Clinical Research On Association Of Ghrelin And Leptin Receptor MRNA Expression In Peripheral Blood Mononuclear Cells With Metabolic Syndrome

BackgroundMetabolic syndrome is a constellation of cardiovascular risk factors,including obesity,hypertension,dyslipidemia and hyperglycemia,which are correlated with cardiovascular disease and diabetes.These risk factors interact and interrelate with each other,directly promoting the development of target organ damage.The mechanism of MS is complex.Recently the role of neuroendocrine system in the pathogenesis of MS has received more and more attention.The adipose tissue, besides its energy-preserving function,has been recognized as one of the most important endocrine organs in human body.Leptin,one of the hormone-like substances it secrets,plays an important role in the regulation of energy balance,as well as fat and glucose metabolism.Hyperleptinemia is the prime drive underlying the downregulation of leptin receptors(Ob-Rs) and/or the disruption of leptin signal transduction,triggering leptin resistance,a process closely related to obesity and diabetes,and possibly the MS.Ghrelin is a new endogenous ligand for the growth hormone secretagogue receptor.It activates the release of growth hormone from the pituitary and also participates in the regulation of food intake and insulin secretion.Interactions of leptin and ghrelin have been demonstrated,which promote the development of MS and the related damage.Doppler ultrasound as the noninvasive diagnostic method has been widely applicated in clinical practice.The high-resolution sonography,providing a direct and dynamic evaluation of artery structure and function,improves the prediction of cardiovascular events greatly.Tissue Doppler imaging,a relatively new echocardiographic technique,offers a more sensitive method for the detection of the left ventricular long-axis myocardial velocity changes,which reflect the alterations in cardiac structure more accurately, and thus for the assessment of cardiac systolic and diastolic function.Based on these knowledge,we investigated the expression levels of Ob-R and ghrelin in patients with MS,observed alterations in structure and function of the heart and carotid arteries,and discussed the relationship between expression of these genes and MS components,as well as its effect on the target organ damage.Objectives(1) To investigate the mRNA expression levels of Ob-R and ghrelin in peripheral blood mononuclear cells(PBMCs) in patients with MS,and to probe into the potential mechanism underlying these changes.(2) To investigate the alterations in structure and function of the carotid artery, as well as the association of Ob-R and ghrelin mRNA expression levels with these changes.(3) To investigate the alterations in structure and function of the heart,as well as the association of Ob-R and ghrelin mRNA expression levels with these changes.Subjects and MethodsAccording to IDF 2005 criteria of MS,we enrolled 48 patients with MS(25 females and 23 males,53.50±9.73 yr of age) and 49 people matched with age and sex (P＞0.05) without MS(35 females and 14 males,50.86±9.24 yr of age).Height(H), weight(W),systolic blood pressure(SBP),diastolic blood pressure(DBP),waist circumference(WC) and hip circumference(HC).Pulse pressure(PP),body mass index(BMI) and waist-hip ratio(WHR) were calculated.Furthermore,fasting blood sample was collected from each subjects after 12-14 hours fast to determine the total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C),and uric acid(UA),fasting blood glucose(FBG),insulin(FINS) and HOMA-IR.Briefly,PBMCs were isolated.Total RNA was extracted using Trizol one-step method.The RT-PCR technique was applied to detect the mRNA expressions of Ob-R and ghrelin in PBMCs.B-mode ultrasonography of the carotid arteries was performed and Ultrasound images were acquired using a 5～10MHz linear array transducer and a commercially available ultrasound machine(Vivid 7 dimension;General Electric Medical Systems,Horten, Norway).ECG data were recorded concurrently,carotid ultrasonic parameters, including lumen diameter(D),intima-media thickness(IMT),plaque index,flow velocity(V),pressure -strain elastic modulus(E_p),normalized E_p,arterial stiffness (β),arterial compliance(AC),resistance index(RI) and pulse index(PI),were measured.Echocardiograms were obtained with a commercially