1.Nucleolin Overexpression Upregulates Hepatic Glycogen Content And Relieves High-Fat Diet Induced Glucose Metabolism Disorder 2.Relation Of Gut Microbes And L-Thyroxine Through Altered Thyroxine Metabolism In Subclinical Hypothyroidism Subjects

Part 1 Nucleolin Overexpression Upregulates Hepatic Glycogen Content and Relieves High-Fat Diet Induced Glucose Metabolism DisorderBackgroundIn 2017,there were more than 449 million diabetics all over the world,and the incidence of diabetes in China was as high as 10.9%,with 120 million patients,ranking first in the world.Type 2 diabetes mellitus(T2DM)is a metabolic disorder that is characterised by defects in glycolipid metabolism.The National Health and Nutrition Examination Survey(NHANES 1999-2000)in the United States shows that more than half of diabetic patients are accompanied by dyslipidemia,of which about 61.6%were accompanied by an increase in triglycerides,and 63.7%were accompanied by a decrease in high-density lipoprotein cholesterol.At the same time,about 70%of non-alcoholic fatty liver patients will have symptoms of glucose metabolism disorders such as insulin resistance or hyperinsulinemia,and about 44%of them are diagnosed as T2DM.The liver is essential for the maintenance of euglycemic homeostasis,which produces glucose during fasting and stores glucose after meals.However,in the state of metabolic diseases such as T2DM,the function of the liver to maintain blood glucose is dysregulated.The glucose content of the liver is the sum of glucose metabolism through gluconeogenesis,glycogenolysis,glycogen synthesis and glycolysis.However,the metabolic mechanism of hepatic glycogen has not attracted widespread attention.It is not clear whether it is possible to alter glycogen metabolism to delay the occurrence of diabetes.Nucleolin(NCL),also known as C23,is the most abundant of hundreds of known nucleolar proteins,accounting for about 10%of the total nucleolar protein.Nucleolin is highly conserved in vertebrates,and there are analogues in animals and yeast.Nucleolin is widely located in the cell,shuttles in the nucleus,cytoplasm and cell membrane,and plays a multi-faceted regulatory role on DNA,RNA and protein metabolism.In addition to participating in chromosome remodeling,DNA replication,RNA Pol I/II transcription,ribosome assembly and other functions,NCL has also been found to have anti-angiogenesis and anti-myocardial ischemia-reperfusion injury and other effects,especially in type 2 diabetic cardiomyopathy,NCL exhibits a protective effect of inhibiting myocardial apoptosis and alleviating myocardial fibrosis.In recent years,it has been reported that NCL can respond to the insulin signaling,but whether NCL participates in the glucose metabolism is not yet known.In this paper,we explored the effect of NCL on the homeostasis of hepatic glucose metabolism.Objectives1.To clarify the changes of hepatic NCL expression in a mouse model of metabolism disorders.2.Through the hepatic NCL overexpression mouse model,clarify the effect of NCL overexpression on hepatic glucose and lipid metabolism,and the improvement effect of NCL overexpression on the glucose metabolic disorder induced by high-fat diet.3.To demonstrates that NCL regulates hepatic glycogen synthesis through the ERK1/2-MNK1-GSK3? signaling pathway in vivo and in vitro experiments.4.To demonstrate that the interaction between NCL and CRTC2 is involved in the regulation of hepatic glucose output by using a variety of agonists/inhibitors and a variety of cell models.Methods1.In this study,several metabolic disorder models were used,including db/db,ob/ob,and mice fed a high-fat diet.During the period,body weight,fasting plasma glucose(FPG)and other metabolic indicators were recorded,and western blot was used to detect the expression of hepatic NCL.And analyzed the correlation between FPG,body weight and NCL.2.A mouse model of hepatic NCL overexpression was constructed by tail vein injection of NCL overexpressing adenovirus,and the control group was injected with control adenovirus.In vivo imaging system evaluates the infection efficiency of the two groups of mice.We records body indicators,and evaluates the NCL overexpression efficiency and the changes in glucose and lipid metabolism in the model mice.3.High-fat diet was used to induced metabolic disorder and chow diet was used as control.On this basis,the high-fat group was further divided into NCL overexpression group and control group.We evaluate the