【作者】 陈娟；

【导师】 孙子林；

【作者基本信息】 东南大学 ， 内科学， 2019， 博士

【摘要】 第一部分:腰臀比与尿糖排泄的相关性研究背景及目的:肾脏在维持全身血糖稳态中的作用越来越受到重视,其主要是通过肾小管葡萄糖重吸收参与调控全身血糖稳态。糖尿病患者肾小管葡萄糖重吸收能力显著高于正常人群。葡萄糖重吸收增强导致进入血循环中的葡萄糖增多,而随尿液排出体外的葡萄糖减少,进而加重高血糖的发生与发展。因此,抑制葡萄糖重吸收,促进尿糖排泄(urine glucose excretion,UGE),继而改善血糖,成为糖尿病治疗的新途径。然而,哪些因素导致糖尿病患者葡萄糖重吸收增强并不明确。已知肥胖可显著增加2型糖尿病的患病风险。腰臀比(waist-tohip ratio,WHR)是评估中心性肥胖的简易指标,WHR增加意味着内脏脂肪沉积,本研究旨在探讨WHR与UGE的相关性,明确WHR增加即腹型肥胖,对UGE的影响。方法:采用分层随机抽样的方法,抽取江苏省6个城市,年龄18至65周岁,既往从未诊断过糖尿病人群共7689人。所有的受试者均给予口服葡萄糖耐量试验(oral glucose tolerance test,OGTT)试验,检测空腹血糖(fasting plasma glucose,FPG),餐后2h血糖(2h plasma glucose,2h-PG),并留取2小时阶段所有的尿液,记尿量,定量检测尿糖浓度。UGE=尿量(dL)×尿糖浓度(mg/dL)。收集基线资料,包括性别、年龄、血压、心率、身高、体重、腰围、臀围等,检测糖化血红蛋白(glycated hemoglobin,HbA1c)、甘油三脂(triglycerides,TG)、总胆固醇(total cholesterol,TC)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)、肌酐、尿素氮(blood urea nitrogen,BUN)等生化指标。采用多元线性回归及Logistic回归分析,校正潜在混杂因素的影响,评估WHR与UGE的相关性。结果:1.排除UGE及HbA1c缺失者,本研究最终共纳入7485名受试者,其中糖耐量正常组(normal glucose tolerance,NGT)3243名,糖尿病前期组(pre-diabetes,PDM)3645名,新诊断糖尿病组(newly diagnosed diabetes,NDD)597名。三组中,NDD组UGE最高。进一步根据WHR进行亚组分析,分为高腰臀比(high waist-to-hip ration,H-WHR)组及低腰臀比(low waist-to-hip ration,L-WHR)组。不管是NGT组还是PDM组,H-WHR受试者UGE显著低于L-WHR受试者(p均<0.05)。此外,NDD组中,H-WHR受试者UGE仍然低于L-WHR受试者,但未达到统计学差异。2.在整个人群中,UGE与年龄、心率、血压、FPG、2h-PG、HbA1c、TC、TG、LDL-C、BMI及WHR呈显著正相关(p均<0.001),然而与HDL-C呈显著负相关(p<0.001)。此外,UGE与肌酐未见显著相关性。3.进一步校正潜在混杂因素对UGE的影响,包括年龄、血压、性别、FPG、2h-PG、TG、TC、HDL-C、LDL-C等,WHR与UGE呈显著负相关(β=-250.901,95%CI:-471.891 to-29.911,p=0.026)。然而,多因素校正后,BMI与UGE的相关性不再显著(p=0.096)。FPG、2h-PG、HbA1c及TG仍与UGE显著正相关。4.以UGE的第75百分位数为分界,将UGE转化为二分类变量。进一步Logistic回归分析显示在控制其他变量后,H-WHR受试者高UGE风险显著降低(OR=0.83,95%CI:0.71-0.97,p=0.018)。结论:H-WHR受试者更倾向于低尿糖排泄。WHR,而非BMI,可能是UGE的一个重要影响因素,提示内脏脂肪沉积可能是影响UGE的重要因素。第二部分:肾脏脂质含量与肾糖阈值的相关性研究背景及目的:H-WHR人群倾向于低尿糖排泄,可能归因于增高的肾糖阈值(Renal threshold for glucose,RTG)。RTG增高意味着肾小管葡萄糖重吸收增强,导致进入循环中的葡萄糖增多,而随尿液排出体外的葡萄糖减少。已知糖尿病患者RTG显著高于正常人群。然而,哪些因素导致RTG升高,目前还无法阐明。内脏脂质沉积是引起各种代谢紊乱的主要驱动力。不管是糖尿病患者还是动物模型,肾脏中均可观察到明显的脂质沉积。那么,肾脏脂质沉积是否会影响RTG值得探究。此外,WHR增加反映内脏脂肪堆积,但却无法直接评估内脏脂质含量。因此,本研究旨在借助核磁共振扫描技术(magnetic resonance imaging,MR)评估肾脏脂质含量,并进一步探讨肾脏脂质含量与RTG的相关性。方法:2017年12月~2018年04月于东南大学附属中大医院内分泌科门诊共招募受试者40例。收集基线资料,包括性别、年龄、血压、心率、身高、体重、腰围、臀围等。所有的受试者均接受3.0 T MR腹部扫描评估肾脏脂质含量。监测24小时血糖,并收集24小时尿液,定量检测尿糖。根据血糖及尿糖值评估RTG。采用简单相关及偏相关分析评估肾脏脂质含量与RTG的相关性。结果:1.本研究有7名受试者数据被排除,其中5名受试者屏气失败,无法配合完成MR腹部扫描,2名中途退出试验,最终纳入分析33名受试者。2.根据BMI分组,其中高BMI(high BMI,H-BMI)组有17人,低BMI(low BMI,L-BMI)组16人。H-BMI受试者肾脏脂肪分数(renal fat fraction,RFF)显著高于L-BMI受试者(3.90%±1.09%vs 2.90%±1.06%,p<0.05)。根据有无糖尿病分组,数据显示糖尿病受试者RFF明显高于非糖尿病受试者(4.18%±0.82%vs 2.25%±0.53%,p=0.001)。此外,以RTG分组,高RTG受试者RFF显著增高(4.57%±0.97%vs 3.08%±1.07%,p=0.001)。3.Pearson相关性分析显示BMI及WHR与RFF呈显著正相关,其中WHR与RFF的相关性最强(r=0.537,p=0.001)。4.WHR与24h-UGE显著正相关(r=0.371,p=0.033),而BMI与24h-UGE无显著相关性(r=0.235,p=0.187)。进一步偏相关分析,校正平均血糖、年龄、性别、糖尿病状态、肾小球滤过率(estimated glomerular filtration rate,e GFR)、血压后,WHR与24h-UGE的正相关关系不再存在,而转为显著负相关(r=-0.405,p=0.045)。此外,校正其他混杂因素后,BMI与24h-UGE之间虽未达到统计学意义,但同样呈现为负相关趋势。5.RFF与24h-UGE呈显著正相关(r=0.396,p=0.022)。然而,校正平均血糖、年龄、糖尿病状态及e GFR后,RFF与24h-UGE呈显著负相关(r=-0.382,p=0.041)。6.BMI、WHR及RFF与RTG均呈显著正相关,其中RFF与RTG相关性最强。