The Correlation Between Adiponectin And Metabolic Diseases And The Mechanism Of Adiponectin Improving Insulin Resistance

In recent years,with the rapid economic development and the impact of the western lifestyle,great changes have taken place in the diet structure of Chinese residents,and the intake of high-fat and high-calorie diet,such as fast food and fat,has significantly increased than before.The dangers of high-fat and high-calorie diet are also emerging.Increased intake of high-fat,high-calorie diets can cause many metabolic diseases such as type 2 diabetes mellitus(T2DM),non-alcoholic fatty liver disease(NAFLD),hyperur-icemia,metabolic syndrome(MS).Insulin resistance(IR)is one of the important pathological bases for the occurrence and development of these metabolic diseases,especially type 2 diabetes.Epidemiological data show that the incidence of type 2 diabetes is increasing year by year.Obesity-related type 2 diabetes is one of the most important causes of morbidity and mortality in the world,posing a major threat to human health,productivity and quality of life,and has become a global public health problem.The occurrence and development of type 2 diabetes is related to many factors,especially the high fat and high calorie diet and the sedentary lifestyle.Obesity and insulin resistance are the core of type 2 diabetes.However,the specific pathogenesis is not very clear.High-fat diet-induced insulin resistance and ectopic lipid deposition are important pathological changes,and it is also a good model to study the pathogenesis and treatment of type 2 diabetes.Our research group has successfully established an animal model of insulin resistance and ectopic lipid deposition induced by high-fat diet feeding in mice in previous basic studies.And we have done some research on the relationship between adipokines,inflammatory factors,oxidative stress,apoptosis process and insulin resistance,especially the relationship between adipokines(adiponectin)and insulin resistance.Adiponectin(APN)is an adipocyte-secreting factor secreted by white adipose tissue and has extensive biological activities.Adiponectin is is a small part of the fat protective factors that can enhance tissue insulin sensitivity,regulate glycolipid metabolism,resist arteriosclerosis,protect vascular endothelium cells,regulate inflammation,and play a vital role in the regulating energy metabolism,exert a protective effect on obesity-related diseases.Although there are many studies on adiponectin and diabetes,obesity,arteriosclerosis,there are few studies on adiponectin intervention.This study first analyzed the association of adiponectin and adiponectin polymorphism with metabolic syndrome,type 2 diabetes and its macrovascular complications in the population with metabolic syndrome and type 2 diabetes.And through the high-fat diet-induced insulin resistance mouse model,to understand the adiponectin intervention on high-fat diet mice insulin resistance and the improvement of lipid deposition in liver and skeletal muscle,to explore the mechanism of adiponectin in improving insulin resistance and glucose-lipid metabolism disorder,and to provide theoretical basis for seeking new therapeutic targets for metabolic diseases,for instance type 2 diabetes mellitus and obesity.The study was divided into four parts:The first part is the correlation study of plasma adiponectin level and gene polymorphism with metabolic syndrome.The second part is the correlation study between plasma adiponectin and type 2 diabetes mellitus.The third part is the effect of adiponectin intervention on ectopic lipid deposition and inflammatory factors in hyperlipidemic insulin resistance mice.The fourth part is the effect of adiponectin on insulin signaling pathway in insulin resistant mice.Part One The correlation study of plasma adiponectin level and gene polymorphism with metabolic syndrome.Objective:To compare the changes in serum adiponectin levels and gene polymorphism in people with metabolic syndrome,to further explore the relationship between adiponectin and metabolic syndrome.Methods:From April 2014 to March 2016,Chinese Han volunteers aged between 25 and 65 years old in Cangzhou,Hebei province were selected.According to the presence or absence of metabolic syndrome,256 cases were divided into two groups:MS Group(n=142)met the following three items or all four criteria:(1)abdominal obesity:waist circumference?90cm for men,?85cm for women.(2)hyperglycemia:fasting blood glucose?6.1mmol/L,or blood glucose 2 hours after dextrose load?7.8mmol/L and/or patients diagnosed with diabetes and have received treatment.(3)hypertension:blood pressure?130/85mm Hg and/or patients who have been diagnosed with hypertension and have received treatment.(4)fasting triglyceride(TG)?1.7mmol/L.