Blocking FSH inhibits hepatic cholesterol biosynthesis and reduces serum cholesterol

Abstract

Menopause is associated with dyslipidemia and an increased risk of cardio-cerebrovascular disease. The classic view assumes that the underlying mechanism of dyslipidemia is attributed to an insufficiency of estrogen. In addition to a decrease in estrogen, circulating follicle-stimulating hormone (FSH) levels become elevated at menopause. In this study, we find that blocking FSH reduces serum cholesterol via inhibiting hepatic cholesterol biosynthesis. First, epidemiological results show that the serum FSH levels are positively correlated with the serum total cholesterol levels, even after adjustment by considering the effects of serum estrogen. In addition, the prevalence of hypercholesterolemia is significantly higher in peri-menopausal women than that in pre-menopausal women. Furthermore, we generated a mouse model of FSH elevation by intraperitoneally injecting exogenous FSH into ovariectomized (OVX) mice, in which a normal level of estrogen (E2) was maintained by exogenous supplementation. Consistently, the results indicate that FSH, independent of estrogen, increases the serum cholesterol level in this mouse model. Moreover, blocking FSH signaling by anti-FSHβ antibody or ablating the FSH receptor (FSHR) gene could effectively prevent hypercholesterolemia induced by FSH injection or high-cholesterol diet feeding. Mechanistically, FSH, via binding to hepatic FSHRs, activates the Gi2α/β-arrestin-2/Akt pathway and subsequently inhibits the binding of FoxO1 with the SREBP-2 promoter, thus preventing FoxO1 from repressing SREBP-2 gene transcription. This effect, in turn, results in the upregulation of SREBP-2, which drives HMGCR nascent transcription and de novo cholesterol biosynthesis, leading to the increase of cholesterol accumulation. This study uncovers that blocking FSH signaling might be a new strategy for treating hypercholesterolemia during menopause, particularly for women in peri-menopause characterized by FSH elevation only.

Fig. 1

Both serum cholesterol levels and the prevalence of hypercholesterolemia are higher in peri-menopausal women than in pre-menopausal ones. a Serum FSH and E2 levels. b Serum TC and LDL-C levels in the women with pre-menopause or peri-menopause. Data are presented as the mean ± SD. c Prevalence of hypercholesterolemia in the recruited women. Data are presented as prevalence. d Cholesterol levels across the spectrum of serum FSH levels. Data are presented as the mean ± SD. e Prevalence of hypercholesterolemia across the spectrum of serum FSH levels. Data are presented as prevalence. The statistical methods for a–e included the Mann–Whitney U-test, Student’s t-test, chi-squared test, One-way ANOVA and chi-squared test, respectively. *P < 0.05, **P < 0.01; ns, not significant. TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol

Fig. 2

FSH increases cholesterol accumulation in vivo and in vitro. a–e C57BL/6 mice were sham-operated (Sham) or ovariectomized (OVX), and then, the OVX mice were supplemented with estrogen (E2) to a normal level, named OVX + E2 mice, and then injected with solvent (normal saline, N.S.) or FSH (L-FSH represents low-dose, 15 IU/kg body weight per day; H-FSH represents high-dose, 30 IU/kg body weight per day) for 2 weeks (n = 12 for Sham group; n = 10 per OVX groups). a Flow-chart. b Mimetic diagram of the serum E2 and FSH levels in the OVX mouse model. Arrows at the X-axis represent the starting points of the mice receiving different treatments. c ELISA analysis of serum E2 and FSH concentrations (n = 10 per group). d Serum TC and LDL-C levels. e Liver TC and FC contents. f Intracellular TC contents in HepG2 cells with FSH stimulation. Data represent three independent experiments. TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; FC, free cholesterol. Data are represented by the mean ± SEM. One-way ANOVA was used for statistical analysis. *P < 0.05 and **P < 0.01

Fig. 3

Blocking FSH reduces serum and hepatic cholesterol accumulation. a–e E2-supplemented OVX mice received solvent (N.S.) or GnRH analog (GnRHa, 25 μg/kg body weight per day), which would lead to pituitary desensitization and to the inhibition of pituitary FSH secretion (n = 6 per group). a Workflow schematic. b Mimetic diagram of the changes following different treatments. Arrows at the X-axis represent the starting points of the mice receiving different treatments. c ELISA analysis of serum E2 and FSH concentrations. d Serum TC and LDL-C levels. e Liver TC and FC contents. f–h The OVX + E2 mice received an intraperitoneal injection of FSHβ antibody (FSHAb) or mouse IgG followed by treatments with FSH or normal saline (N.S.) for 4 weeks (n = 6 per group). f The flowchart. g The serum TC and LDL-C. h The liver TC. i–k The wild-type mice received an intraperitoneal injection of FSHAb (FSHAb group, n = 8) or mouse IgG (IgG group, n = 10) for 8 weeks. i The flowchart. j The serum TC and LDL-C. k The liver TC and FC. TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; FC, free cholesterol. Data are represented by the mean ± SEM. One-way ANOVA or Student’s t-test is used for statistical analysis. *P < 0.05 and **P < 0.01; ns, not significant