校正平均血糖、年龄、性别、糖尿病状态、e GFR和血压,BMI及WHR与RTG的相关性不再显著,然而RFF与RTG仍呈显著正相关(r=0.432,p=0.031)。进一步校正平均血糖、年龄、性别、糖尿病状态、e GFR、血压、BMI及WHR后,RFF与RTG仍呈显著正性相关(r=0.433,p=0.039)。结论:RFF与UGE显著负相关,而与RTG显著正相关,提示肾脏脂质沉积是造成RTG增高的重要因素。肾脏脂质沉积可能促进肾小管葡萄糖重吸收,从而导致尿糖排泄减少。第三部分:大鼠肾脏脂质沉积影响肾小管葡萄糖重吸收关键分子SGLT2的表达背景及目的:脂毒引起胰岛素抵抗及胰岛功能损伤在糖尿病发病机制中的作用已经明确,然而脂毒是否会通过其他途径参与糖尿病发生发展的病理过程目前并不清楚。近年来,肾脏在维持全身血糖稳态中的作用越来越受到重视,其主要是通过肾小管葡萄糖重吸收参与调控全身血糖稳态。已知钠-葡萄糖协同转运蛋白2(sodium-glucose cotransporter 2,SGLT2)是决定肾小管葡萄糖重吸收的关键靶蛋白,即是决定RTG的主导分子。RFF与RTG显著正相关,意味着肾脏脂质沉积是造成高RTG的重要因素。那么,肾脏脂质沉积是否会影响SGLT2表达,目前还并没有研究关注这一领域。因此,本研究旨在通过动物实验明确肾脏脂质沉积与SGLT2表达的联系,并进一步探索脂质合成有关蛋白肝X受体(liver X receptor,LXR)、固醇调节元件结合蛋白1c(Sterol regulatory element-binding proteins,SREBP-1c)与SGLT2表达水平的相关性。方法:SD大鼠随机分为两组,一组正常饲料喂养(control,CON),另一组高脂饲料喂养(high fat diet,HFD)。高脂饲料连续喂养16周构建肥胖大鼠模型,HFD组大鼠体重超过CON组大鼠体重20%认定造模成功。两组大鼠均给予腹腔注射葡萄糖耐量实验(intraperitoneal glucose tolerance test,IPGTT)及胰岛素耐量实验(intraperitoneal insulin tolerance test,IPITT)。代谢笼收集24小时尿液,定量检测尿糖,同时监测血糖。留取血标本检测血脂、肌酐等生化指标。留取肾皮质组织,油红O染色及游离脂肪酸(free fatty acid,FFA)定量检测评估脂质含量,免疫荧光染色、实时荧光定量聚合酶链反应(quantitative real-time PCR,q PCR)和蛋白印迹实验(western blot,WB)检测肾脏SGLT2及脂质合成信号通路LXR/SREBP-1c表达水平。结果:1.HFD组大鼠体重及肾重明显高于CON组大鼠(p<0.05)。监测血糖,两组平均血糖无显著差异(p=0.358)。2.IPGTT及IPITT结果提示HFD组大鼠未达到糖尿病诊断标准,但已经出现糖耐量异常及胰岛素抵抗现象。3.估算大鼠RTG,HFD组大鼠为6.04±0.23mmol/L,高于CON组大鼠RTG 5.88±0.14 mmol/L,然而差异无统计学意义(p=0.33)。4.油红O染色发现,HFD组大鼠肾小管脂质沉积显著,而CON组大鼠肾小管中未见脂滴。肾皮质FFA定量检测,结果显示HFD大鼠肾皮质FFA含量明显高于CON组大鼠(P<0.001)。5.免疫荧光染色、q PCR及WB结果显示HFD组大鼠SGLT2表达水平明显高于CON组。此外,HFD组大鼠脂质合成相关蛋白LXRβ、SREBP1c表达明显增高,然而LXRα表达两组之间无显著差异。6.Pearson相关性分析显示肾皮质FFA含量与SGLT2蛋白表达水平显著正相关。此外,脂质合成有关蛋白LXRβ及SREBP1c表达水平与SGLT2蛋白表达水平同样呈显著正相关。结论:肥胖大鼠肾小管SGLT2表达水平显著上调,这可能与LXRβ/SREBP1c信号通路激活造成的局部脂质沉积有关。第四部分:游离脂肪酸影响肾小管上皮细胞SGLT2的表达背景及目的:肥胖及2型糖尿病人群血液中游离脂肪酸(Free fatty acid,FFA)含量显著高于正常人群,其中棕榈酸(palmitic acid,PA)是循环中含量最丰富的饱和FFA。已知FFA增多超过脂肪组织的储存能力时,遂在非脂肪组织中沉积,从而对组织细胞造成损伤称为脂毒性。高脂饲料喂养构建的肥胖大鼠模型肾小管中脂质沉积显著,且SGLT2表达水平明显高于正常对照组,提示肾脏脂质沉积是造成SGLT2表达上调的重要因素。然而第三部分研究仍无法反向证明改善脂质沉积可以减轻SGLT2表达上调。因此,本研究旨在通过细胞实验进一步验证脂质沉积影响SGLT2表达,并通过抑制脂质合成有关信号通路LXRβ/SREBP1c,改善细胞脂质沉积,观察SGLT2表达是否会减轻,即反向验证脂质沉积是导致SGLT2表达上调的重要因素。方法:人近端小管上皮HK2细胞分组培养,分为PA处理组及正常对照组。油红O染色评估细胞内脂质沉积情况,免疫荧光染色、q PCR及WB实验检测细胞SGLT2、LXR、SREBP1c表达水平变化,同时检测各组细胞的葡萄糖摄取能力。此外,HK2细胞转染si RNA-LXRβ和si RNA-SREBP1,抑制LXRβ和SREBP1c表达,并给予PA处理,再次评估胞内脂质沉积情况,检测SGLT2表达水平,评估细胞葡萄糖摄取能力。结果:1.200μmol/L PA处理HK2细胞24小时后,胞浆内脂质沉积显著,而正常对照组胞内未见红染脂滴。2.免疫荧光染色结果显示正常HK2细胞表达SGLT2蛋白,并且PA处理组细胞平均荧光强度明显高于正常对照组。此外,q PCR及WB结果显示PA处理组SGLT2表达明显上调。与正常对照组相比,PA处理组细胞对葡萄糖的摄取能力显著增强(P<0.05)。3.PA处理组脂质合成相关因子LXRβ、SREBP1c m RNA和蛋白表达明显高于正常对照组,而LXRα表达两组之间无显著差异。5.细胞转染si RNA-LXRβ及si RNA-SREBP1抑制脂质合成有关蛋白LXRβ和SREBP1c表达,可明显改善胞内脂质沉积,且SGLT2表达明显减弱。与PA处理组相比,转染si RNA-LXRβ组及转染si RNA-SREBP1组细胞葡萄糖摄取能力明显减弱。结论:LXRβ/SREBP1c信号通路激活促使细胞内脂质沉积,进而引起肾小管上皮细胞SGLT2表达上调,最终影响细胞葡萄糖摄取能力。脂质沉积是影响SGLT2表达的重要因素。更多还原

