The Influence On Glucose And Lipid Metabolism Of Obesity Mices And Initial Observation On Impossible Effective Target Of Zinc-α₂-glycoprotein

Object:Obesity has emerged as a worldwide health issue, and has close relationship with the occurrence of many disease, including type 2 diabetes, hypertension, coronary heart disease, stroke, dysregulation of lipid metabolism and insulin resistance, etc. It also causes non-alcoholic liver disease, asthma and arthritis. The much mor fat accumulation in adipocytes is the core factor in development of obesity. ZAG ( zinc-α₂-glycoprotein) is a new adipokine which has been shown to induce lypolysis in tumor patients with cachexia and increase the ultilization of fat. But as far as we know, there isn't any reports concerning the relationship between ZAG and fat accumulation in obesity patients. In this study, we constructed pcDNA3.1(-)-mZAG vector containing murine ZAG full coding sequence. We observed the influence of ZAG overexpression on the weight, epididymal fat, glucose and lipid metabolism in obesity mices . We also observed the influence of ZAG overexpression on the expression of FAS mRNA and HSL mRNA in the adipose tissues. Furthermore we compared the serum ZAG level in normal people, obesity patients and obesity patients after weight loss.Methods:1 The construction of pcDNA3.1(-)-mZAG plasmid containing murine ZAG full coding sequence: Total RNA from BALB/c mice were extracted from murine liver, transcribed reversly into cDNA, then amplified and gained high copies of murine ZAG cDNA full sequences. The pcDNA3.1(-)-hZAG plasmid containing human ZAG full coding sequence were performed to high copies. EcoRâ…¤,Hindâ…¢were used to cut the plasmid to have the vector fragments. Then we ligated the murine ZAG cDNA sequences and the vector fragments by T4 lignase and constructed the pcDNA3.1(-)-mZAG plasmid containing murine ZAG coding sequence successfully.2 The identification of pcDNA3.1(-)-mZAG plasmid containing murine ZAG coding sequence in vitro: The plasmid was transfected into murine 3T3-L1 cells by liposome transfection method and gathered the solution after 40 hours , then observed ZAG protein expression of culture solution by western-blotting method.3 The influcence of ZAG overexpression on the weight, epididymal fat, glucose and fat metabolism of obesity mices: The KunMing mices were distinguished to 5 groups: nomal food group, high-fat food group, ZAG overexpression group, pcDNA3.1(+) plasmid control group and QUMEI group. The mices were bred with high-fat food except nomal food group for 4 weeks to make them obesity. Then ZAG overexpression group were administered with the blending of 25ug pcDNA3.1(-)-mZAG plasmid and 40μl liposome by tail venus injection qod for 2 weeks. The influcence of pcDNA3.1(-)-mZAG plasmid on the weight, epididymal fat, glucose and fat metabolism was observed later.4 The expression of FAS,HSL mRNA in murine epididymal adipose tissues: Total RNA was reverse transcribed and then amplified by PCR. The PCR products were compared, murineβ-actin was used as a house-keeping gene.5 Compared the serum ZAG level among the normal people, obesity patients and obesity patients after weight loss: we used human monoclonal ZAG antibody to compare the serum ZAG level among the different BMI people, after weight loss, murineβ-actin was used as a house-keeping protein. The BMI of nomal people were 21.6, 32.6 of obesity patients, and 29.6 of obesity patients after weight loss.Results:1 Murine ZAG coding sequence was amplified successfully and ligated with vector fragments by T4 ligase. The construction of pcDNA3.1(-)-mZAG plasmid containing murine ZAG coding sequence was succeed.2 pcDNA3.1(-)-mZAG plasmid was transfected into murine 3T3-L1 cells by liposome transfection method. It was confirmed that pcDNA3.1(-)-mZAG plasmid could well expressed in murine 3T3-L1 adipocytes..3 The serum ZAG levels of high-fat food group (0.51±0.10), pcDNA3.1(+) plasmid control group (0.54±0.08)and QUMEI group (0.55±0.05) didn't have significant difference. And the serum ZAG levels of three groups all have significant decreased comparing to normal food group (P<0.01) . The serum ZAG level of ZAG-overexpression group( 1.01±0.16) has significant increased comparing to normal food group and high-fat food group (P<0.001) . This suggested that pcDNA3.1(-)-mZAG plasmid could presented ZAG protein in murine.4 During the two-weeks intervention test, the ingested volume of high-fat food group and ZAG-overexpression group are respectively 115.4±13.5g and 115.8±7.5g, all have significant increased comparied with normal food group. It didn't have significant difference between the high-fat food group and ZAG-overexpression group.The weight gain of high-fat food group is 3.7±0.9g , it has significant increased comparing to normal food group(2.7±0.7g). The weight gain of ZAG-overexpression group is 3.0±0.5g, is significant decreased comparied with high-fat food group(P<0.001) and didn't has significant difference with normal food group(P=0.23).The ratio of visceral fat and body weight of high-fat food group is 2.22±0.48% , it has significant increased comparing to normal food group(P<0.05). The ratio of visceral fat and body weight of ZAG-overexpression group is 1.16±0.10%, is significant decreased comparied with high-fat food group and normal food group (P<0.05).The fasting blood glucose level of high-fat food group is 10.87±1.77 mmol/L, it has significant increased comparing to normal food group(P<0.05). The fasting blood glucose level of ZAG-overexpression group is 9.13±1.00 mmol/L, is significant decreased comparied with high-fat food group(P<0.05) and didn't has significant difference with normal food group(P=0.55). The serum low density lipoprotein level of high-fat food group is 1.10±0.24mmpl/L , it has significant increased comparing to normal food group(0.59±0.44mmpl/L). The serum low density lipoprotein level of ZAG-overexpression group is 0.78±0.17mmpl/L, is significant decreased comparied with high-fat food group(P<0.05) and didn't has significant difference with normal food group(P=0.49).5 The FAS mRNA expression in murine visceral adipose tissue of high-fat food group is 4.10±0.64, it has significant increased comparing to normal food group(2.67±0.46)(P<0.001). The FAS mRNA expression in murine visceral adipose tissue of ZAG-overexpression group is 1.54±0.39, is significant decreased comparied with high-fat food group(P<0.001) and normal food group(P<0.001).6 The HSL mRNA expression in murine visceral adipose tissue of high-fat food group is 1.18±0.19, it has significant decreased comparing to normal food group(P<0.01). The HSL mRNA expression in murine visceral adipose tissue of ZAG-overexpression group is 4.39±0.95, is significant increased comparied with high-fat food group(P<0.001) and normal food group(P<0.001).7 The serum ZAG level of normal people is 0.74±0.10, the serum ZAG level of obesity patients is significant decreased comparing to normal people(P<0.05). After the obesity patients lost weights, the serum ZAG level is raised but still has significant difference with normal people(P<0.05). The serum ZAG level and BMI in normal people, obesity people and the obesity patients who lost weights didn't have correlativity(r=0.16, 0.34, 0.25, P>0.05).Conclusion:1 mZAG expression plasmid pcDNA3.1(-)-mZAG was constructed successfully and could express murine ZAG protein in vitro murine preadipocytes 3T3-L3 cells and in vivo mices. This is a convenient tool for ZAG study.2 ZAG could decrease the weight increasing, visceral fat and serum LDL levels, but didn't influence the fasting blood glucose, CHO, TG and HDL in obesity mices. This indicates that ZAG maybe reduce fat accumulation.3 ZAG can inhibit fat synthesis, promote lipolysis through murine adipose FAS and HSL.4 The expression of ZAG maybe has relationship with othe obesity.