can be used to synthesize triglyceride in skeletal muscle cell. Diacylglycerol acyltransferase (DGAT1) is the rate-limiting enzyme in the reaction.In the condition of T2DM, fatty acid and triglyceride accumulation in skeletal muscle cell leads to decreased insulin sensitivity of skeletal muscle. But the molecule mechanism of fatty acids and triglyceride accumulation is not clear. It is not reported whether it is relative to the decreaced fatty acidsβ-oxidation in mitochondria and peroxisome and the increased fatty acids esterification in endochylema.In present experiment, type 2 diabetes mellitus (T2DM) was induced by a high fat diet and streptozocin (STZ). Oral glucose tolerance test, Detection of insulin sensitivity index and Hyperinsulinemic-euglycemic clamp indicated insulin resistance. Observation of skeletal muscle with oil red O staining and other experiments showed fatty acids and triglyceride accumulation in skeletal muscle. The mRNA expression of the enzymes in fatty acidsβ-oxidation in mitochondria and peroxisome (M-CPTⅠ,ACOX1,ACOX3,DBP,LBP) , the key enzyme in triglyceride synthesis( DGAT1), and nuclear receptor PPARαare detected with RT-PCR. The type of the fatty acids in skeletal muscles are analyzed with gas chromatograph. The activity of fatty acidsβ-oxidation are detected with spectrophotometer. The reasons of fatty acids and triglyceride accumulation in skeletal muscle cell are studied, which provides references to researches of etiopathogenisis and prevention of 2 diabetes mellitus.Methods:1 Animals and materialsMale SD rats were divided randomly into control group (Con) and type 2 diabetes mellitus group (DM). Con group and DM group were fed respectively with standard diet and high fat diet. After eight weeks, DM group insulin resistant were confirmed by experiments. One more week later, DM group were injected STZ 27mg/kg in enterocoelia. The fasting blood glucose (FBG) was detected after 72 hours. Finally, the rats with FBG higher than 7.8mmol/L,were defined type 2 diabetes mellitus rats. All the rats were killed after another six weeks. Blood were collected for blood index assay. A part of skeletal muscle tissue was used for morphologic analyses, the other part was used for the extraction of total RNA and lipids.2 Oral glucose tolerance test (OGTT)The rats were perfused with glucose on a empty stomach. The blood glucoses were measured respectively at 0, 30, 60, 120 minutes, then blood glucoses of the two groups were analyzed.3 Detection of insulin sensitivity index (ISI)Fasting blood were collected for detections of insulin and FBG, then the ISI was calculated.4 Hyperinsulinemic-euglycemic clampIn the condition of hyperinsulinemic-euglycemic homeostasis with intravenous injection of insulin and glucose, the insulin sensitivity was evaluated with glucose infusion rate (GIR).5 Detection of blood glucose with glucose oxidase method6 Detection of serum triglyceride with oxidase method7 Observation of skeletal muscle with oil red O staining(ORO)8 Detection of skeletal muscle triglycerideSkeletal muscle was homogenated with isopropanol. Supernatant was imbibed after centrifugalization, then the content of triglyceride was detected.9 Detection of skeletal muscle free fatty acids Skeletal muscle was homogenated with physiological saline. Supernatant was imbibed after centrifugalization, then the content of free fatty acids was detected.10 Detection of skeletal muscle fatty acids with gas chromatograph11 Assay of skeletal muscle genes mRNA expressionSkeletal muscle total RNA was extracted by Trizol regent. The relative content of m-CPTⅠ, ACOX1, ACOX3, LBP, DBP, PPARαand DGAT1 mRNA was measured by RT-PCR.Results:1 OGTTThe blood glucose concentrations (at 0, 30, 60, 120 min) of DM group were respectively higher than those of Con (P<0.01). The result showed that carbohydrate tolerances of DM rats were injured.2 ISIISIs of DM group were lower than those of Con group (-5.33±0.26 vs -4.60±0.14, P<0.01). It indicated that insulin sensitivity of T2DM rats decreased.3 Hyperinsulinemic-euglycemic clampIn homeostasis, GIRs of DM group were lower than those of Con group (20.44±1.99mg/kg.min vs 27.97±1.72 mg/kg.min, P<0.01). It showed that T2DM rats insulin resistance was obvious.4 Blood glucose (BG)BGs of DM group were higher than 7.8mmol/L and obviously higher than those of Con group (15.55±2.69mmol/L vs 5.25±0.52 mmol/L, P <0.01).5 Serum triglycerideSerum TGs of DM group were obviously higher than those of Con group(2.26±0.51 mmol/L vs 1.09±0.20 mmol/L,P <0.01). It indicated that triglyceride metabolisms of T2DM rats were disorder.6 Skeletal muscle with oil red O stainingThe quantity of lipid droplets in skeletal muscle cells of DM group was larger than that of Con group.7 Skeletal muscle triglyceride and free fatty acidsSkeletal muscle triglycerides and free fatty acids of DM group were respectively higher than those of Con group (38.1±7μmol /gtissue vs 9.42±1.84μmol /gtissue, P<0.01; 15.57±2.92μmol/gpro vs 9.80±1.39μmol/g pro, P <0.01). 8 Detection of skeletal muscle fatty acids with gas chromatograph Differences of percentage contents of hexadecanoic acid, octadecylic acid, octadecenoic acid and hexacosoic acid between DM group and Con group were no statistical significance.9 The relative expression of skeletal muscle genes9.1 The relative expression of m-CPTⅠmRNADM group compared to Con group (1.303±0.260 vs 1.437±0.283, P>0.05), the changes of relative expression of m-CPTⅠmRNA, gene of the enzyme of fatty acidsβ-oxidation in mitochondria, were no statistical significance.9.2 The relative expression of LBP,ACOX1 mRNADM group compared to Con group, genes of the enzymes in the first pathway of fatty acidsβ-oxidation in peroxisome, LBP mRNA expression decreased (0.526±0.071 vs 0.812±0.121,P<0.01), and differences of ACOX1 mRNA expressions (1.580±0.171 vs 1.498±0.187,P>0.05) were no statistical significance.9.3 The relative expression of DBP,ACOX3 mRNADM group compared to Con group, genes of the enzymes in the second pathway of fatty acidsβ-oxidation in peroxisome, differences of DBP mRNA expressions (0.746±0.145 vs 0.764±0.116,P>0.05) were no statistical significance, and ACOX3 mRNA expression decreased(0.690±0.124 vs 0.392±0.054,P<0.01).9.4 The relative expression of PPARαmRNADM group compared to Con group, PPARαmRNA expression decreased (0.460±0.044 vs 1.089±0.213,P<0.01). The results indicated that PPARαmRNA expression in T2DM rats skeletal muscle cell was down-regulated.9.5 The relative expression of DGAT1 mRNADM group compared to Con group, gene of the enzyme of triglyceride synthesis, DGAT1 mRNA expression decreased (0.690±0.124 vs 0.392±0.054,P<0.01).Conclusion:1 The increased free fatty acids in skeletal muscle of type 2 diabetes rats might be associated with decreased expression of the genes invoved in peroxisomalβ-oxidation and PPARα2 The triglyceride accumulation in skeletal muscle of type 2 diabetes rats might be associated with the increased DGAT1 mRNA expression.