【作者】 张菁；

【导师】 贾伟平；

【作者基本信息】 上海交通大学 ， 内科学（内分泌与代谢病）， 2019， 博士

【摘要】 小肠分泌的成纤维细胞生长因子19(FGF19)参与调节糖代谢。胆汁酸释放进入肠道继而激活法尼醇受体(FXR)后可诱导FGF19表达。胆汁酸由胆固醇在肝脏合成,除了调节脂类物质的消化和吸收,还可以作为信号分子调节机体代谢。胆汁酸进入肠道后,经过肠道菌群的作用,初级胆汁酸可转化为次级胆汁酸。肠道菌群可能通过影响胆汁酸的组分从而对机体代谢产生影响。近年来发现,肠道菌群参与宿主的糖代谢,与糖尿病发生发展密切相关。本课题聚焦于FGF19、胆汁酸及肠道菌群与糖代谢的关联及机制的研究。研究分为三个部分,主要内容及结果如下:一、FGF19与独立于胰岛素的降糖作用的密切相关目的:FGF19是由远端小肠分泌的一种具有类激素作用的细胞因子,动物研究已证实FGF19可通过独立于胰岛素的方式调节肝糖代谢。本研究旨在研究人血清FGF19水平与葡萄糖自身代谢效能(GE,独立于胰岛素的作用)以及肝糖产生(HGP)的关系。方法:通过静脉葡萄糖耐量试验(FSIVGTT)在31例正常糖调节者(NGT),34例单纯糖调节受损(I-IGT)以及31例单纯空腹血糖受损(I-IFG)受试者中检测并计算GE水平。通过口服糖耐量试验(OGTT)在56例NGT,23例I-IFG,36例混合糖调节受损(CGI)及83例2型糖尿病(T2DM)患者中检测并计算oGE水平。通过高胰岛素正葡萄糖钳夹联合3-~3H标记葡萄糖示踪技术检测14例糖调节正常者的HGP水平。通过高葡萄糖钳夹和高胰岛素正葡萄糖钳夹技术分析14例NGT,10例I-IGT和10例I-IFG者的胰岛素分泌和胰岛素敏感性。结果:血清FGF19与FSIVGTT计算得出的GE呈正相关(r=0.29,P=0.004),该结果也在OGTT中通过oGE得到证实(r=0.261,P<0.001)。FGF19与空腹血糖呈负相关(r=-0.228,P=0.025),校正GE后,此相关性不复存在(r=-0.177,P=0.086)。空腹FGF19与基础肝糖产生呈负相关(r=-0.697,P=0.006)。然而,FGF19与胰岛素分泌和胰岛素敏感性未见显著相关。结论:空腹FGF19与GE正相关,与HGP负相关。人体空腹血糖的升高可能与FGF19的降低及其独立于胰岛素的作用方式有关。二、不同糖耐量人群血清FGF19水平变化与胆汁酸密切关联目的:FGF19参与调节糖代谢,胆汁酸激活FXR后可诱导FGF19表达。临床研究发现血清FGF19水平在I-IFG和T2DM人群中下降。然而,FGF19在不同糖调节人群中的变化原因未知。在本部分研究中,我们分析了不同糖耐量人群口服葡萄糖后血清胆汁酸各组分的变化情况,并探讨FGF19和胆汁酸的关系。方法:本研究使用液相色谱-串联质谱法测定10例NGT、9例I-IGT、10例I-IFG、12例CGI以及24例T2DM患者空腹及OGTT后血清胆汁酸浓度。共检测了6种胆汁酸:鹅脱氧胆酸(CDCA)、胆酸(CA)、脱氧胆酸(DCA)、及其对应的甘氨酸结合型胆汁酸。血清FGF19水平通过ELISA法检测。结果:OGTT后,所有受试者血清FGF19水平在120分钟达峰值。甘氨酸结合型胆汁酸在30分钟达峰值,而游离型胆汁酸水平在口服葡萄糖后无显著增加。空腹血清CDCA水平在I-IFG、CGI以及T2DM患者中显著下降(P<0.05),与空腹血清FGF19水平变化趋势一致。空腹CDCA与空腹FGF19独立相关。基础实验发现给肠细胞系LS174T进行CDCA处理后,可引起FGF19的mRNA水平呈剂量依赖和时间依赖性增加。结论:空腹血清CDCA水平在空腹血糖升高的人群中降低,且与FGF19呈正相关。提示血清FGF19水平在空腹血糖受损人群中下降的原因可能与CDCA降低有关。三、肠道菌群、胆汁酸和FGF19在糖代谢中的关联与机制目的:在本研究第二部分中发现FGF19水平变化与胆汁酸有关。胆汁酸的代谢受到肠道菌群的影响。既往研究显示肠道菌群及胆汁酸与糖尿病密切相关。在本部分研究中,我们从不同代谢状态人群入手,研究肠道菌群、胆汁酸、FGF19与糖代谢之间的关系,并分析其机制。方法:本研究使用宏基因组关联分析比较了52例正常糖耐量正常体重受试者(NL)、52例正常糖耐量肥胖受试者(NAO)、22例T2DM正常体重受试者(TL)和56例T2DM肥胖受试者(TAO)肠道菌群组成。使用液相色谱-串联质谱法,在每组随机选取20例样本作为亚组检测血清胆汁酸水平。找出在各组间有临床意义的“关键菌”,并分析可能介导菌群和临床表型的“关键代谢物”。并进一步通过基础实验研究关键菌和关键代谢产物对糖代谢的调控作用。结果:4组菌群βdiversity多样性分析发现TL和TAO组之间(P=0.012)以及TL和NAO组之间(P=0.03)存在显著差异,而NAO与TAO之间未观察到显著差异。菌群与临床指标的相关性分析发现肠道细菌Akkermansia muciniphila(A.muciniphila)丰度与胰岛素分泌及FGF19呈正相关。进一步研究连接菌群与临床指标的代谢物发现,4组之间有20种胆汁酸水平呈显著差异,其中3β-chenodeoxycholic acid(βCDCA)水平与A.muciniphila丰度、胰岛素分泌和FGF19均呈负相关。给予小鼠口服A.muciniphila后,βCDCA水平降低,胰岛素分泌和FGF15表达增加,糖耐量改善。给胰岛细胞系min6和肠细胞系LS174T分别进行βCDCA处理后,可抑制胰岛素分泌和FGF19的mRNA表达。结论:人体肠道中A.muciniphila丰度减少,与βCDCA水平的增加,胰岛素分泌的降低和FGF19水平的降低密切相关。补充A.muciniphila能通过抑制βCDCA,增加胰岛素分泌和小肠FGF19表达,从而改善糖耐量。更多还原

【Abstract】 The gut-derived hormone Fibroblast growth factor 19(FGF19)could regulate glucose metabolism and is induced by bile acids(BAs)through activating Farnesoid X Receptor(FXR).BAs are synthesized from enzymatic oxidation of cholesterol in the liver,and have long been known to facilitate dietary lipid absorption.Recent years,BAs are gaining increasing recognition as important metabolic signaling molecules.Primary BAs can be transformed to secondary BAs by enzymes in gut bacteria.Gut microbiota may affect metabolism through affecting the components of BAs.It has been found