(5)fasting high-density lipoprotein cholesterol(HDL-C)<1.04mmol/L.The healthy group(n=114)served as the control group.The levels of waist circumference(WC),blood pressure(BP),total cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL-C),low-density lipoprotein(LDL-C),fasting insulin(FINS),fasting blood glucose(FBG),adiponectin(APN)were determined in two groups,and the APN rs121917815 gene polymorphism was detected.Results:1.The body mass index(BMI),waist circumference,systolic pressure(SBP),diastolic pressure(DBP)and fasting insulin levels(FINS)in MS group were apparently higher than in healthy control group,and the levels of HDL were apparently lower than in healthy control group(P<0.05),the difference was statistically significant(P<0.05).2.Serum APN levels in MS group were apparently lower than in healthy control group,the difference was statistically significant(P<0.05).3.The distribution of rs121917815 genotype in 114 subjects in control group was as follows:CC genotype 41(35.962%),CT genotype 36(32.45%),TT genotype 37(32.45%),and The distribution of SNPrs121917815 genotype in142 subjects in MS group was as follows:CC genotype 68(47.89%),CT genotype 42(29.58%),TT genotype 32(22.53%),The gene frequency of CC genotype in MS group was apparently increased compared with the healthy control group,the difference was statistically significant(P=0.025).And the results of relative risk analysis were:OR=1.64,95%CI=(1.05-2.30),suggesting that CC+CT genotype may increase the risk of MS.The gene frequencies of C and T alleles were 51.76%and 48.24%in control group,and the frequencies of C and T alleles in MS group were62.68%and 37.32%,respectively.The results of chi-square test prompted that there were certain differences in the alleles gene frequency distribution of the two groups,the frequencies of C allele at APNrs121917815 in MS group were significantly higher than those in control group,the difference was statistically significant(p=0.019).The results of relative risk analysis in the control group were:OR=1.71,95%CI=(1.03-2.98),suggesting that the risk of MS in C allele carriers may be increased.Summary:1.Plasma APN levels are closely related to metabolic syndrome,and plasma APN levels are significantly decreased in patients with metabolic syndrome.2.The APN polymorphism locus RS121917815 is associated with MS,and carriers of CC+CT genotype and C allele may increase the risk of MS.Part Two The correlation study between plasma adiponectin and type 2 diabetes mellitus.Objective:To compare the changes of plasma APN level in type 2diabetes mellitus and to further explore the association of APN with type 2diabetes mellitus and arteriosclerosis.Methods:From March 2015 to March 2016,inpatients aged 25-65 years in Cangzhou Central Hospital were selected.Among them,34 cases were in the simple type 2 diabetes group(T2DM group),and 34 cases were in the diabetes mellitus combined with macrovascular disease(MVD group)group.Diabetes diagnosis criteria were in accordance with the 1999 WHO Diabetes Diagnosis criteria.The diabetes mellitus combined with macrov-ascular disease group includes cardiovascular and cerebrovascular disease and lower extremity arterial disease(except in the acute phase).At the same time,a healthy control group with matching gender and age was selected from the physical examination center.After the subjects informed consent,measured height,weight and waist circumference on an empty stomach,and calculated body mass index(BMI)=weight(kg)/height2(m2).Rest for 15 minutes,the systolic blood pressure(SBP)and diastolic blood pressure(DBP)on the right arm with a mercury sphygmomanometer.Fasting venous blood samples were keeped,and measured blood glucose,triglyceride,cholesterol,low-density lipoprotein,fasting insulin,glycated hemoglobin and adiponectin levels.Results:1.There were no statistical differences between the three groups in age and gender(P>0.05).Compared with the healthy control group,the levels of BMI,FBG,TC,TG,FBG,and FINS in the T2DM group and MVD group were increased,and there were statistical differences(P<0.05).2.Plasma APN level of the MVD group and T2MD group was lower than the healthy control group,and the level of plasma APN in MVD group was lower than that in T2MD group.The Pai-1 level in MVD group and T2MD group was higher than that in control group,and the levels in MVD group were apparently higher than those in T2MD group,the differences were statistically significant(P<0.05).3.Pearson correlation analysis shows that,as shown in table 2,the plasma APN level in the T2DM group was negatively correlated with BMI,FBG,FINS and TG levels(P<0.05),but a positive correlation with ISI(P<0.05).The plasma APN level of the MVD group was negatively correlated with the levels of BMI,SBP,FBG,FINS and TG(P<0.05).In the T2DM group and MVD group,the plasma level of APN was negatively correlated with the plasma level of PAI-1(P<0.05).4.Multiple stepwise regression analysis of plasma APN levels and other parameters.The plasma APN level was used as the