Study Of Islet β Cell Function With Early-onset Type 2 Diabetes And The Association Of Angiopoietin-like Protein 4 With Insulin Resistance And Coronary Artery Disease

PART1.STUDY OF ISLET β CELL FUNCTION WITH EARLY ONSET TYPE 2 DIABETESBackground Diabetes is a serious global health issue and has become one of the most common epidemics worldwide,the number of diabetes patients in the world in 2015 estimated at 415 million,and is expected to 642 million in 2040.Five million people died of diabetes in 2015.The International Diabetes Federation(IDF)estimated that 7%of the world’s population between 25 and 79 years old had diabetes in 2010,by 2030 the figure will rise to 8%.Type 2 diabetes(T2DM)is the most common type of diabetes.Early onset type 2 diabetes(EOD)has become an increasingly serious public health problem in developing countries and regions,especially in Asia,where social and economic development is developing rapidly.A recent national investigation completed in China showed that the prevalence of diabetes and pre-diabetes in individuals aged 18-39 years was 45%.Among the people with diabetes,more than 90%have T2DM.Previously,the concept that insulin resistance is assumed to be a major pathophysiological feature of T2DM.T2DM never develops unless β cells fail to compensate insulin resistance became widely believed.However,since Butler et al.have reported reduced β cell mass in both lean and obese individuals with T2DM,lots of studies have indicated that the first-phase insulin secretion is the earliest detectable defect in β cell function,and impaired β cell function precedes insulin resistance in the pathogenesis of T2DM.Impaired β cell function is regarded as a key factor in the progression from glucose intolerance to overt type 2 diabetes.T2DM is a progressive disease characterized by continuing loss of β cell function.However,the development of β cell hypofunction is poorly understood with regards to type 2 diabetes.In obese but non-diabetic patients,fatty acids could inhibit the insulin secretion,prior to the occurrence of T2DM,the insulin-secretory response to nutrient overload is impaired in subjects predisposed to developing the disease,suggesting lipotoxicity is not restricted to the late stages of Type 2 diabetes,but may actually play a role in early disease progression.When insulin resistance develops,for instance as a result of obesity,the β cell mounts a compensatory response that involves coordinated increases in β cell mass,insulin biosynthesis and insulin secretion.Insulin secretion is usually increased two to three-fold to compensate insulin resistance in obese non-diabetic individuals.On the other hand,only 0%to 50%increase in β cell mass in obese non-diabetic individuals has been observed by histological analyses,implying that insulin secretion per β cell i.e.,β cell workload,increases in the face of obesity.When the increase in β cell workload causes overwork of β cells,β cells may become dysfunctional and undergo apoptotic cell death.Metabolic abnormalities of diabetes result in microvascular circulatory abnormalities,impairment of endothelial function and endothelial cell apoptosis.This gradual development of microvascular circulatory abnormalities and endothelial cell dysfunction were the cornerstones of what is clinically known as microcirculatory complications.Diabetes is associated not only with diabetic microangiopathy such as retinopathy,nephropathy and neuropathy,but also with a 2-to 4-fold increase in risk of cardiovascular disease。EOD patients were exposed to hyperglycemia longer than that of late onset diabetes(LOD)patients,and the incidence of complications increased,The course of disease in EOD patient progresses rapidly,and it is easy to combine multiple metabolic disorders,and develop diabetic complications.EOD subjects had early onset of islet βcell failure.Glucagon-stimulating C-peptide test(GST)is commonly used to assess islet β cell function in clinical,C-peptide is a widely used to measure islet β cell function.It is secreted from pancreatic β cells at an equimolar ratio to insulin however excreted at a more constant rate over a longer time,half-life of C peptide is about 20 to 30 minutes,while insulin only 3 to 5 minutes.6 minutes after Glucagon intravenous injected,the patient reach maximum C peptide regardless of blood glucose level,so the determination of C peptide after GST of can better reflect the aspect of islet β cell function.Therefore,we analyzed the clinical characteristics between the EOD and LOD group.GST was used to assess islet β cell function followed up for 3 years,the evolution of islet function and influence factors were studied,providing the basis for optimal treatment and prevention in early onset of type 2 diabetes.Objectives We analyzed the clinical characteristics between EOD and LOD group.GST was used to assess islet(3 cell function followed up for 3 years,the evolution of islet function and