effect of high-fat diet model and the efficiency of NCL overexpression,then record metabolic indicators,and reveal whether NCL overexpression can improve glucose and lipid metabolism disorders.4.The NCL over-expression plasmid was constructed to upregulate the expression of NCL in cell models.We used western blot to detect proteins in ERK pathway,AMPK pathway and proteins about glycogen metabolism;ERK specific inhibitor U0126 was used to evaluate whether the function of NCL was regulated by the ERK signaling pathway.5.The immunoprecipitation method was used to clarify the interaction between NCL and CRTC2.To clarify that combination of NCL and CRTC2 participates in the molecular mechanism of hepatic glucose homeostasis regulation by using CRTC2 agonists FSK,palmitic acid and others.Results1.Increased expression of NCL in mouse models with abnormal glucose and lipid metabolism such as obesity and diabetes.In 12-week-old male leptin receptor-deficient mice(db/db)and leptin-deficient mice(ob/ob),compared with the control mice,an increase in the expression of nucleolin protein in the liver was observed.The mice were fed a normal diet or a high fat diet.The changes in glucose tolerance and insulin tolerance were observed at the 4,and 8 weekends.It was found that at the end of the 4th week,mice fed a high fat diet showed impaired glucose tolerance compared with controls.Western blot showed that the expression of nucleolin protein in the liver of mice fed a high-fat diet for 4 or 8 weeks increased.It is suggested that nucleolin may be involved in the regulation of hepatic glucose metabolism,providing experimental evidence for the study of the role of NCL in the regulation of liver glucose metabolism.2.The body weight,blood glucose,blood lipid and systemic metabolic rate of NCL overexpressing mice in the liver had no significant changes.Among them,the insulin sensitivity of male mice and hepatic glycogen content was increased.(1)Analysis of body weight,body composition and analysis of systemic metabolismThere was no significant difference in body weight and food intake between female and male mice.There was no statistical difference in liver weight,epididymal fat weight,and the liver weight index and fat weight index.The circadian rhythm of NCL overexpressing mice was conformed with the characteristics of normal mice.Compared with the control group,the oxygen consumption,carbon dioxide production,night RER and EE of the overexpression group were reduced.(2)Glucose tolerance test,insulin tolerance test:After female nucleolin overexpressing mice,there is no significant difference between the glucose tolerance test and the insulin tolerance test.In male mice overexpressing nucleolin,the glucose tolerance curve peaked at 30 minutes.In the insulin tolerance test,the curve of overexpression mice was significantly lower than the control,and the blood glucose was not restored at 120 minutes.It is suggested that overexpression of nucleolin enhances the insulin sensitivity of male mice.(3)NCL was related to hepatic glycogen metabolismPAS staining showed that the hepatic glycogen content of NCL overexpression mice increased significantly.There was no statistical difference between hepatic triglyceride content and cholesterol content,but hepatic glycogen content measurement showed that glycogen content increased in the overexpression group.It suggested that NCL may be related to liver glycogen metabolism.3.Overexpression of NCL could significantly alleviate glucose metabolism disorders induced by high-fat diet.In the evaluation of the high-fat diet modeling,the high-fat group gained weight compared with the general diet group,showing the characteristics of impaired glucose tolerance,insulin resistance,and high serum cholesterol level at the fourth weekend.Compared with the control mice fed with high-fat diet,the body weight of the over-expressing NCL group was decreased.The impaired glucose tolerance was significantly improved,and the insulin sensitivity was significantly enhanced in the NCL overexpressing mice fed high-fat diet.4.NCL participates in regulating the phosphorylation of liver glycogen synthase through the ERK pathwayIn the NCL overexpression mice,we detected that the phosphorylation level of AKT serine 473 in the AKT signaling pathway had a decreasing trend,but it was not statistically significant.However,the phosphorylation level of ERK 1/2 increased significantly,and the phosphorylation level of Serine 6 at