Expression, Transcriptional Regulation And Effect On NFALD Of Human FTO Gene

Part Ⅰ Analysis of serum FTO level and related factors inobese and T2DM patientsObjective:To explore the relation of serum FTO to adiposity and glucose metabolism inobesity and T2DM individuals.Methods:1. The experiment designs the groups as fellow: normal weight subjects withnormal glucose regulation (NW-NGR, n=96), over-weight/obesity subjects withnormal glucose regulation (OW/OB-NGR, n=67), T2DM and its subgroups ofT2DM with normal weight (NW-T2DM, n=153) and T2DM withover-weight/obesity (OW/OB-T2DM n=116).2. Serum FTO was detected by double antibody sandwich ELISA in fasting statusin pationes.The levels of Glucose, lipids, insulin and anthropometricalparameters such as BMI, fat content (Fat%), waist circumference (WC), waisthip ratio (WHR) and waist to height ratio (WHtR) were measured. Meanwhileinsulin sensitivity was assessed by (Homeostasis model assessment of insulinresistance index) HOMA-IR.3. Correlation analysis was performed in order to investigate the relationshipbetween FTO and the detected parameters.Results：1. FTO levels in OW/OB-NGR, NW-T2DM, and OW/OB-T2DM were significantly higher than that of NW-NGR [32.74±2.65ng/L,28.34±1.96ng/L,39.22±3.48ng/L vs.23.66±1.62ng/L, P <0.05]. There was no difference inserum FTO levels between OW/OB-NGR and NW-T2DM, but the serum FTOLevels in OW/OB-T2DM was significantly higher than that of NW-T2DM (P <0.01) and OW/OB-NGR (P <0.05).2. In the linear correlation analysis, serum FTO were positively correlated withBMI, WC, Fat%, WHtR, WHR, TG, TC (P all <0.01); and also positivelycorrelated with FINS, HOMA-IR, LDL-C (P all <0.05); negatively correlatedwith HDL-C (P <0.05). Multiple stepwise regression analysis revealed BMI,WC, WHtR, WHR, TG were the significant impact factor influencing the of theserum FTO level.Conclusions:1. Serum FTO is not only strongly related to abdominal obesity and insulinresistance, but also associated with blood glucose and lipids.2. BMI, WC, WHtR, WHR and TG were most significant independentdeterminants for serum FTO concentration. As such, the serum FTO levelswould be a candidate metabolic marker obesity, especially abdominal obesity. Part Ⅱ Identification and characterization of humanFTO gene promoterObjective:The Polymorphisms of FTO gene association with obesity have been identified inlarge genome-wide association studies in multiple populations. However, the basal mechanism of regulation of human FTO gene is not understood. In this part weinvestigate the transcriptional regulation of human FTO gene and identify its corepromoter region．Methods:1. PCR was used to generate5’stepwise deletion constructs of the FTO promoter.2. The human cell line HEK293and Hela were transfected with serial reporterconstructs. The promoter activities were analyzed using dual luciferase reporterassay.3. Bioinformatics analysis prediction of transcription factor binding sites on theputative promoter regions.4. The modulatory role of transcription factors were identified by mutagenesis,over expression and RNA interference. The binding of transcription factors withpromoter region were confirmed using EMSA and ChIP.Results:1. The core promoter region was located at-201/+34by luciferase activity analysis.2. Three Sp1binding sites, a potential Foxa2(-14~-26bp) and an upstreamstimulatory factor (USF) binding site was identified in the-201bp fragmentupstream of the transcription start site (TSS).3. Furthermore, using mutagenesis, overexpression and RNA interference, weidentified the Foxa2as a negative regulatory element to the expression of thehuman FTO. One of the Sp1binding site was crucial for the basal activities ofthe promoter and the FTO expression. The USF binding site did not affect theFTO promoter activity.4. EMSA and ChIP assays were performed to confirm Sp1and Foxa2binding tothe FTO promoter.Conclusions: 1. The human FTO core promoter region was located in the+34to-201bp．2. The Sp1played a positively role in the regulation of FTO; Foxa2binds to thepromoter and inhibits the expression of FTO. Part Ⅲ FTO gene enhanced oxidative stressand lipidosis in NAFLDObjective:In this part we elucidate the expression and function of FTO in experimentalnonalcoholic fatty liver disease.Methods:1. Sprague–Dawley rats were fed with standard rat chow for a week and dividedinto two groups. NAFLD induced by high-fat diet for12weeks (n=12, D12492)or control subjects with normal chow diet (n=12, D12450B). Recorded bodyweight of rats weekly and sacrificed all the subjects at the end of the12week after12h of fasting.2. The liver index (liver weight/body weight×100%), blood glucose, serumbiochemical markers, serum lipid levels and malondialdehyde, superoxidedismutase levels were measured and compared in two groups. Tissues ofliver were dissected and stained with hematoxylin and eosin (HE) and observedvia light microscopy. Applied the technique of real-time PCR and Westernblot to analyze the expression of Fto in rats liver.3. The recombinant human FTO over-expressed plasmids were constructed, thenstably transfected into hepatic L02cells. Hepatocyte steatosis model was induced by oleic acid for24h, detected the lipid content by oil red O stainingand oxidative stress markersResults：1. NAFLD rat model was successfully established by feeding high-calorielaboratory chow for12weeks. Micrograph of histological section showed asevere fatty liver.2. The body weight and the liver index of experimental rats with HFD wasnotably heavier than the NFD group. Levels of ALT and AST were significantlyraised under HFD feeding. MDA content elevated but the SOD levels declinedwere observed in the model compared with control. The expression levels of FTOmRNA and protein were up-regulated in NAFLD rats.3. Human FTO recombinant plasmid was constructed successfully and confirmedby sequencing and restriction digesting. The transfected L02cell strain wereestablished and employed with oleic acid to induce cells steatosis. Adipogenesiswas assessed by oil Red O staining. Higher rise of lipid levels and lipidaccumulation were observed in the FTO overexpression group. MDAconcentration increased by30%, in constrast, the activity of anti-oxidative stressindicator SOD significantly decreased than control.Conclusion:We demonstrate that increased FTO levels in experimental NAFLD are involved inoxidative stress and hepatic lipidosis.