EFFECTS OF VITAMIN C ON CARDIAC AUTONOMIC FUNCTION, OXIDATIVE STRESS AND DYSLIPIDAEMIA IN FRUCTOSE-ALLOXAN DIABETIC RATS

Abstract

Cardiac Autonomic Neuropathy (CAN) is characterised by cardiac autonomic dysfunction and is the principal cause of mortality in type-2 diabetes. Oxidative stress has been implicated in the aetiology of CAN, thus antioxidants are possible therapeutic agents for CAN. Although, Vitamin C (VC) is a readily available and water soluble antioxidant, there is a dearth of information on its effects on CAN in diabetes. Therefore, this study was designed to investigate effects of vitamin C on cardiac autonomic function in fructose-alloxan diabetic rats. Thirty rats were grouped equally into Control, Fructose (20% w/v), Fructose +150 mg/kg (i.p.) alloxan. Fructose solution was freely administered via gavage for 2-weeks before a single alloxan injection. Thereafter, rats were observed for fourteen weeks. Response to metformin and Insulin tolerance test were done. Fasting Plasma Insulin (FPI) and Fasting Blood Glucose (FBG) were measured using ELISA and spectrophotometry. Homeostasis Model Assessment (HOMA) was used to evaluate Insulin Resistance (IR), Insulin sensitivity and Beta cell function. Further, 72 rats were grouped equally into: control, fructose (20% w/v), diabetic (fructose-alloxan), diabetic + VC, diabetic + VC+metformin and diabetic metformin. The VC (1g/kg/day) and metformin (500 mg/kg/day) were administered orally for 6-weeks. Each group was further divided into two (n=6). Cardiac autonomic function tests were done on one sub-group using electrocardiography based Heart Rate variability (HRV) after anaesthesia with urethane (1g/kg, i.p) and non-invasive blood pressure. Rats in the other sub-group were bled and sacrificed after anaesthesia to harvest organs for histology and histomorphometry. Oxidative stress markers, haematology, apolipoproteins and atherosclerotic plaques were determined using spectrophotometry, hemocytometry, immunoturbidimetry and histochemistry. Data were analysed using ANOVA and Fishers LSD post-hoc test at α0.05. Administration of alloxan to rats pre-treated with fructose elevated FBG (378.3±40.0 vs control 60.8±2.5 mg/dL), which was not reduced significantly by insulin (351.8±30.4 mg/dL) but significantly decreased in response to metformin (259.8±38 mg/dL). Diabetic heart rate and blood pressures were significantly increased compared to control (392.6±8.9 vs 310.0±15.6 bpm, MBP 189.7±4.1 vs 93.6±3.8 mmHg) and VC group (327±22.0 bpm, 97.4±5.4 mmHg). The HRV analysis revealed reduced total power (290.4±0.5 ms²), Low Frequency (LF; 126.5±4.6m²), High Frequency (HF;108.4±7.0 ms² ), RMSSD (65.1±1.0 ms²) in diabetic group compared to control (45,159.3±7839.2, 5434.0±877.0, 19,574, 1±4019.1, 107.5± 11.5ms²) and VC group (44,927.8±7831.5, 5338.7±1191.8, 14,634.1±4865.8, 98.3±13.3 ms²). Normalised-LF and LF/HF ratio were significantly (p<0.01) greater in diabetic than control and VC group. Diabetic rats showed decrease in glutathione (47.8%), VC (35.7%) and apolipoprotein-A1 (25.0%) but these variables were increased by 29.0%, 2.1% and 34.6%, respectively upon VC administration. Diabetic group showed increase in FPI (8.2.± 1.4 vs 4.8±0.3 IU), IR (10.3±3.0 vs 0.58±0.0), SOD (82.5%), LPO (102.2%), NO (111.4%), MPO (40.5%), neutrophils (14.1%), monocytes (14.8%), apolipoprotein B (62.5%) and intima-media thickness (107.4±9 vs 78.87±2.3um), but VC group showed no significant difference from control. Atherosclerotic-amyloids were present in diabetic heart and aorta but regressed following VC administration. Vitamin C ameliorated cardiac autonomic dysfunction in diabetic rats via mechanisms related to reduction of oxidative stress, leucocyte activation, vascular inflammation and dyslipidaemia.