Follicle-stimulating Hormone Induces Hepatic Cholesterol Disorder And The Mechanism

Background:The life span of sexually mature females can be divided into three stages based on self-reported bleeding patterns,that is,pre-menopause,peri-menopause and post-menopause.During menopause,the prevalence of dyslipidemia also increases,which is the maj or risk factor of cardio-cerebrovascular disease.The classic view assumes that the underlying mechanism of dyslipidemia is attributed to an insufficiency of estrogen.However,women during peri-menopause,at which time estrogen levels remain relatively unperturbed and only the FSH level increases,also suffer from a significant increase in total cholesterol(TC)and low-density lipoprotein cholesterol(LDL-C).This cholesterol accumulation begins 5 years before the final menstrual period(FMP),and substantially increases within 1 year of the FMP.Hormone replacement therapy(HRT)can decrease the serum cholesterol level,however,estrogen use can be associated with increased risks for breast cancer and coronary heart disease.The relatively poor alternatives for menopause related dyslipidemia therapies,particularly for early and rapidly progressing cholesterol accumulation,makes the advent of new preventative strategies very desirable.FSH,a glycoprotein polypeptide hormone,is produced by the anterior pituitary gland,and its main function is widely considered to be in the regulation of sex hormone synthesis in the gonads.During peri-menopause,because of compensation for failing ovaries,FSH increases earlier than estrogen decreases.Strong correlations exist between rising serum FSH levels and osteoporosis and obesity after menopause.Blocking FSH reduced body fat levels and increased the bone density in menopause.Furthermore,pre-menopausal women with high basal FSH levels>7 IU/L displayed greater cholesterol levels than those,with basal FSH levels<7 IU/L.In the study by Song et al,postmenopausal women with a higher serum FSH(? 78.3 IU/L)had higher serum TC and LDL-C levels than those with relatively lower FSH levels(40-78.3 IU/L),and ovariectomized mice had high serum FSH and lipid levels and reduced hepatic LDL receptor expression.Together,all these data showed that FSH participated in the pathogenesis of menopause related dyslipidemia.However,whether FSH is a valuable therapeutic target for dyslipidemia has been unexplored.The function of FSH is mainly mediated through FSH receptors(FSHRs).FSHR is a transmembrane receptor that interacts with FSH and is a G protein-coupled receptor(GPCR).Published data demonstrate that FSHR couples with Gsa,which is involved in the differentiation and maturation of granulosa cells,or Gia,which regulates bone mass in osteoclasts and fat accumulation and redistribution in adipocytes.Arrestins are a small family of proteins,which are recruited by activated GPCRs through binding to GPCRs and act to uncouple the receptors from G protein subunits.It has been reported that ?-arrestin-2 also acts as a multifunctional adaptor following GPCR activation in metabolic processes.The liver plays a pivotal role in maintaining cholesterol homeostasis,including cholesterol biosynthesis,uptake,conversion,transport and esterification.It is known that the biosynthesis of cholesterol starts from acetyl coenzyme A,in which 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR)acts as the rate-limiting enzyme.It has been proven that cyclic AMP response element-binding protein(CREB)and sterol regulatory element binding protein(SREBP)-2 are two important transcription factors that can bind with cAMP-responsive element(CRE)and sterol-response element(SRE),respectively,in the HMGCR promoter.SREBP-2 is an isoform of the SREBP family,which preferentially activates genes involved in cholesterol synthesis.This project was to study the cholesterol metabolism disorder in peri-menopausal women,through the epidemiological investigation and a variety of mice models.We found that high levels of serum FSH increases serum cholesterol levels and liver cholesterol biosynthesis,and that blocking FSH signaling prevents cholesterol accumulation induced by elevated FSH or high cholesterol diet.We studied the metabolism of FSH regulating hepatic cholesterol biosynthesis and that of blocking FSH reducing cholesterol accumulation in vivo and in vitro,respectively.The results demonstrated FSH,binding with hepatic FSHRs,activated the Gi2a/?