【Abstract】 Part Ⅰ: Association of waist-to-hip ratio with urine glucose excretionBackground and objectives: The role of the kidney in glucose homeostasis has gained much more attention.The kidney contributes to glucose homeostasis largely through renal tubular glucose reabsorption.It has been reported that the capacity of renal tubular glucose reabsorption in particptants with diabetes is significantly higher than that of particptants with normal glucose tolerance.Increased tubular glucose reabsorption leads to an increase in the amount of glucose entering the blood circulation,thereby contributing to the progression of hyperglycemia.Therefore,promoting urine glucose excretion(UGE)through inhibition of renal glucose reabsorption has become an attractive approach for the treatment of diabetes.However,it is not clear that which factors that may contribute to increased renal glucose reabsorption.It is well known that obesity significantly increase the risk of type 2 diabetes.This study aimed to investigate the association of waist-to-hip ratio(WHR),a simple measure of abdominal obesity,with UGE determined in subjects without previous history of diabetes.Methods: A multistage,stratified sampling method was used to select a representative sample of individuals aged between 18 and 65 years in the general population from 6 cities in Jiangsu Province.A total of 7689 residents without previously diagnosed diabetes participated in this survey.A 75?g oral glucose tolerance test was used to diagnose diabetes.All urine samples were collected within 2 h of oral glucose loading to measure UGE.Information on age,sex,blood pressure,heart rate,height,weight,waist circumference,hip circumference was collected.Glycated hemoglobin(HbA1c),triglycerides(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),creatinine,blood urea nitrogen(BUN)were detected.Multiple linear regression analysis and multivariate logistic regression analysis were performed to adjust the impact of potential confounding factors and explore the correlation between WHR and UGE.Results: 1.There were 7485 participants included in the final analysis after the exclusion of 204 participants whose data on HbA1 c or UGE were missing.597 individuals were newly diagnosed with diabetes mellitus(NDD),3645 individuals had prediabetes(PDM),and 3243 individuals had normal glucose tolerance(NGT).NDD group exhibited significantly higher UGE than NGT group and PDM group.A further subgroup analysis according to WHR was conducted.Participants were divided into high WHR(H-WHR)group and low WHR(L-WHR)group.Individuals with H-WHR exhibited significantly lower UGE compared to those with L-WHR,in either NGT group or PDM group.In NDD group,individuals with H-WHR also showed lower UGE than those with L-WHR,however,no statistical significance was observed.2.UGE was positively associated with age,heart rate,blood pressure,FPG,2h-PG,HbA1 c,TC,TG,LDL-C,BMI,and WHR(all p < 0.001),whereas negatively associated with HDL-C in the overall population.In addition,there was no significant correlation between UGE and creatinine.3.To identify the association of WHR with UGE,and eliminate the influence of potential confounders,a multiple linear regression analysis with UGE as a dependent variable was performed.WHR was negatively associated with UGE after adjustment for age,heart rate,genders,blood pressure,FPG,2h-PG,TG,TC,HDL-C,LDL-C,and BUN(β =-250.901,95% CI:-471.891 to-29.911,p = 0.026).However,there