Effect Of Oxidative Stress Mediated By Mitochondria On The Pathogenesis Of Skeletal Myopathy In Diabetic Rats

Objectives:1. To explore the effect of oxidative stress mediated by mitochondria on skeletalmyopathy in diabetic rats2. To elucidate the regulation of UCP3 expression in mitochondrial oxidative stress3. To explore the possible mechanism of insulin resistance aggravated bymitochondrial oxidative stress4. To assess the effect of antioxidantα-LA treatment and its potential pathwayMethods:1. Intervention given to type 1 diabetic rats1.1 Type 1 diabetic rat model was induced by a single injection of streptozotocin(45mg/kg body weight) via tail vein.1.2 Four groups of rats were used: control, diabetes, diabetes with insulin treatment,diabetes withα-LA treatment. Rats in each group were sacrificed at three timepoints: 4w, 8w, 12w after the onset of diabetes. PZI was administrateddaily(6U/kg body weight) via hypodermic injection in Insulin group, andα-LAwas given as a daily intraperitoneal injection(100mg/kg body weight).1.3 Biochemical assays: blood glucose, HbA1C, lipid profiles and FFA wereexamined for rats in each group.1.4 The pathological changes of rat skeletal muscle in each group were detected byHE and Masson staining.1.5 Transmission electron microscopy was used for ultra-structural analysis of skeletalmuscle.1.6 The mitochondrion in skeletal muscle was extracted by differential centrifugation,followed by the examination of MDA content(using TBA method), Mn-SODactivity and CAT concentration by spectrophotometer.1.7 Expression of Caspase-3 and Bcl-2/Bax were determined byimmuno-histochemical technique.1.8 Expression of Mn-SOD and UCP3 mRNA and protein were determined bySQ-RT-PCR and Western blotting assay respectively.2. Treatment of type 2 diabetic rats2.1 Insulin resistance with hyperlipidemia and type 2 diabetic rat model was inducedby low dose streptozotocin(30mg/kg body weight) combined with high energy intake.2.2 Experimental rats were randomly divided into four groups: normal control, highenergy diet, type 2 diabetes and diabetes withα-LA administration.2.3 Insulin level in plasma was tested by radioimmunology, and insulin sensitivitywas assayed by capillary method.2.4 Expression of GLUT-4 in skeletal muscle was examined byimmunohistochemistry.2.5 Before sacrifice, animals were anesthetized with chloral hydrate (100mg/kg, ip),the carotid artery was cannulated to measure arterial systolic pressure anddiastolic pressure, and other indexes were as the same as above in type 1 diabeticrats.Results:1. Intervention in type 1 diabetic rat1.1 The incidence of STZ-induced diabetes was 86.4%.1.2 Compared to control group, the body weight of rats in group diabetes, insulintreatment andα-LA administration were significantly decreased, while bloodglucose and HbA1C were statistically higher than those of control rats (P＜0.01).The levels of body weight, HbA1C and plasma glucose showed no differencebetween DM andα-LA group(P＞0.05). Rats in insulin treatment group hadsignificantly lower glucose and HbA1C level and markly higher body weight thanthose of rats in diabetes andα-LA group(P＜0.01). There were no statisticaldifferences in plasma lipid and FFA(P＞0.05).1.3 The indexes related mitochondrial oxidative stress in control rats were unchanged.In diabetic rats: the content of GSH declined gradually with the progression ofdisease; at 4 weeks, the activities of Mn-SOD and CAT showed no differences,but a significant reduction at 8 weeks, 12 weeks; MDA concentration increasedstatistically(P＜0.05). Compared to diabetes group, GSH content, CAT andMn-SOD activities inα-LA and insulin treatment group were improved, whileMDA decreased. Insulin treatment could enhance this effect.1.4 Compared to normal control, we observed comprehensive pathological change indiabetes group such as skeletal muscle atrophy, widen interspace anddisarrangement of muscle fibres. At 4 weeks the increased quantity ofmitochondria, compensatory swollen mitochondria were seen from ultra-structuredetection. With the progression of diabetes, we could see more obvious changessuch as muscle fiber reduction, swelling cellular nucleus, skeletal muscle fibersconfused rank, verge of muscle bundle coarse, Severity myolysis and myorrhexis.Collagen content in skeletal muscle of DM group was significantly higher,resulting from Masson staining, which were counteracted byα-LA administration.Distinct muscle atrophy was not seen in insulin group.1.5 The expression of Bcl-2 in diabetes group was greatly decreased compared tocontrol rats, on the contrary, the level of Bax increased gradually, leading to thedecline of the Bcl-2/Bax ratio. The expression of Cas-3 enhanced as time went by.The treatment of insulin andα-LA can statistically ameliorate this abnormality to a certain extent.1.6 Compared to control group, the m RNA and protein expression of Mn-SOD andUCP3 in diabetes group was slightly higher,but not significantly(P＞0.05);meanwhile deteriorated at 8w and 12w(P＜0.05). The intervention with insulin andα-LA could upgrade the mRNA and protein expression of Mn-SOD and UCP3 tosome degree.2. Intervention in type 2 diabetic rats:2.1 The incidence of type 2 diabetes was 73.3%.2.2 remarkably higher body weight, TG,TC,FINS,FFA and HOMA-IR and lowerKITT were observed in diabetic rats after 4w high energy diet, compared tonormal control(P＜0.05), blood glucose was mild higher but had nosignificance(P＞0.05). After STZ injection, diabetic rats showed increased bloodglucose, TG,TC,FINS,FFA, HOMA-IR, SBRDBP and lower KITT and bodyweight(P＜0.05),α-LA treatment could ameliorate the changes mentioned above.All indexes in high energy diet group were in the middle of diabetes and normalcontrol.2.3 The activities of Mn-SOD and CAT declined, and MDA concentration increasedstatistically (P＜0.05) in high energy diet group and diabetes group,α-LA couldimprove these abnormalities(P＜0.05).2.4 Mild muscle atrophy, fat infiltration and degeneration were seen in type 2 diabeticrats resulted from LM and EM.α-LA could partially reverse this disarrangement.2.5 Compared to normal control, the expression of Bcl-2 in diabetes group decreased,and Bax increased gradually, leading to the decline of the Bcl-2/Bax ratio. Theexpression of cas-3 enhanced at the same time, but unfortunately, there were nostatistical significance in the indexes above(P＜0.05),α-LA could amelioratethis abnormality to a certain extent. No distinct abnormal alteration were seen inhigh energy diet group.2.6 Expression of protein GLUT-4 was significantly reduced in diabeticgroup(P＜0.05), and slightly declined in high energy diet group(p＞0.05),compared to control group.α-LA could elevate the expression of GLUT-4.Conclusions:1. Mitochondrial oxidative stress in skeletal muscles of type 1 diabetic rats wasevidently increased, sequenced by apoptosis and obvious and prevalent atrophy.2. Insulin resistance in type 2 diabetic rats could be deteriorated by mitochondrialoxidative stress in skeletal muscles, which in turn aggravated skeletal myopathy.3. Antioxidants could alleviate the injury of mitochondrial oxidative stress in skeletalmuscles by enhancing the activity of Mn-SOD and CAT, and improve insulinresistance by increasing GLUT-4 in insulin transduction pathway.