the same condition of the total amount of protein of glycogen synthase decreased.After using the ERK signaling pathway blocker U0126,the phenomenon that NCL overexpression reduces the phosphorylation level of glycogen synthase serine 641 position no longer exists.This phenomenon suggests that NCL can participate in the regulation of hepatic glycogen synthase phosphorylation through the ERK pathway.5.NCL has an interaction relationship with CRTC2Through the method of immunoprecipitation tandem mass spectrometry,our NCL can bind to some molecules involved in the regulation of glucose and lipid metabolism,such as CRTC2.The function of NCL in regulating hepatic glycogen may be related to the interaction of CRTC2.Through IP experiments,we verified that NCL can interact with CRTC2,and the binding site is located in the nucleus.Interestingly,under the action of palmitic acid,we found that the interaction between NCL and CRTC2 weakened.It is suggested that the disorder of hepatic glucose metabolism caused by high-fat diet may be related to the changes in the interaction relationship between NCL and CRTC2.6.Conformation analysis of NCL and CRTC2 bindingThe homology modeling of NCL and CRTC2 was carried out by using T-Tasser method.After protein docking with ClusPro,it was found that the potential positions of NCL and CRTC2 protein binding were:RGG domain region(650-710 amino acids)of NCL;130-145 and 290-330 amino acids of CRTC2.Conclusions1.The expression of NCL is increased in mouse models with abnormal glucose and lipid metabolism such as obesity and diabetes.2.NCL is involved in the regulation of hepatic glucose metabolism.NCL overexpression enhances the insulin sensitivity and increases the liver glycogen content;and it can significantly protect from the glucose metabolism disorder caused by high-fat diet.3.NCL regulates GS through the ERK1/2-MNK1-GSK3? signaling pathway,thereby promoting hepatic glycogen synthesis.4.NCL participates in the regulation of hepatic glucose homeostasis by interacting with CRTC2 in the nucleus.Part 2 Relation of Gut Microbes and L-Thyroxine Through Altered Thyroxine Metabolism in Subclinical Hypothyroidism SubjectsBackground Subclinical hypothyroidism(SCH)is considered the early stage of hypothyroidism,with increased level of thyroid stimulating hormone(TSH)and normal level of free thyroxine(FT4)which will progress to clinical hypothyroidism at certain point The global prevalence of SCH is 4-10%,and morbidity has raised up to 16.7% in China.SCH has multiple potential risks.Multiple clinical researches indicate that SCH is highly related to several metabolic diseases including obesity,hypertension,dyslipidemia,non-alcoholic fatty liver disease and cardiovascular disease.L-thyroxine replacement therapy(LRT)is usually only recommended for severe SCH patients(TSH>10 mlU/L)by either international or national guidelines.Thyroid dysfunction is always associated with disorders of serum lipid profiles,especially hypertriglyceridemia and hypercholesterolemia.TTie traditional view reckon thyroid dysfunction as a causal role of dyslipidemia.The main reason might be due to abnormal regulation of thyroxine or thyrotropin to lipid metabolism of targeted tissues,such as liver or adipose.Recently,reports had established the potential effects of excess lipid deposition to thyroid function.However,the relationship between mild SCH and lipid profile is not yet explored.Our previous studies reported that mild SCH people could also benefit from LRT.However,the dosage of L-thyroxine required to maintain TSH stability varies significantly among individuals,which can not be explained by the difference of age,blood glucose level or lipid profiles.Our hypothesis is that the individual difference of dosage maybe related to gut microbes.In liver,thyroxine the reaction of thyroxine with glucuronic acid facilitates thjroxine entering intestine with bile.Thyroxine reenters liver via the portal vein after decomposed by p-glucuronidase from intestinal anaerobe and reabsorbed by the gut,which is called the entero-hepatic circulation.This process is vital in maintaining the normal thyroxine level and function.Gut microbes is a generally recognized ''endocrine factor55,which is important in thyroxine metabolism and L-thyroxine absorption.The intestinal anaerobe can rapidly decompose thyroxine and ensure the reentering of thyroxine to the circulating system.Gut microbes also involve in the intestinal storage and release