-arrestin-2/Akt/FoxO1/SREBP-2/HMGCR pathway,leading to the increase of cholesterol accumulation.This study uncovers the effect of blocking FSH on hepatic cholesterol biosynthesis and serum cholesterol,and reveals the molecular mechanism of the menopausal cholesterol disorder.This finding may be helpful in devising therapy-decision strategies,as well as in exploring novel hormone-based therapies,for hypercholesterolemia during the menopausal transition.Objectives:1.To determine the effect of FSH on menopausal disorder through epidemiological investigation and ovariectomized mouse models.2.To determine the existence of functional FSHR on the surface of liver cells,and to reveal the important role of FSH on the metabolic disorder of cholesterol related to menopause.3.To study the metabolism of cholesterol disorder in hepatocytes induced by FSH in vivo and in vitro.4.To explore the signaling pathway of cholesterol biosynthesis by FSH in the liver.Methods:1.Epidemiological investigation:154 pre-menopausal and 124 peri-menopausal female subjects were eligible for inclusion in our study.The basic information(age,weight,BMI,etc.),serum hormone(E2 and FSH)levels and serum lipid profiles were analyzed by linear and nonlinear correlation equations and other statistical methods,to explore the correlation between serum FSH and serum lipid.2.2.Animal model(1)OVX+E2 mouse model:Nine-week-old female mice were randomly divided into four groups:1)sham-operation(sham),2)bilateral ovariectomy(OVX)with diet-supplemented oestradiol(E2)(OVX+E2),3)OVX+E2 with low-dose FSH(OVX+E2+L-FSH)and 4)OVX+E2 with high-dose FSH(OVX+E2+H-FSH).(2)GnRHa mouse model:The OVX+E2 mice received an intraperitoneal injection of solvent(N.S.)or GnRH analog(GnRHa)daily.(3)FSHAb ovariectomized mouse model:The OVX+E2 mice were injected with FSH or normal saline(N.S.)following the daily administration of FSH? antibody or mouse IgG for 4 weeks.(4)FSHAb mouse model:Nine-week-old female mice received intraperitoneal injections of either FSH? antibody or mouse IgG at 100 ?g/d daily.(5)Fshr knockout mouse model:The 4-week-old Fshr-/-mice were fed a 0.26%estradiol valerate-supplemented diet to maintain normal estrogen levels.Nine-week-old Fshr-/-mice and their Fshr+/+ littermates received ovary removal surgery,following estrogen supplementation,and then receiving treatment with recombinant FSH was conducted by daily intraperitoneal injection for 2 weeks.(6)Fshr knockout high-cholesterol diet-fed mouse model:Seven-week-old female wild-type mice and Fshr KO micewere fed a normal-cholesterol diet(NC)or high-cholesterol diet(HC)for 20 weeks.(7)Fshr siRNA mouse model:OVX+E2 mice were injected with Fshr siRNA adenovirus every seven days for three times,and 7 days later the first time,all mice were treated with FSH or vehicle daily.(8)Ldlr knockout mouse model:Ldlr knockout female mice were randomly divided into three groups:1)sham-operation(sham),2)bilateral ovariectomy(OVX)with diet-supplemented oestradiol(E2)(OVX+E2),3)OVX+E2 with high-dose FSH(FSH)for 2 weeks.3.Cell culture:1)HepG2 cells and were routinely maintained in MEM/EBSS and RPMI 1640,respectively,supplemented with 10%fetal bovine serum 2)Hepatocytes were isolated from C57BL/6J male mice using the two-step collagenase perfusion protocol.The isolated mouse hepatocytes were then cultured at 80%-90%confluence in DMEM media containing 10%FBS in rat-tail collagen type I coated p]ates.4.Serum hormone measurements:The levels of serum E2 and FSH were measured using commercial immunoassay kits.Absorbance at 450 nm was determined using an ELISA microtiter plate reader.5.Blood samples from the animals were obtained for analyses of blood lipid and liver function using Olympus AU5400 autoanalyzer in Clinical Laboratory of Provincial Hospital Affiliated to Shandong University.6.Intracellular TC determination:Cholesterol was extracted from cells using a Cholesterol assay Kit according to the manufacturer's instruction,and the cholesterol content was normalized by the corresponding protein content.7.VLDL production rate:To measure the VLDL production rate,overnight-fasted mice were injected i.p.with 1 g/kg BW poloxamer 407 in PBS.Blood samples were collected immediately prior to inj ection and at 1,2,3,4,and 6 hours following inj ection.Cholesterol and triglyceride concentration were measured enzymatically 78.iTRAQ labeling and NanoLC-MS/MS analysis:Whole protein samples were isolated