influence factors were studied,providing the basis for optimal treatment and prevention in early onset of type 2 diabetes.Methods Patients with type 2 diabetes were recruited from the Department of Endocrinology of Huashan Hospital affiliated to Fudan University(Shanghai,China)between 2003 and 2009.A total of 48 patients with EOD and 55 patients with LOD were selected for the present study.Diabetes was diagnosed according to the World Health Organization plasma glucose criteria.In addition,anti-insular cellular antibody,anti-glutamate decarboxylase antibody and anti-insulin antibody all are negative.Patients were excluded if they had type 1 diabetes or unknown diabetes type.Disease course was no more than 5 years in all subjects and all patients received oral blood glucose lowering drugs based on alimentary control.Participants underwent routine physical examinations including the following measurements:Height,weight,waist circumference,hip circumference and resting blood pressure.After an overnight fast,venous blood was sampled for measurement of plasma glucose(FPG),glycated hemoglobin A1c(HbAlc),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-c),low-density lipoprotein cholesterol(LDL-c),triglyceride(TG),ICA,GAD A and IAA.C-peptide(CPRO)levels were measured at fasting state.Glucagon stimulation test was performed by intravenous loading of 1 mg glucagon.The acute response to glucagon was measured by the C-peptide levels 6 min after glucagon challenge(CPR6).WHR,BMI,FPQ HbAlc,TC,HDL-c,LDL-c,TG,CPRO and CPR6 were measured at the beginning of the experiment,thereafter,these indexes were examined once every year for 3 years.ResultsBaseline general characteristics in the EOD and LOD groups At the baseline,subjects with EOD had lower levels of SBP,DBP,BMI,CPRO,CPR6 and greater levels of HbAlc and TG compared to subjects with LOD(all P<0.01).There was a decreasing trend in the EOD group.HbAlc,CPRO,CPR6 levels were 10.88±2.32%,1.72±0.76 nmol/1 and 2.96±1.67nmol/l,respectively,when the subjects were recruited,and these were decreased to 8.39±1.67%,1.45±0.64 nmol/l and 2.44±0.84nmol/l after 3 years.Compared with the baseline,significant differences were detected in these indexes at 36 months(P<0.01).Furthermore,a decreasing trend was similarly observed in the LOD group.At the baseline,HbAlc,CPRO and CPR6 levels were 9.83±1.46%,2.89±1.19 nmol/l and 6.66±2.43 nmol/l,respectively,which decreased to 8.29±1.31%,2.16±0.86nmol/l and 5.20±1.56 nmol/l after 3 years.Thus,these indexes showed significant changes during the 3-year follow-up(P<0.01).Comparison of p-cell function failure in the EOD and LOD groupsA total of 14 subjects(29.17%)in the EOD group and 6 subjects(10.91%)in the LOD group were up to the>50%reduction standard 3 years later,and there was significant difference between the two groups(x2=5.861,P<0.05).Effect of WHR and BMI on CPRO and CPR6 Pearson correlation analysis indicated a positive correlation between CPRO and WHR(P<0.01),HbAlc(P<0.05)in EOD group after a 3-year follow-up.Furthermore,there was a positive correlation between CPRO and BMI(P<0.05)in the LOD group.Conclusions Compared with patients in LOD group,there are much higher HbAlc and TG in EOD group on baseline,After 3-year follow-up study,islet beta cell function with EOD decreased more rapidly,declining islet beta cell function with EOD is positive associated with HbAlc,WHR,which highlight the importance of protecting islet beta cell function by modulating blood sugar,blood lipids and improve insulin resistance.PART 2.INVERSE ASSOCIATION OF SERUM ANGIOPOIETIN-LIKE PROTEIN 4 WITH INSULIN RESISTANCE AND CORONARY ARTERY DISEASEBackground Coronary artery disease(CAD),refers to the occurrence of atherosclerosis in coronary arteries,caused by coronary lumen stenosis and occlusion,resulting in myocardial ischemia,hypoxia,and even necrosis of coronary artery.At present,it is generally believed that atherosclerosis is the result of a variety of complex factors.In the early stage of atherosclerosis,mononuclear cells mediate lipid oxidation through the impaired endothelial cells,translating into the foam cells after lipid phagocytosis,which is the characteristic pathological cells of early atherosclerotic plaque.The incidence of CAD is increasing,and the age of onset tends to be younger,so the prevention and treatment of CAD is a task which brooks no delay.Angiopoietin-like protein 4(ANGPTL4)is a multifunctional signal protein which was discovered in the recent years.ANGPTL4 is an inhibitor of lipoprotein lipase(LPL),which reduces lipid absorption by inhibiting LPL,thereby reducing the formation of form cells.Studies have shown that ANGPTL4 plays an important role in glucose and lipid metabolism.Objectives In this study,serum ANGPTL4 levels were measured by enzyme-linked immunosorbent assay.CAD was diagnosed by coronary angiography,and insulin resistance was assessed by the homeostasis model assessment method