dependent variable,while other parameters were assigned as independent variables.In the regression equation of MVD group,BMI and SBP,TG and PAI-1 were independent variables.In the T2DM group,BMI,TG and PAI-1 were independently correlated with plasma APN levels.In the MVD group,BMI,FCP,PAI-1 and FBG were independently correlated with plasma APN levels.Summary:1.The plasma APN level and PAI-1 level are closely related to type 2diabetes.The plasma APN level of patients with type 2 diabetes decreases,while PAI-1 increases.2.Plasma APN levels and PAI-1 are closely related to the complications of type 2 diabetic macroangiopathy and atherosclerosis.Plasma APN levels further decrease in type 2 diabetic macroangiopathy patients,while PAI-1further increases.Part Three The effect of adiponectin intervention on ectopic lipid deposition and inflammatory factors in hyperlipidemic insulin resistance mice.Objective:To explore the effects of adiponectin on blood glucose,insulin,insulin sensitivity index,blood lipids,inflammatory factors,and lipid deposition in liver and skeletal muscle of insulin resistance mice induced by high-fat diet.Methods:C57BL/6J mice were randomly divided into control group(CON)10 mice and high fat group(HFD)20 mice.Normal control group was given normal diet,high-fat diet group was given high-fat diet,each group of mice were given the same calorie diet.The weekly body weight and food intake were recorded during the feeding period.After 8 weeks,insulin resistance model was evaluated by intraperitoneal glucose tolerance test(IPGTT).10 mice from HFD group were randomly selected as adiponectin intervention group(HFD+APN).The adiponectin intervention group was injected exogenous recombinant adiponectin intraperitoneally at a dose of15?g/kg per day.CON and HFD groups were given corresponding dose of0.9%sodium chloride solution intraperitoneally for 6 weeks.After 6 weeks of intervention,the blood glucose values at 0 min,15 min,30 min,60 min and120 min were measured again by IPGTT test,and area under curve(AUC)of glucose was calculated.Serum samples were collected to measure fasting blood glucose(FBG),total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C),fasting insulin(FINS),adiponectin(APN),interleukin-6(IL-6),tumor necrosis factor factor(TNF-?)and insulin sensitivity check index(QUICKI)were measured by enzyme linked immunosorbent assay(ELLSA).The liver tissue samples were collected for H&E staining,and the skeletal muscle tissues were stained with oil red to observe the pathological changes and lipid content of liver and skeletal muscle tissues.Results:1 Establishment of insulin resistance model in mice induced by high-fat diet.After 8 weeks of high-fat diet intervention,the body weight of HFD group was apparently higher than the CON group(P<0.05).The average daily food intake of HFD group was apparently higher than the CON group(P<0.05).IPGTT test was performed at the end of 8 weeks.Compared with the CON group,the HFD group had significantly higher blood glucose levels at 0min,15 min,30 min,60 min,and 120 min(P<0.05).And the area under curve of glucose in HFD group was significantly increased(P<0.05),it is suggested that a high-fat-induced insulin resistance model has been successfully established.2 Changes of body weight and food intake and insulin resistance parameters in mice after 6 weeks of adiponectin intervention.The body weight of mice in HFD group was apparently higher than that of the mice in CON group(P<0.05).After 6 weeks of adiponectin intervention,the body weight of mice in HFD+APN group decreased.after 4weeks of intervention,but there was no significant difference compared with HFD,the daily food intake of mice in the HFD group was apparently higher than that in the CON group(p<0.05).There was no difference in daliy food intake between HFD+APN group and HFD group.IPGTT test was conducted at the end of 6 weeks.Compared with the CON group,the HFD group had obviously higher blood glucose levels at 0 min,15 min,30 min,60 min,and120 min(P<0.05).Compared with HFD group,the blood glucose level of mice in the HFD+APN group decreased at 30 min,60 min,and 120 min(P<0.05).Compared with CON group,the area under the curve in HFD group was significantly increased(P<0.05),and that the area under the curve in HFD+APN group was significantly decreased(P<0.05).Compared with the CON group,mice in the HFD group had apparently higher fasting blood glucose and insulin levels(P<0.05),while the fasting blood glucose and insulin levels of HFD+APN group were obviously lower than HFD group(P<0.05).The QUICKI value of the HFD group was apparently lower than that of the APN group(P<0.05).After the intervention of adiponectin,the QUICKI value was apparently increased(P<0.05).3 Comparison of blood lipid levels of mice in each group.The levels of TG,TC,LDL-C and HDL-C in HFD group were obviously higher than in CON group(P<0.05).TG,TC and