Experimental Study On The Effection Of L-carnitine On Type2Diabetic Mice By Gastrointestinal Administration

Subject:To replicate a type2diabetic mice model induced by high-fat high-glucose diet and streptozotocin(STZ). To develop a HPLC-FL method determine L-carnitine(LC) and its acyl esters levels in type2diabetic mice plasma and liver homogenate.To select the effective dose of L-carnitine on type2diabetic mice by gastrointestinal administration and explore the mechanism by studying neuropathy and liver disease.Methods:①Replication type2diabetic mice model:Male Kunming SPF3-week-old mice fed high-fat high-glucose diet were injected intraperitoneally with STZ(100mg/kg, i.p.) twice at the age of6and9weeks.24hours later blood glucose levels were determined to confirm the development of diabetes (>12mmol/L).②Experimental Design:Male Kungming mice were randomly divided into five groups:control group, diabetic group, LC treatment group (250mg/kg,125mg/kg,62.5mg/kg, q.d.,p.o.). Blood glucose levels were determined every week.TG levels in plasma and liver homogenate were determined by enzyme method, liver and fat weights were recorded with Electron balance, relative liver weights were calculated (liver weight/body weight×100%), morphology of hepatocytes was observed by eyes and light electron microscopy, ultrastructure of sciatic nerves and hepatocytes was observed by transmission electron microscope, insulin and IGF-1levels in plasma, IGF-1levels in sciatic nerves homogenate and FFA levels in liver homogenate were determined by ELASA after4weeks. Tail-flick latency of mice were recorded once a week from0to8weeks after model induction through immersing the tail into49℃water. Treatment with LC (125mg/kg) from5to8weeks after model induction, record the tail-flick latency once a week.③L-carnitine(LC) and its acyl esters levels in type2diabetic mice plasma and liver homogenate were determined by HPLC-FL. The analytes were extracted by protein precipitation and then fluorescence derivatizated with1-aminoanthracene (1-AA). L-carnitine(LC) and its acyl esters Acetyl-L-cainitine(ALC) and Propionyl-L-cainitine(PLC) levels in plasma and liver homogenate were determined by HPLC-FL. Statistical analysis was done by SPSS17.0software.RESULT:①Type2diabetes was induced by combination of high-fat high-glucose diet-fed and low-dose STZ injection intraperitoneally twice.24h after the second STZ injection, the blood glucose levels above12mmol/L were considered diabetic. ②Compared with those in control group,the relative liver weights were significantly greater(p<0.05), TG in plasma and liver homogenate was significantly greater(p<0.01) and FFA in liver homogenate was significantly greater(p<0.05) in model group.In the diabetic group, obvious fatty degeneration, mitochondrial swelling and crista disorder were observed in liver; delamination of myelin lamellae, atrophy and deletion of axon and deformed nerve fibers were observed in sciatic nerves.The insulin and IGF-1levels in plasma, IGF-1levels in sciatic nerves homogenate were no significant differences in diabetic mice compared to control mice (p>0.05). After therapeutic treatment with LC(250mg/Kg、125mg/Kg), decreased relative liver weights, TG levels in plasma and liver homogenate(p<0.01, p<0.05) increased insulin levels in plasma(p<0.01) were observed. Less hepatocyte steatosis, clearer crista and fewer glycogen granules in the mitochondria were observed in liver.Remarkably ameliorated the splits of myelin lamellae, atrophy and deletion of axon and the swellings and ruptures of mitochondria were observed in sciatic nerves. Compared with those in control group,the tail-flick latencies in diabetic mice were significantly shortened from3to6weeks after model induction(p<0.05). LC treatment group (250mg/kg,125mg/kg) mice showed stable tail-flick latency,which were significantly prolonged compared to model group mice(p<0.05).The tail-flick latencies of LC treatment group (62.5mg/kg) were shortened at4weeks after model induction compared to control group mice(p<0.05),but they were prolonged compared to model group mice(p<0.05). The tail-flick latencies of LC delayed-treatment group were shortened from2to4weeks after model induction compared to control group mice(p<0.05). After therapeutic treatment with LC(125mg/Kg) one week, tail-flick latencies were no significant differences compared to control mice (p>0.05),which were prolonged compared to model group mice(p<0.05).③LC and ALC and PLC levels in plasma and liver homogenate could be determined simultaneously HPLC-FL methods.The PLC levers were under the detection limit.LC and ALC levels in plasm and LC levels in liver homogenate were significant decreased in diabetic mice compared to control mice(p<0.05). Administration of LC increased plasm LC and ALC levels in type2diabetic mice(p<0.01, p<0.05).CONCLUSION:This method was successful to replicate type2diabetic mice model. The HPLC-FL methods were developed to determine L-carnitine(LC) and its acyl esters Acetyl-L-cainitine(ALC) and Propionyl-L-cainitine(PLC) levels in type2diabetic mice plasma and liver homogenate. LC and ALC levels were significant decreased in type2diabetic mice with neuropathy and liver disease. Gastrointestinal administration of LC(125mg/Kg) ameliorated diabetic neuropathy and liver disease via increasing LC and ALC and insulin levels,decreasing TG levels in type2diabetic mice.