of thyroxine,which is important in maintaining the stability of thyroxine level and decreasing the demand of L-thyroxine.Gut microbes can also regulate the absorption of trace elements related to thyroxine metabolism and in turn affect thyroxine metabolism.Apart from the regulation of internal thyroxine metabolism,gut microbes can also affect the absorption of external L-thyroxine.With the increase of gut microbes,the demand of L-thyroxine increases.While the use of antibiotics can reduce the adjustment of L-thyroxine.The dysfunction of intestine caused by gut microbes can also lead to the malabsorption and increased demand of L-thyroxine.ObjectivesThis study is a cross-sectional study which based on an open-labeled,randomized,controlled trial(LRT-2013)conducted in Ningyang.The clinical data and stool samples of mild SCH subjects were collected,and the subjects were grouped by lipid profiles and the L-thyroxine treatment to explore the relationship among gut microbes,dyslipidemia and LRT treatment in order to provide new insight of the diagnose and treatment of SCH.MethodsThis study collected the clinical data and stool samples of mild SCH subjects.And the subjects were grouped by lipid profiles and the L-thyroxine treatment.The changes of gut microbes were examined by high-throughput sequencing of 16 S rDNA extracted from the gut microbes,and analyzed via alpha diversity analysis,principal component analysis(PCA)? principal co-ordinates analysis(PCoA),canonical correspondence analysis(CCA)and relative abundmice analysis.The detailed grouping methods are as follows:1.Mild SCH subjects were randomly divided as 1.5:1 ratio into intervention group(LRT group)and control group(NC group)to receive L-thyroxine treatment or not;2.In intervention group,subjects were divided into low-dose group(L group),mediumdose group(M group)and high-dose group(H group)by the dosage of L-thyroxine treatment;3.In intervention group,subjects were also divided into developed group(L-D group)and non_developed group(L-ND group)according to whether the L-thyroxine dosage increased during follow-up;4.All mild SCH subjects were divided into the STG(SCH with high serum triglyceride level)group,STC(SCH with high serum cholesterol level)group,SM(SCH with both high serum triglyceride and cholesterol level)group,and S(SCH with normal serum lipid profile)group according to the guidelines of Chinese adult dyslipidemia prevention and treatment(2016 edition).Results1.No significant difference was observed among STG group,STC group,SM group and S group by alpha diversity analysis,PCA or PCoA.2.No significant difference was observed between LRT group and NC group by alpha diversity analysis,PCA or PCoA.3.No significant difference was observed among L group,M group and H group by alpha diversity analysis.While H group exhibited a clustered tendency when compared with M group and L group by PCA,especially in PCI axis of genus and fmnily levels.4.No significant difference was observedbetween L-D group and L-ND group by alpha diversity analysis.While L-ND group exhibited a clustered tendency when compared with L-D group by PCA and PCoA.5.The relative abundance analysis of certain bacteria associated with thyroxine metabolism revealed that:(1)The relative abundance of the Odoribacter and Enterococcus showed discrepancies with statistical significance among L group,M group and H group.The relative abundance of the Odoribacter and Enterococcus increased significantly in L groupwhen compared with L group;(2)The relative abundance of theRuminococcus,and Anaerotruncus in L-ND group are significantly increased when compared with L-D group.6.In all SCH subjects or LRT group,the angle between serum cholesterol and Lthyroxine dosage was shown as a sharp angle,which implied the synergistic effect of these two factors on shaping the gut microbial profile.In the NC group,serum cholesterol and self-secreted T4 was shown as an obtuse angle,which implied opposite effects in the regulation of the gut microbial community.Conclusions1.Dyslipidemia is not significant enough to induce obvious changes in gut microbes of mild SCH subjects;2.The individual variation of L-thyroxine dosage to maintain normal TSH level may be related to gut microbes;3.Gut microbes may affect the L-thyroxine dosage of mild SCH subjects and SCH progression by interfering with thyroxine metabolism;4.Serum cholesterol and L-thyroxine may function synergistically in shaping gut microbial profile.