The Effectiveness And Mechanisms Of Pentamethylquercetin For Ameliorating Metabolic Syndrome

PartⅠ:Establishment of the neonatally MSG-induced MetS mice modelObjective:To establish a neonatally MSG-induced MetS mice model.Methods:Healthy Kunming mice were purchased from animal center of Tongji Medical College of Huazhong University of Science and Technology at 10-week old. After a 2-week acclimation period,12-week-old virgin female mice were mated with male mice as 1 tol. From day 2 to day 8 of lactation, a solution of MSG dissolved in saline (3mg/g body weight/d and 10μl/g body weight/d) or equipotent vehicle (saline) was given subcutaneously every day to the male pups using a microsyringe. On day 21, after weaning, All treated mice were divided into 2 different groups randomly (n=10):Saline group and MSG group and all groups were allowed to eat ad libitum. From weaning until 18 weeks of age, body weight and food intake were recorded once/week for the duration of the study. At 18 weeks of age, body weight, body length and waist circumference was measured in live mice, blood samples were collected from the orbital vein of experimental animals for measuring serum glucose, triacylglycerol, total cholesterol and insulin. Then, mice were sacrificed after an overnight fast, and fat mass and some organs were weighted, then, together with serum stored at -80℃until analyzed. Western blot was used to detect the phosphorylation protein level of AMPK, ACC and GLUT4 abundance in the muscle of MSG mice.Results:(1) MSG mice had significant obesity. Compared with Saline mice, MSG mice emerged a profound central obesity body characteristics, including the increase of body weight (elevate 18%), waist circumference (elevate 26%), Lee index (elevate 17%)and fat content (elevate 734%). The results have statistics difference. (2) MSG mice have emerged hyperglycaemia. Based on the MSG mice fasting blood glucose level examination, we found that the blood glucose level of MSG mice was significantly elevated, and compared with Saline mice, The results have statistics difference (p<0.05). (3) MSG mice have emerged lipid metabolism disorder. The serum triacylglycerol (TG) and total cholesterol (TC) levels have emerged different degrees of elevation (TG:52%, TC:49%). (4) MSG mice had significant hyperinsulinemia and insulin resistance. The serum insulin level of MSG mice was significantly elevated (p<0.001). By calculating the HOMA of insulin resistance (HOMA-IR), We found that MSG mice have significant insulin resistance compared with Saline mice (p<0.001). (5) In muscles of MSG mice phosphorylation levels of AMPK and ACC were decreased, and GLUT4 abundance downregulated.Conclusion:Neonatally MSG-induced MetS mice model well mimic the pathophysiological characteristics of MetS in humans, and modeling success rate of close to 100%. It is a readily available and low-cost animal model of MetS, and will provide a platform for the new therapeutic drugs of MS. PartⅡ:Anti-metabolic syndrome effects of PMQ in neonatally MSG-induced MetS mice modelObjective:To study the effects of PMQ in neonatally MSG-induced MetS mice model.Methods:On day 21, male MSG mice and Saline mice were weaned and after adaptive feeding for two weeks, at 5 weeks of age, Saline mice were set into the normal control group (Control, n=10). MSG mice were divided into five different groups randomly (n=10) as follows:Vehicle, PMQ 5 mg/kg, PMQ 10 mg/kg, PMQ 20 mg/kg and RSG 5 mg/kg. All mice were administrated by gastric gavage once/day for 13 weeks. Body weight and food intake were recorded once/week for the duration of the study. At 18 weeks of age, body weight, body length and waist circumference was measured in live mice, blood samples were collected from the orbital vein of experimental animals for measuring serum glucose, triacylglycerol, total cholesterol and insulin. Then, mice were sacrificed after an overnight fast, and fat mass and some organs were weighted, then, together with serum stored at -80℃until analyzed.Result:PMQ treatment could induce different degrees decrease of body weight, waist circumference, Lee index and fat content in MSG mice compared with Vehicle mice. PMQ (20 mg/kg) decreased body weight by 75%, waist circumference by 89, Lee index by 92% and fat content by 58%. The results have statistics difference (p<0.01). The results show that PMQ has significant anti-obesity effect in MSG mice. Meanwhile, PMQ could improve glucose and lipid metabolism disorders in MSG mice. PMQ (5,10,20 mg/kg) could decrease blood glucose by 69%,64%,55% in MSG mice respectively. For lipid metabolism, PMQ had different degrees of reduction effects in serum triacylglycerol and total cholesterol levels of MSG mice. Moreover, PMQ could significantly improve hyperinsulinaemia in a dose-dependent manner (PMQ 5 by:65%, PMQ 10 by:58%, PMQ 20 by:39% vs Vehicle) and further improved insulin resistance in MSG mice. The results have statistics difference.Conclusion:PMQ has a comprehensive anti-metabolic syndrome effects in neonatally MSG-induced MetS mice model.PartⅢ:The possible mechanisms of PMQ in the prevention of MetSObjective:To study the mechanisms of PMQ in the prevention of MetS in vivo and vitro.Methods:C2C12 skeletal muscle cells were differentiated to mature C2C12 myotubes and the C2C12 myotubes were incubated with PMQ (1-10μM) for 24 hours. MTT assay was used to detect cell viability and glucose consumption assay was used to assess the glucose uptake ability in C2C12 myotubes after PMQ treatment. Then, we assessed the effects of PMQ on the expression of certain genes that are known to play a critical role in fatty acid oxidation in C2C12 myotubes by RT-PCR. We next performed western blot experiment in vivo and vitro to measure the protein level of AMPK, ACC and GLUT4 in skeletal muscle of MSG mice and in C2C12 myotubes.Results:The MTT data suggested that PMQ had no cytotoxicity for C2C12 myotubes in our experimental system, as it did not influence cell viability at concentrations ranging from 1 to 10μmol/1. The glucose consumption results showed that, in C2C12 myotubes, PMQ treatment increased glucose consumption in a dose-dependent manner and PMQ (1-10μmol/1) increased glucose consumption by 19%-65% respectively. RT-PCR results show that the mRNA expression of several genes specific to fatty acid oxidation, including PGC-la, PPARa, MCAD, ACO and CPT-1b, were increased in C2C12 myotubes after PMQ treatment for 24 h. Meanwhile, PMQ treatment (1-10μmol/1) activated AMPK, increased ACC phosphorylation and GLUT4 abundance in skeletal muscle of MSG mice and in C2C12 myotubes. Time course experiments showed that the effect of PMQ on phosphorylation of AMPK lasted for at least 16 h in C2C12 myotubes.Conclusion:PMQ can ameliorate MetS at least in part via stimulation of AMPK activity.