1.The Study On The Effects Of CRTC2 On Hepatic Cholesterol Synthesis Via FoxO1/SREBP-2/HMGCR Pathway 2.the Study On The Effects Of TSH On Hepatic Gluconeogenesis Via CRTC2

Background:Disorders of glucose and lipids coexist and interact.High lipids incduce disturbance of glucose.Hyperlipidemia induces the abnormal liver function,then leads to the disturbance of hepatic gluconeogenesis,glycogen synthesis and glycogenolysis.Hypercholesterolemia also induces the injury of beta cells in the pancreas,then aggravates the status of diabetes mellitus.Meanwhile,abormal of glucose aggravates the disturbance of lipids.In type 2 diabetes and high-normal serum glucose level patients,cholesterol absorption efficiency is low,and cholesterol synthesis is elevated.The levels of TG,TC and LDL-C are lower in better glucose control groups compared with poorer groups.The levels of TG and TC are positively related to HbAcl.CRTC2 is a key regulator of fasting glucose metabolism.However,recent study showed that CRTC2 also involved in the TG metabolism.3T3 L1 cells stably expressing CRTC2 shRNA exhibited the lower triglycerides contents than the control.And CRTC2 mutant mice had lower circulating triglycerides levels compared with the wild type mice.Thus,CRTC2 may be the molecule which regulates the glucose and lipids,simultaneously.These indicated that wheter CRTC2 regulated both glucose and lipids,simultaneously.FoxOs,a member of the forkhead family of transcription factors,recognize a specific regulatory element termed insulin response element(IRE)on the promoter.There are four major isoforms in mammals named FoxO1,Fox03,Fox04,and Fox06,and they play an important role in various cellular signaling pathways in the energy metabolism,stress resistance,and longevity.As in the case of CRTCs,FoxOs is located in cytoplasm by the phosphorylation status.Stimulus induced the dephosphorylation of FoxOs and entried into nucleus.FoxO1,as a member of FoxOs,mainly regulated hepatic gluconeogenesis.FoxO1 binded the sites(IRE)on the promoters of G6Pase and PEPCK to induce to increase of gluconeogenesis.Recently,Fox06,another member of the FoxOs,was found in hepatic gluconeogenesis.Mice with hepatic FoxO1 ablation led to increase of VLDL secretion,cholesterol,and plasma free fatty acids.FoxO1 and Fox03 could suppress the sterol-regulatory element binding protein(SREBP)-2 promoter activities,however IRE-1 mutation abolish the effect.These indicated that the molecule involved in glucose metabolism aslo regulated the lipids metabolism and these provided the possibility that we explore the mechanism of coexistence of glucose and lipids.SREBP-2 is the master regulator of cholesterol biosynthesis.SREBP-2 as a precursor is synthesized in endoplasmic reticulum,which is processed to mature nuclear form(nSREBP-2)in golgi apparatus.nSREBP-2 enteries into nucleus then controls expression of genes involved in cholesterol homeostasis,including 3-hydroxy-3-methylglutaryl-CoA reductase(HMGCR),the rate-limiting enzyme in cholesterol biosynthesis.The regulation of SREBP-2 includes transcriptional and post-translational levels.The translational levels of SREBP-2 can be regulated by feed-forward which it was recognized by nSREBP-2 via binding the SRE sequence in the promoter of SREBP-2.And it aslo ca be negatively regulated by FoxO1 via IRE sequence in the promoter of SREBP-2.In the present study,we observed that CRTC2 enhanced the hepatic cholesterol synthesis and further determined the mechanism:CRTC2 regulated tranccriptional SREBP-2/HMGCR pathway via FoxO1,at least partially,involved in the cholesterol metabolism.Objectives:1.To determine the high expression of CRTC2 and lipid-related genes in hypercholesterolemia patients and high cholesterol diet mouse model.2.To determine the effect of CRTC2 on lipids especially hepatic new cholesterol in Ad-CRTC2 and CRTC2-KO mouse model.3.To test the upregulation of HMGCR by CRTC2 in vivo and in vitro.4.To determine upregulation of HMGCR by CRTC2 dependent on SREBP-2 in vivo and in vitro.5.To determine that CRTC2 can reduce the nucleic FoxO1,leading to regulate the transcriptional SREBP-2.Methods:1.Human liver samples:Patients in Shandong Provincial Hospital affiliated to Shandong University undergoing surgery of hepatic hemangioma.2.Cell cultureHepG2 cells,CHO cells,BNL cells and HEK293 cells:cells were cultured according to guidance from ATCC.3.Animal model(1)Cholesterol diet mouse model:Male C57BL/6J mice(6-8 weeks)were given cholesterol diet,another group were given a chow diet.