were no significant association between BMI and UGE after multivariable adjustment(p = 0.096).In addition,FPG,2h-PG,HbA1 c,and TG were still positively associated with UGE in this model.4.Furthermore,a binary logistic regression analysis was performed to identify the factors associated with the risk of high UGE.The result showed that H-WHR was significantly associated with a decreased odds ratio of high UGE in the multi-adjusted model(OR = 0.83,95% CI: 0.71-0.97,p = 0.018).Conclusion: Individuals with H-WHR were more likely to have low UGE.This study suggests that WHR,but not BMI,may be an important determinant of UGE,which means that the pattern of fat distribution,not overweight or obesity by itself,may be an important determinant of UGE.Part Ⅱ: Association of renal fat amount with renal threshold for glucoseBackground and objectives: Individuals with H-WHR were more likely to have low UGE,probably due to increased renal threshold for glucose(RTG).Increased RTG,which indicates renal glucose reabsorption is increased,leads to an increase in the amount of glucose entering the blood circulation,while a decrease in UGE.Accumulating evidences have demonstrated that RTG is increased in individuals with diabetes.However,little research has been designed to investigate factors that influence RTG.Visceral lipid deposition is the main driver of various metabolic disorders.Significant renal lipid deposition has been reported in hyperglycemic animals and humans.However,it is unclear that whether the renal lipid deposition may affect RTG.In addition,increased WHR reflects visceral fat accumulation,but it can not directly assess visceral fat amount.Therefore,this study aimed to evaluate renal fat amount using magnetic resonance imaging(MR)and further explore the correlation between renal fat amount and RTG.Methods: A total of 40 subjects were enrolled in the Department of Endocrinology,Zhongda Hospital Affiliated to Southeast University from December 2017 to April 2018.Baseline data were collected,including gender,age,blood pressure,heart rate,height,weight,waist circumference,hip circumference,etc.All participants underwent 3.0 T MR abdominal scan to assess renal fat amount.24-hour blood glucose was monitored and 24-hour urine smaples were collected to measure UGE.RTG was evaluated based on blood glucose and UGE.Simple correlation and partial correlation analyses were performed to investgate the correlation between renal fat amount and RTG.Results: 1.Seven subjects in this study were excluded.Five of them failed holding their breath during MR scanning and two withdrew from this study.Finally,a total of 33 subjects were included in this study.2.All subjects were devided into two groups when based on BMI.17 subjects with high BMI(high BMI,H-BMI)and 16 subjects with low BMI(low BMI,L-BMI).The renal fat fraction(RFF)of subjects with H-BMI was significantly higher than that of subjects with L-BMI(3.90% ± 1.09% vs 2.90% ± 1.06%,p < 0.05).In addition,subjects with diabetes had significantly higher RFF than that of non-diabetic subjects(4.18% ± 0.82% vs 