using a standard protocol.The denatured and digested peptides were then labeled with the iTRAQ Reagents 4-plex Kit.The MS analysis was performed using a NanoLC system equipped with a Triple TOF 5600 Plus mass spectrometer.The functional annotation of the detected proteins was performed using the GO database.9.RT2 PCR array:Cholesterol metabolism-related genes were measured by RT2 Profiler PCR Array system according to the manufacturer's instructions.The real-time PCR was performed using a 40-cycle 2-step PCR protocol on a Light Cycler 480.10.Nuclear run-on(NRO)RT-qPCR:The cell nuclei were isolated and NRO transcription was performed in the presence of bromouridine.Labeled nascent transcripts were purified by immunoprecipitation,and the transcript levels were determined by RT-qPCR.11.Measurement of de novo Cholesterol Biosynthesis:The mice were sacrificed 20 minutes after injection,and 50 mg of the liver was rinsed in ice-cold phosphate-buffered saline.The[1-14C]-acetate content within the lipid fraction of each tissue was calculated from the measured disintegrations/minute(dpm).The results were adjusted by the hepatic whole protein level.The de novo cholesterol biosynthesis was expressed as the amount of[1-14C]-acetate incorporated into sterols per gram of protein in the liver tissue.12.Assay of HMGCR activity:The concentration of microsomes was determined by the BCA method.The HMGCR activity was accessed using the HPLC-MS/MS method.13.Competitive FSH-FSHR binding assay:The competition assay to detect the specific binding of FSH and FSHR was measured by radioimmunoassay.14.Immunofluorescence were adopted to determine the expression and intracellular distribution of FSHR protein.15.Plasmid construction and transfection and dual luciferase activity assays:The plasmids were transfected into cells.After transfection with a corresponding plasmid for 24 hours following the manufacturer's instructions,HepG2 cells were treated with FSH for an additional 6 hours.,The cells were subsequently harvested,and the luciferase activity was measured using a dual-luciferase reporter assay system(Promega).pRL-TK was used as a control to normalize the luciferase activity.16.GloSensor cAMP assay:The Glosensor plasmid was transfected into the HepG2 cells.Twenty-four hours later,the cells were plated on 96-well plates at a density of 20,000 cells per well.The cells were maintained in culture medium for an additional twenty-two hours and were then incubated with 100 ?l of CO2 independent medium with 2%v/v GloSensorTM cAMP Reagent and 10%FBS for two hours.The cAMP signal was monitored on a luminescence counter.17.Luciferase reporter analysis in vivo:OVX mice were infected with adenoviruses,HMGCR-Luc or SREBP-2-Luc,and RSV?-gal by tail-vein injection.Five days after the delivery of the adenovirus,the mice were imaged under ad libitum feeding conditions.Prior to imaging,the mice were intraperitoneally injected with 50 mg/kg pentobarbital and 100 mg/kg sterile firefly D-luciferin.The mice were imaged with the LB983 Imaging System and analyzed with Living Image software.18.Silencing of genes using siRNA:small interfering RNAs(siRNAs)targeting the human SREBP-2 and Ga subunits gene was designed and transfected into cells.19.RNA isolation and real-time quantitative RT-PCR:Total RNA from cells and mice liver tissue was isolated and real-time quantitative RT-PCR was used to determine relative mRNA expression of mouse Fshr,Hmgcr,Hmgcs,Srebp2,Cyp7a1,Ldlr,Abca1,Abcg1,Srb1,Acat2??-actin(Actb);? FSHR,HMGCR,SREBP2,Gsa,Gi1?,Gi2?,Gi3?,G12?,G13?,Gq? and ?-actin(ACTS).20.12.Protein extraction and western blotting:Protein was extracted from hepatocytes and tissues.Western blotting were adopted to determine the expression of FSHR,HMGCR,LDLR,CYP7A1,ABCA1,SRB1,ACAT2,p-Ser133 cyclic AMP response element-binding protein(CREB),CREB,SREBP-2,p-Ser473 Akt,Akt,Gi2a,?