(2)Adenoviruses of CRTC2,lentivirus of SREBP-2 RNAi and the corresponding control adenoviruses and lentivirus were delivered to male C57BL/6J mice by tail vein injection.(3)CRTC2-KO mouse model:According the related reference,we got the wild-type mice and knockout of mice by breeding heterozygote mice and determined the genotype by PCR.And all mice lived in SPF environment of the experimental animal center.After about 7 weeks mice were given cholesterol diet(4%cholesterol)for 18 weeks or different dose of cholesterol diets(0.02%,1%,4%cholesterol)for 3 days.(4)CRTC2-/-mice fed a high cholesterol diet for 14 weeks,then the mice were iniected with adenoviruses of CRTC2 by tail vein injection.(5)Luciferase mouse modelAdenoviruses of CRTC2,HMGCR-Luciferase reporter and the corresponding control adenoviruses were delivered to male C57BL/6J mice by tail vein injection.Adenoviruses of SREBP-2-Luciferase reporter were delivered to male CRTC2-KO mice(CRTC2+/+ and CRTC2-/-)with 18 weeks high cholesterol diet by tail vein injection.4.Real time-PCR:was adopted to determine the expression of CRTC2,HMGCR,FDPS,SS,SREBP-2,LDLR,PEPCK,G6P,FAS,SREBP-1C,ABCA1,ACAT2,CYP7A1,amd FoxO1 mRNA.5.Protein extraction and Western Blotting analysis:were adopted to determine the expression of CRTC2,HMGCR,SREBP-2,PEPCK,CREB,p-CREB,FoxO1,Lamb and beta-actin protein.6.Immunofluorescence:was adopted to determine the expression of HMGCR protein in HepG2 cells,H&E staining detected lipid droplet accumulation in mouse liver.7.CRTC2 and FoxO1 silence:were adopted to CRTC2 expression in HepG2 cells.8.Blood samples from the animals were obtained for analysis of lipids and glucose function using Mindrary autoanalyzer in Laboratory of Endocrinology and Metabolism in Shandong Provincial Hospital affiliated to Shandong University.9.Intracellular cholesterol contents and liver lipids(toal cholesterol,free cholesterol and triglyceride)were measured using acolorimetric assay.10.HepG2 cells was tested the cholesterol staining by Filipin assay.11.Dual luciferase activity assays:DNA fragment corresponding to the HMGCR and SREBP-2 promoter was fused to a luciferase gene and and the mutation of HMGCR-Luc(CER and SRE,respectively)and SREBP-2(CER-Like,IRE-1 and IRE-2,respectively).Then the resultant luciferase reporter constructs were transfected into cells.12.In vivo Imaging and Analysis:For imaging,mice were injected IP with 50 mg/kg nembutal.Mice were imaged on the IVIS 100 Imaging System,and analyzed with Living Image software.13.Tolerance tests:Glucose tolerance tests(OGTT)were performed to evaluate the glucose metabolism.14.Hepatic new cholesterol contents were meatured according to related reference.15.CHIP was used to analyze the binding of FoxO1 to SREBP-2 promoter.Results:1.The high expression of CRTC2 in hypercholesteremia patients.Hypercholesteremia patients had high serum LDL-C levels,however,the serum TG,HDL-C levels were no significant difference between hypercholesteremia patients and control group.Liver FTC and TTC contents were higher in hypercholesteremia patients.Real-time PCR results showed that hepatic CRTC2,SREBP-2 and HMGCR levels were higher in hypercholesteremia patients than control group.However,the levels of SREBP-la,LXR,FoxO1,HMGCS and LDLR were no significant difference.2.The high expression of CRTC2 in HC-diet mouse model.Mice fed high cholesterol diet had higher liver/body ratios,abnormal FBG and serum lipids.Meanwhile,the hepatic TTC and FTC contents were also increased.The expression of the key genes associated with gluconeogenesis(PEPCK)and CRTC2 were increasd in liver of HC-diet mouse.3.The effect of CRTC2 on serum and hepatic lipids,especially hepatic new cholesterol.Ad-CRTC2 mice exhibited