2.25% ± 0.53%,p = 0.001).Moreover,RFF was significantly increased in subjects with high RTG(4.57% ± 0.97% vs 3.08% ± 1.07%,p = 0.001).3.The results of pearson correlation analysis showed that BMI and WHR were significantly and positively correlated with RFF.The correlation between WHR and RFF was the strongest(r = 0.537,p = 0.001).4.WHR was positively correlated with 24h-UGE(r = 0.371,p = 0.033),while BMI was not significantly associated with 24h-UGE(r = 0.235,p = 0.187).Furthermore,after adjusting for mean blood glucose,age,gender,diabetes status,estimated glomerular filtration rate(e GFR),and blood pressure,WHR was negatively associated with 24h-UGE(r =-0.405,p = 0.045).In addition,after adjusting for other confounding factors,there was a negative correlation trend between BMI and 24h-UGE,but there was no statistical significance.5.RFF was positively correlated with 24h-UGE(r = 0.396,p = 0.022).However,after adjusting for mean blood glucose,age,diabetes status,and e GFR,RFF was significantly and negatively correlated with 24h-UGE(r =-0.382,p = 0.041).6.BMI,WHR and RFF were positively correlated with RTG,and the correlation between RFF and RTG was the strongest.After adjusting for mean blood glucose,age,gender,diabetes status,e GFR and blood pressure,BMI and WHR were no longer significantly associated with RTG.However,RFF was still positively correlated with RTG(r = 0.432,p = 0.031).Moreover,after adjusting for mean blood glucose,age,gender,diabetes status,e GFR,blood pressure,BMI,and WHR,RFF was still positively correlated with RTG(r = 0.433,p = 0.039).Conclusion: RFF was negatively correlated with UGE and positively correlated with RTG,suggesting that renal lipid deposition may be an important factor for increased RTG.Renal lipid deposition may promote renal tubular glucose reabsorption,thereby resulting in reduced urine glucose excretion.Part Ⅲ: Effect of lipid deposition on renal SGLT2 expressionBackground and objectives: The role of lipotoxicity in the pathogenesis of diabetes mellitus by inducing insulin resistance and islet function damage has been well confirmed.However,it is still unclear whether lipotoxicity will participate in the pathogenesis of diabetes mellitus through other approaches.In recent years,much more attention has been paid to the role of kidney in glucose homeostasis.It has been well known that the kidney contributes to glucose homeostasis largely through glucose reabsorption.Sodium-glucose cotransporter 2(SGLT2)is the key protein that determines renal glucose reabsorption and the dominant molecule that determines RTG.The second part found RFF was significantly and positively correlated with RTG,suggesting that the renal lipid deposition might be an important factor for RTG.However,it is unclear that whether renal lipid deposition will affect SGLT2 expression.To date,there is no study focuse on the association of renal lipid deposition with SGLT2 expression.Therefore,this study aimed to clarify the effect of renal lipid deposition on SGLT2 expression through animal experiments,and furtherly explore the association of