available ultrasound machine(Vivid 7;GE Vingemed Ultrasound,Horten,Norway) with a 1- to 3- MHZ phased array transducer.The M-mode echocardiography,two-dimensional echocardiography,pulsed-Doppler echocardiography and tissue Doppler echocardiography were performed respectively.Cardiac ultrasonic parameters, including ascending aorta diameter(AO),left atrium(LA) diameter,left ventricle(LV) diameter,interventricular septum(IVS) thickness,LV posterior wall thickness,left ventricle mass(LVM),left ventricle mass index(LVMI),left atrium index,left ventricular ejection fraction(LVEF),early diastolic mitral inflow velocity(E),late diastolic mitral inflow velocity(A),E/A,systolic mitral annular velocity(V_s),early diastolic mitral annular velocity(V_e),late diastolic mitral annular velocity(V_a),V_e/ V_a and E/V_e,were measured.All statistical analyses were performed using the SPSS 13.0 statistical program. Normally distributed data were expressed as means±SD or±SEM as indicated,and non-normally distributed data are presented as medians(quartile range).Continuous variables were compared between groups by unpaired Student's t test.The associations between categorical ones was analyzed using the chi-square test.Correlation coefficients between the variables were determined using the Pearson's or Spearman's method,meanwhile multiple regression analysis was performed to determine relationships between variables of interest.A P value＜0.05 wasconsidered statistically significant.Results(1) Comparison of clinical and biochemical characteristics of the subjectsThere were no significant differences in age and gender between MS group and control group.Compared with the controls,the MS group showed significantly higher systolic blood pressure(SBP),diastolic blood pressure(DBP),pulse pressure(PP), W,BMI,WC,HC,WHR,TC,TG and LDL-C,UA,FBG,FINS,HOMA-IR, but lower HDL-C(P＜0.001,respectively).(2) Comparison of carotid ultrasonic parametersThe mean IMT(P＜0.001),maximum IMT(P=0.044),D_d(P =0.011),plaque index (P=0.003) of the MS group were significantly higher than those of the control group. Furthermore the MS group showed significantly decreases in V_s(P=0.002),V_d (P=0.005),V_m(P=0.004),RI(P＜0.001) and AC(P＜0.001),with significantly increases in PI(P＜0.001),E_p(P＜0.001),normalized E_p(P＜0.001) andβ(P＜0.001).(3) Comparison of cardiac ultrasonic parametersThe IVSd(P＜0.001),LVPWd(P＜0.001),LVM(P＜0.001),LVMI(P＜0.001), LA(P＜0.001),AO(P＜0.001) of the MS group were significantly higher than those of the control group.Furthermore the MS group showed significantly decreases in E/A (P＜0.001),TVI-E(P=0.004),TVI-E/A(P＜0.001),TVI-S(P=0.001),with significantly increases in mitral-A(P=0.002).(4) Comparison of gene expression parametersThe mRNA expression levels of Ob-R(0.74±0.48 vs.0.92±0.33,P=0.0375) and ghrelin(2.12±1.04 vs.2.55±0.96,×10^-2,P=0.037) in PBMCs of the MS group were significantly lower than those of the control group.(5) Correlation of Ob-R mRNA and ghrelin expression levels with clinical and biochemical characteristics,and correlation between these two expression levelsOb-R mRNA expression level was significantly correlated positively with age (P=0.026),SBP(P=0.005),DBP(P=0.003),BMI(P=0.018),WC(P=0.013),TG (P=0.030),FINS(P=0.020) and HOMA-IR(P=0.026),and negatively with HDL-C (P=0.011).Ghrelin mRNA expression level was significantly correlated positively with WHR(P=0.039) and FBG(P=0.023).Ob-R mRNA expression level was significantly correlated positively with that of ghrelin.(6) Effects of the MS components on Ob-R mRNA and ghrelin expression levelsStepwise multiple linear regression model for Ob-R mRNA expression level was as follows:Ob-R mRNA=0.243×HDL-C—0.206×age—0.251×BMI.The HDL-C (P=0.025),age(P=0.046),BMI(P=0.021)were enrolled,suggesting the association of Ob-R mRNA with HDL-C,age and BMI.Stepwise multiple linear regression model for ghrelin mRNA expression level was as follows:ghrelin mRNA = 0.260×HDL-C—0.312×WHR.The HDL-C(P=0.018) and WHR(P=0.016) were enrolled,suggesting the association of ghrelin mRNA expression level with HDL-C and WHR.