higher body weight and FBG levels,abnormal glucose tolerance and high serum cholesterol level(P<0.05).H&E staining showed increased fat accumulation in the hepatic intracellular vacuoles of Ad-CRTC2 mice.Liver lipids showed that FTC levels increased.Similarly in HepG2 cells the TC content increasd after transfected overexpressed CRTC2.Contray,CRTC2-KO mice exhibited reduced FBG levels,OGTT levels and smaller liver and decreased at accumulation and hepatic FTC contents in HC-diet group.However,the cholesterol levels in serum and liver were recovered in CRTC2-/-mice injected with overexpressed CRTC2.The hepatic new cholesterol contents were increased in Ad-CRTC2 mice compared with control group(P<0.05).4.The upregulation of HMGCR induced by CRTC2 in vivo and in vitro.Ad-CRTC2 mice exhibited higher genes of hepatic HMGCR,FDPS,SS,ABCA1,and PEPCK.Similar results in overexpressed CRTC2 HepG2 cells.In vivo imaging results showed the Ad-HMGCR-luc activity increased in Ad-CRTC2 mice.And the protein of HMGCR and PEPCK in Ad-CRTC2 mice liver were increased compared with control group.CRTC2-KO mice showed the decreased genes and protein of HMGCR in HC diet.Smilarly siRNA CRTC2 reduced the HMGCR by IF.5.The upregulation of HMGCR induced by CRTC2 was dependent on SREBP-2.CRTC2 could increase the HMGCR-luc activity.However,CRTC2 decreased the HMGCR-luc(SRE-muation and CRE-muation)activity.The gene and protein of SREBP-2(P,N)were increased in the liver of Ad-CRTC2 mice.Similar results in HepG2 cells overexpressed CRTC2.Interesting,CRTC2 can increase the SREBP-2(P)and SREBP-2(N)in a dose dependent manner.In contray,knockout of CRTC2 reduced the genes and protein of SREBP-2.SiRNA of SREBP-2 decreased the increase of SREBP-2 induced by CRTC2.6.CRTC2 upregulated transcrptional SREBP-2 via reducing the location of FoxO1 in nuclei.In vivo imaging showed the decreased activity of SREBP-2-luciferase in CRTC2-/-mice compared with CRTC2+/+ mice.CHIP results showed that overexpression CRTC2 induced the decrease of binding of FoxO1 to SREBP-2 promoter in cells.The actinomycin D can inhibite the increase of genes of SREBP-2 induced by CRTC2.The activity of SREBP-2-luciferase mutated with CRE was decreased,but partly.Meanwhile,SiRNA of FoxO1 induced the increase of SREBP-2.CRTC2-/-mice exhibited high FoxO1 nuclei protein.And the nuclear protein of FoxO1 was decreased in BNL and HepG2 cells overexpressed CRTC2.Dual luciferase reporter showed that the activity was increased in SREBP-2-luciferase with mutation of IRE1 sequence,rather than IRE2 mutation.7.CRTC2 regulated SREBP-2 activity in various cholesterol diets.Activation of SREBP-2 was prominent under low-cholesterol conditions and was diminished by high cholesterol levels in the diet.CRTC2 knockout greatly reduced SREBP activation in 0.02%cholesterol diet.However,CRTC2 knockout had a minimal effect on SREBP-2 activation at a 4%cholesterol levels.Knockout of CRTC2 not only prominently reduced the mRNA levels of SREBP-target genes(HMGCR,HMGCS and LDLR)but also ameliorated gene expression of SREBP-2 at low cholesterol levels.However,CRTC2 knockout had a minimal effect on the SREBP-2 gene at the 4%cholesterol levels.The effects of CRTC2 knockout on liver FTC and TTC levels were maximal at low cholesterol concentrations.Conclusions:1.Hypercholesteremia patients and mice fed with HC diet exhibited increased CRTC2.2.CRTC2 can regulate lipids in serum and liver,especially hepatic new cholesterol via HMGCR.3.CRTC2 modulated hepatic cholesterol synthesis via SREBP-2/HMGCR pathway.4.CRTC2 regulated hepatic SREBP-2 by FoxO1 recognizing the IRE1 sequence located in SREBP-2 promoter.Background:Subclinical hypothyroidism(SCH)is a common disease and the incidence of SCH is 4-10%.SCH is associated with hypertension,hyperlipidemia,metabolic syndrome,atherosclerosis and cardiovascular disease.However,recent study showed that SCH is correlation with diabetes.The incidence of SCH was 4-17%in patients with diabetes.Moreover,diabetic patients with SCH aggravated the development of diabetes.Previous reports indiciated that diabetes and prrediabetes with SCH patients had a