that intestinal flora participates in host glucose metabolism and is closely related to the development of diabetes.This study focuses on the associations and mechanism of FGF19,BAs and gut microbiota with glucose metabolism.The study is divided into three parts,the contents and main findings are as follows:1.The close relationship between FGF19 and insulin-independent glucose regulationOBJECTIVE:The ileum-derived FGF19 plays important roles in hepatic glucose homeostasis through insulin-independent pathways in animals.In this study,we analyzed the association of FGF19 with glucose effectiveness(GE,insulin-independent effects),as well as hepatic glucose production(HGP)in Chinese subjects.RESEARCH DESIGN AND METHODS:GE was measured by frequently sampled intravenous glucose tolerance test(FSIVGTT)in 31 normal glucose tolerance(NGT),34 isolated-impaired glucose tolerance and 31 isolated-impaired fasting glucose(I-IFG)subjects.The oral glucose tolerance test-derived surrogate of GE(oGE)was determined in 56 NGT,23 I-IFG,36 combined glucose intolerance(CGI)and 83 type 2 diabetes(T2DM)subjects.HGP was assessed by labelled([3-~3H]-glucose)hyperinsulinemic-euglycemic clamp in 14 NGT subjects.Insulin secretion and sensitivity were calculated by the hyperglycemic clamp and hyperinsulinemic-euglycemic clamp in 14NGT,10 I-IGT and 10 I-IFG subjects.RESULTS:FGF19 positively correlated with GE(r=0.29,P=0.004)as determined by FSIVGTT.The result was further confirmed by oGE(r=0.261,P<0.001).FGF19 was negatively associated with FPG(r=-0.228,P=0.025),but the association no longer existed after adjusting for GE(r=-0.177,P=0.086).FGF19 was negatively associated with basal HGP(r=-0.697,P=0.006).However,no significant association between FGF19 and insulin secretion and sensitivity were found.CONCLUSIONS:FGF19 levels are associated positively with GE and negatively with HGP.The increase of FPG in human is at least partially due to the decrease of FGF19 in an insulin-independent manner.2.The relationship between FGF19 levels and BAs in subjects with different glucose tolerance stateOBJECTIVE:FGF19 could regulate glucose metabolism and is induced by BAs through activating FXR.FGF19 was found to decrease in subjects with I-IFG and T2DM.However,the reason for the change of FGF19 in subjects with different glucometabolic status remained unclear.Here,we investigated the kinetics of serum BA composition after oral glucose challenge in different glucose tolerance state,and analyzed the correlation of FGF19 and BAs.RESEARCH DESIGN AND METHODS:Serum free BAs including chenodeoxycholic acid(CDCA),cholic acid,deoxycholic acid,and respective glycine conjugates were measured by liquid chromatography-mass spectrometry in 10 NGT,9 isolated-impaired glucose tolerance,10 I-IFG,12CGI and 24 T2DM subjects.Serum FGF19 levels were determined by ELISA.RESULTS:After OGTT,serum FGF19 peaked at 120min in all subjects.Glycine conjugated BAs peaked at 30min,while free BAs did not elevated significantly.Consistent with the decrease trend in FGF19 levels,fasting serum CDCA