(7) Correlation of Ob-R mRNA and ghrelin expression levels with carotid ultra-sonic parametersOb-R mRNA expression level was significantly correlated negatively with IMT_mean(P=0.005),which denoted the structure of the carotid artery.Meanwhile Ob-R mRNA expression level had a significantly inverse correlation with E_p (P=0.042),normalized E_p,(P=0.027),β(P=0.035) and PI(P=0.034),and a significantly positive correlation with AC(P=0.028).Ghrelin mRNA expression level was significantly correlated negatively with IMT_max(P=0.035),which denoted the structure of the carotid artery.Meanwhile ghrelin mRNA expression level had a significantly inverse correlation withβ(P=0.032) and normalized E_p(P=0.019).(8) Effects of the Ob-R mRNA expression level on carotid artery structure and function:Stepwise multiple linear regression model for IMT_mean was as follows: IMT_mean = 0.269×LDL-C—0.285×(Ob-R mRNA).LDL-C(P=0.011) and Ob-R mRNA(P=0.004) were enrolled,suggesting the association of IMT_mean with Ob-R mRNA expression level.(9) Effects of the ghrelin mRNA expression level on carotid artery structure and function:Stepwise multiple linear regression model for IMT_max was as follows: IMT_max =-0.269×HDL-C+0.315×age—0.278×(ghrelin mRNA).HDL-C(P=0.007), age(P=0.002) and ghrelin mRNA(P=0.005) were enrolled,suggesting the association of IMT_max with ghrelin mRNA expression level.Stepwise multiple linear regression showed that there was no correlation between ghrelin mRNA expression level and carotid artery function alteration.(10) Correlation of Ob-R mRNA and ghrelin expression levels with cardiac ultra-sonic parametersOb-R mRNA expression level was significantly correlated negatively with mitral-A(P=0.008),and positively with E/A(P＜0.001),TVI-E(P=0.002),TVI-E/A (P=0.009),TVI-S(P=0.027),which denoted the function of the heart.Ghrelin mRNA expression level was significantly correlated positively with TVI-E(P=0.026) and TVI-S(P=0.003).(11) Effects of the Ob-R and ghrelin mRNA expression on cardiac functionEffects of the Ob-R mRNA expression level on left ventricular diastolic function: Stepwise multiple linear regression model for E/A was as follows:E/A = -0.250×SBP—0.252×age—0.301×BMI+0.226×(Ob-R mRNA).SBP(P=0.030),age (P=0.013),BMI(P=0.007) and Ob-R mRNA(P=0.023) were enrolled,suggesting the association of E/A with Ob-R mRNA expression level.Effects of the ghrelin mRNA expression level on left ventricular systolic function:Stepwise multiple linear regression model for TVI-S was as follows:TVI-S=-0.233×TG—0.226×BMI +0.315×(ghrelin mRNA).TG(P=0.039),BMI(P=0.048) and ghrelin mRNA (P=0.005) were enrolled,suggesting the association of TVI-S with ghrelin mRNA expression level.Conclusions(1) The mRNA expression levels of Ob-R and ghrelin in PBMCs of the MS group were obviously decreased.With regard to these changes,aging,elevated BMI and low HDL-C were independent risk factors for the Ob-R mRNA expression reduction, elevated WHR and low HDL-C were independent risk factors for the ghrelin mRNA expression reduction.Ob-R mRNA expression level was significantly correlated with that of ghrelin.All the above indicated that leptin resistance and ghrelin expression reduction may play an important role in the pathogenesis of MS.(2) Significant alterations to carotid arteries of the MS patients include thickened IMT, increased plaque index,distended vessel diameter,reduced elasticity and augmented stiffness.(3) The decreased mRNA expression levels of Ob-R and ghrelin in PBMCs were independent risk factors for the IMT thickening in MS.The decline in Ob-R mRNA expression level in PBMCs was an independent risk factor for the elasticity reduction in MS,which indicated that leptin resistance and ghrelin expression reduction may play an significant role in the development of early atherosclerosis lesions.(4) Left ventricular structure and function were impaired in patients with metabolic syndrome,which were characterized as LV remolding and fragmental and global diastolic dysfunction.The left ventricular global systolic function was demonstrated normal,while tissue Doppler imaging revealed a reduced myocardial velocity,a process possibly happened earlier than the development of global systolic dysfunction in MS.(5) The decline in Ob-R mRNA expression level in PBMCs was an independent risk factor for left ventricular diastolic dysfunction,and the ghrelin mRNA expression reduction was an independent risk factor for the early impairment of left ventricular systolic function,which indicated that leptin resistance and ghrelin expression reduction may play an significant role in the impairment of cardiac structure and function.