higher prevalence of diabetic nephropathy than that without SCH patients.The normalization of TSH resulted in decrease of level of fasting insulin,fasting and postprandial glucose in SCH patients.Thus,it is very important for diabetic patients with SCH to explore the mechanism of TSH in regulation of glucose metabolism.CRTC2 is a crual regulator to modulate the glucose metabolism and palys an important role in mainting the glucose homostasis.The increased cAMP levels induced by glucagon activate the CRTC2,then leading to enhance the expression of key gluconeogenic enzymes,such PEPCK and G6P.In refeeding,the circulating glucose and insulin levels are low,glucagon results in the dephosphorylation of CRTC2,then it translocates the nucleus where it activates the transcription of several genes in gluconeogenesis.In fasting,CRTC2 activity is controlled by insulin signaling which leads to CRTC2 phosphorylation and exclusion from the nucleus,thus it can no longer affect gene expression.SCH is a biochemical diagnosis defined as an elevated serum TSH level and a normal serum free thyroxine(FT4)concentration.Previously reported TSH is positive to serum glucose levels.Our previous study showed that TSH induced the increase of cAMP levels to result in the abnormal lipids metabolism.Up to now,the mechanism of TSH impacts the glucose levels,especially the relationship between TSH and CRTC2 is unclear.Our study confirmed that THS upregulated the expression of hepatic CRTC2 dependent on the' THSR.The detailed molecular mechanism of TSH regulating glucose levels is TSH binds to the TSHR located in liver cell membrane to result in increase of cAMP levels,then dephosphorylates CRTC2 and translocates the nucleus where it activates the transcription of PEPCK and G6P and upregulated hepatic gluconeogenesis.Objectives:1.To establish the SCH mouse model and determine the upregulation of glucose metabolism and key enzymes of gluconeogenesis in SCH mouse model.2.To determine the effect of TSH on hepatic CRTC2 expression in SCH mouse model and HepG2 cells treated with TSH.3.To determine the effect of CRTC2 on gluconeogenesis induced by TSH using the primary hepatocytes from CRTC2-KO mouse and SiRNA of CRTC2.4.To explore the mechanism of TSH in regulation of CRTC2 dependent on TSH using the TSHR-KO mice and siRNA of TSHR.5.To explore the mechanism of TSH in regulation of CRTC2 via cAMP/PKA pathway using forskolin and H89.6.To identify the effect of TSH on CREB:CRTC2 complex in TSHR-KO mice liver.Methods:1.Cell culture(1)HepG2 cells:cells were cultured according to guidance from ATCC.(2)Primary mouse hepatocytes:Cells were digested and cultured according to related to references.2.Animal model(1)SCH mouse model:Male mice were given methimazole,a drug that inhibites thyroid hormone synthesis[MMI,0.08 mg/kg BW-d]in the drinking water,another group were given a corresponding volume of vehicle(control group)for 14 weeks.(2)TSHR-KO and CRTC2-KO mouse model:We got the wild-type mice and knockout of mice by breeding heterozygote mice and determined the genotype by PCR.And all mice lived in SPF environment of the experimental animal center.3.Real time-PCR:to analyze the expression of PEPCK,G6P,TSHR and CRTC2 mRNA.4.Protein extraction and Western blotting analysis:to determine the expression of hepatic PEPCK,G6P,TCRTC2,p-CRTC2,CREB,p-CREB and beta-actin protein.5.Immunohistochemistry and Immunofluorescence:to determine the expression of CRTC2 in liver of SCH mouse model and HepG2 cells.6.TSHR,CREB and CRTC2 silence:to silent the expression of TSHR,CREB and CRTC2.7.Tolerance tests:glucose tolerance tests(OGTT)and pyruvate tolerance tests(PTT)were performed to evaluate the glucose metabolism.8.Glucose Oxidase Method:to determine content of glucose in HepG2 cells.9.Dual luciferase activity assays:a DNA fragment corresponding to the PEPCK promoter was fused to a luciferase gene and the resultant luciferase reporter construct was transfected into cells.Dual luciferase activity assays were adopted to detect PEPCK promoter activity and mutant CRE sequence of PEPCK promoter activity.10.Tolerance tests:Glucose tolerance