signaling pathway related to lipid synthesis liver X receptor(LXR)/sterol regulatory element binding protein 1c(SREBP1c)with SGLT2 expression levels.Methods: SD rats were randomly divided into two groups,one group was given a normal diet(control,CON)and another group was given a high fat diet(HFD).The obese rats were constructed by feeding with high fat diet continuously for 16 weeks.Successful models were defined as the weight of HFD group was more than 20% of that of CON group.Both groups were given an intraperitoneal glucose tolerance test(IPGTT)and an intraperitoneal insulin tolerance test(IPITT).24-hour urine smaples were collected using metabolic cage to quantitatively detect UGE.Blood lipids,glucose,and creatinine were measured.Oil red O staining and free fatty acid(FFA)quantitative assessment were used to assess the renal fat amount.Immunofluorescence staining,real-time fluorescence quantitative polymerase chain reaction(q PCR),and wstern blot were conducted to determine renal SGLT2,LXR,SREBP1 c expression levels.Results: 1.The body weight and kidney weight of rats in HFD group were significantly higher than those in CON group(p < 0.05).Blood glucose was monitored and there was no significant difference in the mean blood glucose between the two groups(p = 0.358).2.The results of IPGTT and IPITT showed that the rats in the HFD group did not meet the diagnostic criteria for diabetes,but already had abnormal glucose tolerance and insulin resistance.3.The RTG of rats in HFD group was 6.04 ± 0.23mmol/L,higher than that in CON group 5.88 ± 0.14mmol/L,but the difference was not statistically significant(p = 0.33).4.The results of oil red O staining showed that significant renal tubular lipid deposition was observed in the HFD group,but no lipid deposition was found in the kidney of rats in the CON group.Quantitative detection of renal cortical FFA showed that the FFA levels in HFD group were significantly higher than that in CON group(P < 0.001).5.The results of immunofluorescence staining,q PCR and WB exhibited that the expression of SGLT2 in HFD group was significantly higher than that in CON group.In addition,the expression of lipid synthesis related proteins LXRβ and SREBP1 c in HFD group were markedly higher,while there was no significant difference in LXRα expression between the two groups.Pearson correlation analysis showed that the FFA levels,LXRβ and SREBP1 c protein expression levels were positively correlated with SGLT2 protein expression levels.Conclusion: Compared with CON group,the m RNA and protein expression of SGLT2 in HFD group was significantly upregulated,which may be related to renal lipid deposition caused by activation of LXRβ/SREBP1 c signaling pathway.Part Ⅳ: Effect of free fatty acids on SGLT2 expression in tubular epithelial cellsBackground and objectives: The levels of free fatty acid(FFA)in the blood of individuals with obesity or type 2 diabetetes are significantly higher than that in the normal population.Palmitic acid(PA)is the most abundant saturated FFA in the circulation.It is well known that when FFA exceeds the