of insulin resistance index(HOMA-IR).The association of serum ANGPTL4 level with insulin resistance and CAD as well as the number of stenotic vessels was investigated.Methods1.The study population consisted of 336 consecutive non-diabetic patients(229 men and 107 women)who underwent coronary angiography from January 2014 to December 2014 for suspected or known CAD in the Department of Cardiology,Yantai Yuhuangding Hospital.The mean age of the total subjects were 60.7±7.6 years old.The exclusion criteria were as follows:1)myocardial infarction or coronary intervention history,2)heart valve disease,3)decompensated heart failure,4)severe liver or kidney failure,5)hypothyroidism or hyperthyroidism,6)malignancy patients,7)hematological patients;8)patients taking glucocorticoid or lipid-lowering treatment,9)history of surgery or trauma.2.Coronary angiography was applied to all subjects with Judkins technique.Angiograms were evaluated by an experienced cardiologist.A presence of clinically significant CAD was defined as the presence of more than 50%luminal diameter stenosis in>1 major epicardial vessels(left circumflex artery,left anterior descending artery and right coronary artery).The extent of CAD was assessed using the number of vessels with ≥50%stenosis.Specifically,subjects were grouped as follows:non-CAD(n = 93),one vessel disease(n = 79)and multi-vessel(≥2 vessels)(n =164).3.Height,body weight and resting blood pressure were measured according to a standard protocol on admission to the hospital.Body mass index was calculated.Fasting venous blood sample(at least 8 hours overnight fast)was drawn from all subjects on the morning following admission.Serum samples were stored in-80℃ for the determination of serum ANGPTL4 level.In addition,levels of fasting plasma glucose(FPG),triglycerides(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-c),low-density lipoprotein cholesterol(LDL-c),fasting insulin and C-reactive protein(CRP)were also quantified.The levels of serum ANGPTL4 were measured by enzyme-linked immunosorbent assay using ANGPTL4(Human)ELISA Kit from Abnova following the manufacturer’s instructions.The homeostasis model assessment of insulin resistance(HOMA-IR)was used to assess insulin sensitivity,and was calculated.4.Analyses were performed using SPSS software version 20.0(SPSS,Inc.,Chicago,IL,USA).Descriptive statistics was presented as mean±standard deviation for continuous variables and as percentages for categorical variables.Differences in variables between non-CAD and CAD subjects were determined by the unpaired student’s t text.While Chi-square analysis was used for inter-group comparisons of categorical variables.The correlation of serum ANGPTL4 with other clinical parameters employed partial correlation analysis and multiple stepwise regression analysis.Logistic regression analysis was performed to assess the independent relationship between serum ANGPTL4 and CAD.Differences with p<0.05 were considered as statistically significant.Results1.There was no difference of serum ANGPTL4 between men and women(P>0.05).Compared with non-CAD subjects,those with CAD had higher levels of FPG,CRP,frequency of current smoking and lower levels of serum ANGPTL4(29.54 ± 7.67 vs.33.17 ± 8.72,P<0.001)as well as HDL-c(All P<0.05).In addition,when subjects were divided into three groups according to the number of stenotic vessels,both patients with one vessel(30.19 ± 6.53 vs.33.17 ± 8.72,P = 0.013)and patients with multi-vessel disease(28.37 ± 8.14 vs.33.17 ± 8.72,P<0.001)had lower levels of serum ANGPTL4 than those without CAD,but there were no differences in serum ANGPTL4 levels between patients with one vessel and multi-vessel disease(30.19 ±6.53 vs.28.37 ± 8.14,P = 0.083).2.After adjustments for gender and age,serum ANGPTL4 showed negative associations with BMI,LDL-c,fasting insulin,HOMA-IR,CRP and positive with HDL-c(P<0.05 for all).Next,we performed the multiple stepwise regression analysis to identify the independent variables of serum ANGPTL4.The independent variables were gender,age,BMI,FPG,TC,TG,HDL-c,LDL-c,HOMA-IR,CRP,systolic blood pressure(SBP),diastolic blood pressure(DBP),current smoking.The results indicated that CRP,HOMA-IR as well as BMI were negatively correlated with serum ANGPTL4 levels(P<0.05 for all).3.Multiple logistic regression analyses were used to estimate the association between serum ANGPTL4 level and CAD.After adjustments for the potential confounding factors,including gender,age,BMI,FPG,TC,TG,HDL-c,LDL-c,HOMA-IR,CRP,SBP,DBP,current smoking and ANGPTL4,besides CRP and gender,serum ANGPTL4 level[OR(95%CI):-0.128(0.631-0.826),P = 0.029)showed an independent association with the presence of CAD.Conclusion Our findings suggested that decreased serum ANGPTL4 was significantly and inversely associated with the presence and extent of CAD in Chinese non-diabetic population.And insulin resistance was independently associated with serum ANGPTL4.