tests(OGTT)were performed to evaluate the glucose metabolism.11.Analysis of GPa activity in HepG2 cells.Results:1.Abnormal gluconeogenesis induces high glucose levels in SCH miceMice were fed methimazole for 14 weeks to provide a model of SCH.Compared to controls,SCH mice exhibited similar serum free T4(FT4)and free T3(FT3)levels and higher TSH levels after fed methimazole for 14 weeks.Metabolism cage result showed that SCH mice is consistent with clinical patitents with SCH.OGTT and PTT showed significantly abnormal glucose levels in SCH group than control group.PEPCK and G6P mRNA levels were higher in SCH liver than control mice both in fasted and in re-fed states(P<0.01).Correspondingly,the hepatic PEPCK and G6P protein levels were significantly increased in SCH mice.The GPa avtivity was higher in HepG2 cells upon TSH treatment.2.TSH promotes dephosphorylation of hepatic CRTC2Immunohistochemistry and Western blotting showed the CRTC2 protein levels,the active form,were significantly increased in SCH mice.Glucagon and TSH all increased the CRTC2 expression of HepG2 cells,however,the effect of TSH was reduced by Insulin.The immunofluorescence results showed that TSH increased the expression of CRTC2 not only in the cytoplasm but also in the nucleus.Further,TSH promoted CRTC2 dephosphorylation at Ser171 and Serl36 in HepG2 cells.3.CRTC2 involves in TSH-induced glucose gluconeogenesisThe PEPCK and G6P gene and protein levels were significantly decreased in Crtc2-/-mouse primary hepatocytes compared with Crtc2+/+ mouse primary hepatocytes with or without treatment with TSH,respectively.TSH induced a dramatic increase in the major gluconeogenic enzymes,PEPCK and G6P compared with in control cells However,the increase of G6P and PEPCK stimulated by TSH was significantly attenuated by Crtc2 siRN A(P<0.01),but not completely.Nextly,we observed that luciferase activity of PEPCK sharply increased in response to TSH.Pre-incubated with Crtc2 siRNA prevented this increase in luciferase activity but did not completely abolish it4.TSH-stimulated increase of CRTC2 is dependent on TSHR in vitro and vivoThe CRTC2 protein levels were significantly decreased in the livers of Tshr-/-mice compared with Tshr+/+ mice(Fig 4A).In Tshr+/+ mouse primary hepatocytes,TSH stimulated CRTC2 protein levels in a dose-dependent manner.However,there was no increase in CRTC2 in Tshr-/-mouse primary hepatocytes upon TSH treatment.Transfection of HepG2 cells with a Tshr siRNA(Tshri)significantly reduced TSHR mRNA expression compared with control cells,and the Tshr siRNA also decreased CRTC2 mRNA levels even upon the TSH in HepG2 cells.5.TSH regulates hepatic CRTC2 via cAMP/PKA pathwayAfter forskolin treatment,the protein levels of hepatic CRTC2,PEPCK and G6P were increased.Similar to forskolin,TSH increased the CRTC2,PEPCK and G6P expression.However,pre-treatment with SQ resisted the action of TSH.Similarly,treatment with H89(a selective and potent inhibitor of PKA)in HepG2 cells,partly inhibited TSH-induced expression of CRTC2,PEPCK and G6P(Fig 5B).Further,H89 was administered to Tshr knockout mice,H89 decreased PEPCK and G6P mRNA and protein expression in Tshr+/+ and Tshr-/-mice compared PBS treatment,respectively.Meanwhile,H89 increased the p-CRTC2(S171)proteins in cytoplasm and decreased the CRTC2 in nuclei.6.TSH regulates hepatic CREB:CRTC2 complexTSH increased the expression of p-CREB(S133)in a dose dependent manner.SiRNA CREB induced the obviously decreased PEPCK-luciferase activity;Mmoreover the upregulation induced by TSH was also alleviated by siRNA CREB.Overexpression CRTC2 increased the activity of PEPCK-luciferase,however,mutant CRE sequence in PEPCK promoter decreased the activity of PEPCK.Co-IP results showed that the CREB:CRTC2 complex was lower in Tshr-/-mice liver than in Tshr+/+ mice liver.Conclusions:1.SCH exhibited abnormal glucose metabolism.2.TSH increased the expression of CRTC2 via dephosphorylation and TSH regulated hepatic of PEPCK and G6P via CRTC2.3.TSH increased the hepatic CRTC2 via TSHR/cAMP/PKA pathway.4.TSHR knockout inhitited the expression of hepatic CREB:CRTC2 complex.