-arrestinl/2,LMB1 and GAPDH.21.Statistical analysis:The data are expressed as the means ± SEMs.Statistical analyses were performed using either two-tailed impaired Student's t tests(two groups)or one-way ANOVA tests(more than two groups).A p<0.05 is considered to be statistically significant.Results:1.Serum FSH is positively correlated with TC levels and prevalence ofhypercholesterolemia in women.The basic clinical characteristics of the 154 premenopausal and 124 peri-menopausal female subjects.Compared with the premenopausal women,the estrogen(E2)levels were similar in the peri-menopausal females,however,the level of FSH,as well as the levels of TC and LDL-C were all significantly increased.Furthermore,the serum levels of TC and LDL-C and the prevalence of hypercholesterolemia gradually increased along with the increase of the serum FSH levels.Moreover,the serum FSH levels were positively correlated with the serum TC and LDL-C levels even after adj usting for E2 together with traditional confounding factors.2.FSH increases serum and hepatic cholesterol accumulation in vivo and in vitro.We generated an ovariectomized(OVX)mouse model and maintained E2 level at the normal level by exogenous estrogen supplementation(OVX+E2).Under this condition,the mice were injected with exogenous FSH.Compared with the controls,the mice that received FSH demonstrated not only higher FSH levels but also normal E2 levels.Moreover,the serum levels of TC and LDL-C,as well as the liver concentrations of TC and free cholesterol(FC),were significantly elevated following FSH treatment.Furthermore,VLDL cholesterol production rate was significantly increased in the mice that received FSH.However,the body weights and serum aspartate transaminase(AST)levels did not significantly change.Similarly,FSH also increased the intracellular cholesterol content in vitro.3.Blocking FSH reduces serum and hepatic cholesterol accumulation.First,we established an animal model that exhibited a low serum FSH level.GnRHa inhibits the secretion of FSH in the body.The results showed the mice that received GnRHa displayed significant decreases in the levels of FSH,serum TC,LDL-C,and the contents of liver TC and FC.Furthermore,a polyclonal antibody against FSH was used to block FSH signaling.An FSH? antibody(FSHAb)has been proven to inhibit OVX-induced bone loss and body fat accumulation in mice.Blocking FSH could also effectively reduce the serum TC and LDL-C levels and the liver TC levels in OVX mice.There was no significant difference in the serum levels of E2 and FSH between antibody-and goat IgG-treated mice.More importantly,we also found that the suppression of FSH could decrease cholesterol under normal conditions.The FSHAb was used to block the interaction of FSH with the mouse FSHR by intraperitoneal injection for 8 weeks.The serum levels of E2 and FSH were not significantly different between the antibody-and IgG-treated mice.However,the mice that received antibody showed significant decreases in the serum cholesterol levels and hepatic cholesterol content.4.Liver expresses functional FSHR.The function of FSH is mainly mediated through FSHR.We found that there was FSHR expression in human liver tissues,and immunofluorescent staining results indicated that FSHR was localized on the plasmalemma of hepatocytes.Using a competitive binding assay,we proved that there the hepatic FSHR was functional.5.Fshr deficiency resists hypercholesterolemia induced by FSH or high-cholesterol diet.We performed ovariectomy operations in both Fshr-/-mice and Fshr+/+ littermates,and all mice were subsequently injected with FSH and supplemented with E2.The serum E2 and FSH levels were similar;however,the serum TC and LDL-C levels,as well as the liver TC and FC contents,were significantly reduced in the Fshr-/-mice.Furthermore,FSH increased the intracellular TC levels in primary hepatocytes from Fshr+/+ mice,which was blocked in Fshr-/-hepatocytes.Moreover,an adenovirus-based shRNA was used to specifically silence the expression of the hepatic Fshr gene via tail vein injection in OVX+E2 mice.The results showed that the effects of FSH on cholesterol were reversed when the Fshr gene was silenced.We also assessed the serum cholesterol levels in OVX mice without exogenous E2 supplementation in Fshr-/-mice and Fshr+/+ littermates,and the results showed that Fshr deficient mice had significantly lower levels of serum cholesterol than Fshr+/+ mice.We established a hypercholesterolemic mouse model via high-cholesterol diet(HC)feeding.HC feeding significantly increased the serum TC and LDL-C levels and the liver TC and FC contents as well as the hepatic protein levels of HMGCR.However,the effect of HC feeding was partially blocked in the Fshr-/-mice(E2-supplemented).6.FSH enhances nascent transcription of HMGCR and de novo cholesterol biosynthesis.First,we performed iTRAQ-based quantitative proteomic analysis to investigate hepatic proteome alterations and found significant