LDL-C levels of HFD+APN group were apparently lower than HFD group(P<0.05),there was no significant change in HDL-C.4 The effect of adiponectin on inflammatory markers in mouse model of each group.The serum and liver tissue TNF-?and IL-6 levels of HFD group were significantly higher than CON group(P<0.05).The serum and liver tissue TNF-?and IL-6 levels of mice in the HFD+APN group were apparently lower than those in the HFD group(P<0.05).Serum and liver APN levels in HFD group were significantly lower than those in CON group(P<0.05),and serum and liver APN levels in HFD+APN group were significantly higher than those in HFD group(P<0.05).5 Morphological comparison of liver and skeletal muscle of mice in each group.5.1 liver H&E stainingIn the CON group,the structure of liver tissue was clear and complete,hepatocyte cytoplasm was uniformly red stained,there were few lipid vacuoles and no obvious fatty degeneration.In the HFD group,the liver tissue structure of HFD group was disordered and blurred,and lipid droplets of different sizes could be seen in hepatocyte cytoplasm,and the nucleus was squeezed to the edge of the cell by lipid droplets,presenting obvious steatosis.Compared with HFD group,hepatocyte steatosis and lipid vacuoles were significantly reduced in HFD+APN group.5.2 Skeletal muscle oil red stainingThere were no lipid droplets in the cytoplasm of the skeletal muscle of mice in the CON group,but there were more lipid droplets in the skeletal muscle cells of mice in the HFD group.The nuclei were located below the sarcomembrane and the glycogen particles were accumulated.The lipid droplets in the HFD+APN group were less than those in the HFD group.Summary:1.High-fat diet can induce weight gain in mice with lipid abnormalities,lipid deposition in skeletal muscle and liver,and inflammatory response leading to insulin resistance.2.Adiponectin intervention can change the lipid deposition of liver and skeletal muscle of high-fat diet mice,reduce inflammation,and then improve insulin resistance and dyslipidemia.Part Four The effect of adiponectin intervention on insulin signaling path way in insulin resistant mice.Objective:To study the effect of APN on IRS/PI3K/Akt signaling pathway in insulin resistant mice,and to explore the mechanism of adiponectin in improving insulin resistance in high-fat-induced mice.Method:Animal grouping and specimen collection are the same as the first part.Western blot technology was used to detect the insulin receptor substrate 1/2(IRS1/2),p-IRS^Tyr1/2,and phosphatidylinositol kinase(PI3K),p-PI3-K,serine/threonine kinase(also known as protein kinase B,Protein kinase B,Akt)and p-Akt expression,it also detects the protein levels of adiponectin signaling pathway Adipo R1,adenosine monophosphate activated protein kinase(AMPK)phosphorylation level.Results:1.The expression of related indicators of insulin signaling pathway after adiponectin intervention.Among the three groups,the liver,skeletal muscle,adipose tissue IRS1/2,Akt,PI3K and their phosphorylation levels of mice on high-fat diet were lower than those in the CON group(P<0.05).Adiponectin intervention was followed by 6 weeks,in the HFD+APN group,the levels of p-IRS^Tyr1/2,p-Akt,and P-PI3K increased significantly(P<0.05).2..The expression of Adipo R1/AMPK protein after adiponectin interventionAmong the three groups,the levels of Adipo R1 and p-AMPK in the liver,skeletal muscle,and adipose tissue of the mice on the high-fat diet were gradually reduced compared with the CON group(P<0.05).Adiponectin intervention for 6 weeks,in the HFD+APN group,the level of Adipo R1,p-AMPk was significantly higher than that of the HFD group(P<0.05).Summary:1.Adiponectin regulates the IRS-1/PI3K/Akt pathway by up-regulating the expression levels of IRS1/2,PI3K,and Akt in the liver,skeletal muscle,and peri-renal adipose tissue of high-fat diet mice,thereby improving insulin resistance and glucose metabolism disorders.2.Adiponectin can up-regulate adiponectin receptors and p-AMPK,strengthen the oxidation process of fatty acids,thereby improving lipid deposition in liver,skeletal muscle,and peri-renal adipose tissue.Conclusions:1.The decrease of plasma adiponectin level is closely related to the occurrence of metabolic syndrome,type 2 diabetes and diabetic macrovascular complications;adiponectin gene polymorphism is related to metabolic syndrome.2.Adiponectin intervention can change the lipid deposition of liver and skeletal muscle of high-fat diet mice,reduce inflammation,and then improve insulin resistance and dyslipidemia.3.The mechanism of adiponectin intervention on insulin resistance in the liver,skeletal muscle,and perirenal adipose tissue of high-fat diet mice is to up-regulate the expression levels of insulin signaling pathways IRS1/2,PI3K,and Akt in these tissues,and at the same time up-regulate Adipo R1/AMPK pathway,and strengthen the oxidation process of fatty acids.