storage capacity of adipose tissue,it will deposit in non-adipose tissue,thereby causing damage to the tissue and cells,which is called lipotoxicity.Obvious lipid deposition was observed in the kidney of obese rats.In addition,SGLT2 expression was significantly up-regulated in the obese rats.The result of the third part suggested that the renal lipid deposition might be an important factor for the overexpression of SGLT2.However,the result of the third part still could not identify that improving lipid deposition could alleviate the up-regulation of SGLT2.Therefore,this study aimed to furtherly verify the effect of lipid deposition on SGLT2 expression by cell experiments,and confirm whether SGLT2 expression will be alleviated by improving celluar lipid deposition through inhibiting signaling pathway related to lipid synthesis LXRβ/SREBP1 c,and finally reveal lipid deposition is an important factor for overexpression of SGLT2.Methods: Human proximal tubular epithelial cells(HK2 cells)were cultured in different groups,PA treatment group and normal control group.Oil red O staining was used to evaluate the intracellular lipid deposition.The expression levels of SGLT2,LXR and SREBP1 c were determined by immunofluorescence staining,q PCR and WB.Moreover,the glucose uptake of each group was measured.In addition,in order to inhibit signaling pathway related to lipid synthesis,HK2 cells were transfected with si RNA-LXRβ and si RNA-SREBP1 c,then the cells were treated with PA again.Intracellular lipid deposition,SGLT2 expression levels,and glucose uptake were evaluated again.Results: 1.After treatment with 200 μmol/L PA for 24 hours,obevious lipid deposition in HK2 cells was observed.2.The result of immunofluorescence staining showed that normal HK2 cells expressed SGLT2 protein.In addition,the average fluorescence intensity of PA-treated cells was significantly higher than that of normal control group.Moreover,the results of q PCR and WB also showed that the expression of SGLT2 in PA treatment group was significantly up-regulated compared to normal control group.Furthermore,compared with the normal control group,the PA treatment group exhibited a significant increase in glucose uptake(P < 0.05).3.The expressions of LXRβ and SREBP1c m RNA and protein in PA treatment group were significantly higher than those in the normal control group.However,there was no significant difference in LXRα expression levels between the two groups.5.After transfection with si RNA-LXRβ and si RNA-SREBP1,the intracellular lipid deposition was significantly improved compared with PA treatment group.Moreover,SGLT2 expression was obviously reduced.In addition,compared with the PA treatment group,glucose uptake was significantly attenuated in HK2 cells transfected with si RNA-LXRβ and si RNA-SREBP1.Conclusion: Activation of LXRβ/SREBP1 c signaling pathway promotes intracellular lipid deposition,which in turn leads to upregulation of SGLT2 in renal tubular epithelial cells,ultimately affecting glucose uptake.Lipid deposition is an important factor for overexpression of SGLT2.更多还原