levels in subjects with I-IFG,CGI and T2DM were significantly lower than NGT subjects(P<0.05).Fasting serum CDCA was independently associated with FGF19.CDCA strongly upregulated FGF19 mRNA levels in LS174T cells in a dose-and time-dependent manner.CONCLUSIONS:Fasting serum CDCA levels were decreased in subjects with increased fasting plasma glucose and positively correlated with FGF19,suggesting that the decrease of FGF19 in subjects with I-IFG was at least partially due to their decrease of CDCA acting via FXR.3.The association of gut microbiota,BAs and FGF19 with glucose metabolism and the mechanism of regulationOBJECTIVE:In the second part of this study,it was found that the change of FGF19 levels were correlated with BAs.The metabolism of BAs is affected by the gut microbiota.Emerging evidence has linked the gut microbiome and BAs to T2DM.Here we study the relationship between gut microbiota,BAs,FGF19 and glucose metabolism in subjects with different state of metabolism,and investigated its mechanism of regulation.RESEARCH DESIGN AND METHODS:We performed a metagenome-wide association study and compared the gut microbiota composition in a cohort of 52 normal lean subjects(NL),52 normal obese subjects(NAO),22 T2DM lean subjects(TL)and 56 T2DM obese subjects(TAO).We measured serum BAs levels in a subgroup with 20 subjects per group by liquid chromatography-mass spectrometry.We identified clinically important“specific gut microbes”and investigate the possible“metabolites linking human metabolic phenotype and microbiota”.We then conducted experiments to study the influence of the“specific gut microbes”and“metabolites linking human metabolic phenotype and microbiota”on metabolism.RESULTS:The community-level microbiota diversity(βdiversity)among the four groups showed that there was a significant shift between TL and TAO group(P=0.012),as well as between TL and NAO group(P=0.03),while NAO and TAO groups shared highly similar gut microbial community profiles.The association study between microbiota and clinical indices showed that Akkermansia muciniphila(A.muciniphila)abundance was positively correlated with insulin secretion and FGF19levels.We then investigated the“metabolites linking human metabolic phenotype and microbiota”and found that 20 bile acids(BAs)were significantly different among the four groups.Of all the 20 BAs,3β-chenodeoxycholic acid(βCDCA)was found to be negatively correlated with A.muciniphila abundance,insulin secretion and FGF19 levels.Consistently,supplementation with A.muciniphila reduced plasmaβCDCA concentration,increased insulin secretion and FGF15 expression,and alleviated glucose intolerance in mice.Treatment ofβCDCA to min6 cells and LS174T cells led to decreased insulin secretion and FGF19 mRNA expression.CONCLUSIONS:The decrease of A.muciniphila abundance in human gut correlated with the increase ofβCDCA,and the decrease of insulin secretion and FGF19.A.muciniphila could stimulates insulin secretion and FGF19 expression by inhibition ofβCDCA,which led to the improvement of glucose tolerance.更多还原