differences in the sterol metabolic process in the comparison of the FSH-treated mice with the control mice.Then,the an RT-PCR array results showed significant changes in the genes related to cholesterol biosynthesis,transport,and esterification in the FSH-treated mice.Furthermore,we found the expression of HMGCR significantly increased in the FSH-treated mice.Moreover,live image analysis indicated an increase in HMGCR transcription in the mice treated with adenovirus that contained an HMGCR luciferase reporter plasmid(Ad-HMGCR/luc)followed by FSH inj ection.Actually,HMGCR activity is instrumental in controlling de novo cholesterol biosynthesis.Moreover,both the activity of HMGCR in vivo and the rate of hepatic de novo cholesterol biosynthesis also increased in a dose-dependent manner with FSH treatment.Using the run-on assay,we found that FSH increased the nascent transcription of HMGCR in hepatocytes.FSH also induced HMGCR expression in mouse primary hepatocytes and human HepG2 cells.Actinomycin D,a transcriptional inhibitor,could block the effect of FSH on HMGCR transcription.In FSHAb-treated mouse models and Fshr deficiency mouse model,the results indicated that transcription and expression of SREBP-2 and HMGCR was significantly blocked.We also investigated the effect of FSH in LDL receptor deficiency(Idlr-/-)mice.The results showed that the serum TC and LDL-C levels,as well as the liver contents of TC and FC,were significantly elevated in the FSH treated ldlr-/-mice.The expressions of the mRNAs and proteins of HMGCR and SREBP-2 were also increased with FSH injection.Moreover,the cholesterol accumulation induced by FSH can also be blocked by the specific FSH antibody(FSHAb)previously employed.7.SREBP-2/FoxO1 is indispensable in the effect of FSH on HMGCR.Data showed that FSH upregulated SREBP-2 maturation(SREBP-(N)),not CREB phosphorylation(p-CREB),in a time-dependent manner.As CREB is a cAMP-response element binding protein,we also found there was no significant change in the cAMP levels after FSH stimulation.Using a luciferase reporter assay,we found that FSH could significantly increase the HMGCR luciferase activity in cells transfected with plasmids that contained the CRE and SRE sites(hHMGCR/luc)or the mutated CRE site(muCRE-hHMGCR/luc)but not in cells transfected with plasmids that contained the SRE mutated site(muSRE-hHMGCR/luc).Moreover,the effect of FSH on HMGCR disappeared when SREBP-2 was suppressed by shRNA.FSH increased the nascent transcription of SREBP-2 using the run-on assay.In addition,FSH also increased the activity of the SREBP-2 promoter luciferase reporter(SREBP-2/luc)in vivo and in vitro.There are 2 IRE sites(IRE1 and IRE2)in the SREBP-2 promoter that can be recognized by the Forkhead box protein O1(FoxO1).The results indicated that mutations in either IRE1 or IRE2 could completely block the effect of FSH on SREBP-2.The chromatin immunoprecipitation(ChIP)assay showed that FSH reduced the binding capacity of FoxO1 to the SREBP-2 promoter.Western blots showed that the transfer of FoxO1 from the plasma into the nucleus was significantly attenuated in FSH-treated mice.8.FSH activates hepatic cholesterol biosynthesis via Gi2?/?-arrestin2/Akt pathway.Data indicated that the ability of FSH to increase the HMGCR expression and SREBP-2 maturation was prevented by a specific p-Akt inhibitor(MK2206)or PI3K inhibitor(LY294002).We used a group of siRNAs to silence the corresponding Ga isoforms.Intriguingly,only when Gi2a was silenced,the effects of FSH on HMGCR transcription and expression,as well as on SREBP-2 maturation,were interrupted,?-arrestins,including ?-arrestinl(ARRB1)and ?-arrestin 2(ARRB2),were ubiquitously expressed and participated in many metabolic processes through activation of Akt.In our study,data demonstrated that when ARRB2 was knocked down,the effects of FSH on Akt phosphorylation and HMGCR expression were interrupted.Conclusion:1.FSH plays an important role in regulating the metabolism of cholesterol disorder associated with menopause.2.Blocking FSH can revise cholesterol disorder and reduce cholesterol accumulation in the body,which may provide a new therapeutic strategy for treating menopausal dislipidemia.3.FSH increases cholesterol accumulation in the body through inducing hepatic cholesterol biosynthesis.4.FSH regulates cholesterol biosynthesis might through the Gi2?/?-arrestin2/Akt/Fox01/SREBP-2/HMGCR signaling pathway.