Fig. 4

Functional FSH receptors (FSHRs) are localized to the hepatocyte surface. a (Up) Primers were designed depending on different domains (I, II and III) of FSHR. (Down) Representative RT-PCR bands of FSHR from livers of human or mouse are shown, with RT-PCRs using ovary tissue as positive controls. b FSHR in situ hybridization images of the human ovary and liver. Cell nuclei are stained by nuclear fast red, and the blue granules indicate FSHR RNA. c Representative Western blotting results show the bands of mouse and human FSHR proteins using antibodies corresponding to regions within (Up) the C-terminus, or (Down) N-terminus of FSHR, 90 µg protein per lane. d Representative super-resolution images of immunostaining of FSHR (red) and DAPI (blue) staining of nuclei in mouse and human liver tissues. Scale bars, 5 μm. e FSH-FSHR binding assay in mouse (NCTC1469) and human (HepG2) hepatocytes, as well as in positive control (ovary cells, CHO). Data represent no less than three independent experiments. Data are shown as the mean ± SEM. Student’s t-test is used for statistical analysis. **P < 0.01

Fig. 5

Fshr deficiency resists hypercholesterolemia induced by FSH or high-cholesterol diet. a–c Ovariectomized Fshr^-/- and Fshr^+/+ mice received FSH treatment for 2 weeks after equalizing their serum estrogen levels. n = 10 per group. a The flowchart. b Analysis of serum TC and LDL-C levels, c liver TC and FC contents. d Intracellular TC contents were measured in mouse primary hepatocytes from Fshr^-/- mice and Fshr^+/+ littermates treated with FSH (10 ng/mL), respectively. e–i The Fshr^-/- mice and Fshr^+/+ littermates were fed with a normal cholesterol (NC) diet or high-cholesterol (HC) diet, and serum estrogen levels in all Fshr^-/- mice were normalized, n = 4 for Fshr^-/- mice with NC diet; n = 8 for other three groups. e The flowchart. f Serum TC and g serum LDL-C levels and h liver TC and i liver FC content. TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; FC, free cholesterol. Data are represented by the mean ± SEM. One-way ANOVA or Student’s t-test was used for statistical analysis. *P < 0.05, **P < 0.01; ns, not significant

Fig. 6

FSH enhances nascent transcription of HMGCR and hepatic de novo cholesterol biosynthesis. a–e C57BL/6 mice were sham-operated (Sham) or ovariectomized (OVX), and then, the OVX mice were injected with solvent (normal saline, N.S.) or FSH (L-FSH represents low-dose, 15 IU/kg body weight per day; H-FSH represents high-dose, 30 IU/kg body weight per day), with estrogen (E2) maintained at normal levels. a The iTRAQ-based quantitative proteomic analysis of liver tissues compared the H-FSH group to the N.S. group (n = 6 per group). b mRNA levels and c Protein levels of molecules related to cholesterol metabolism in the liver (n = 10 for the N.S. and H-FSH groups, n = 8 for the L-FSH group). d Live imaging of hepatic HMGCR-luciferase (Ad-HMGCR/luc) activity in OVX + E2 mice after injection of Ad-HMGCR/luc via tail vein followed by injection of solvent (N.S.) or FSH (30 IU/kg) (n = 6 per group). e (Up) Detection of the in vivo activity of hepatic HMGCR, the rate-limiting enzyme in cholesterol biosynthesis, using the HPLC-MS/MS method (n = 5 for Sham group, n = 6 for other groups). (Down) Hepatic de novo cholesterol biosynthesis was measured by the amount of [1- ¹⁴C]-acetate incorporated into sterols per gram of protein in the liver tissue (n = 6 for Sham, N.S and L-FSH groups; n = 4 for OVX group; n = 5 for H-FSH group). f The nascent transcription of HMGCR in HepG2 cells treated with FSH (10 ng/mL) by run-on assay. g Representative immunoblotting of molecules related to cholesterol metabolism in mouse primary hepatocytes treated with FSH (10 ng/mL). h–j Experiments were performed in the HepG2 cell line. h qPCR analysis of HMGCR in HepG2 cells treated with different FSH doses and for different time periods. i Representative Western blotting analysis of HMGCR in HepG2 cells treated with FSH. j qPCR analysis of the HMGCR mRNA level in HepG2 cells treated with 7.5 ng/mL actinomycin D (transcriptional inhibitor, Act D) prior to FSH (10 ng/mL) treatment. Data are shown as the mean ± SEM. No less than three independent experiments were performed. One-way ANOVA or Student’s t-test is used for statistical analyses. *P < 0.05 and **P < 0.01; ns, not significant

Fig. 7

SREBP-2/FoxO1 is indispensable for the regulation of HMGCR by FSH. a Representative immunoblotting of SREBP-2 and p-CREB in HepG2 cells treated with FSH (10 ng/mL) for different times. b The cAMP levels in HepG2 cells treated with Forskolin (50 μM) or FSH (10 ng/mL) for different times. Forskolin (50 μM) was used as a positive control. P < 0.001. c (Left) The human reporter vector containing CRE and SRE sites in the HMGCR promoter (hHMGCR/luc) and vectors containing CRE motif mutant (muCRE-hHMGCR/luc) or SRE motif mutant (muSRE-hHMGCR/luc) were designed. (Right) HepG2 cells were transfected with these vectors for 24 h and were subsequently treated with FSH (10 ng/mL), the relative luciferase activity was assayed. Forskolin (50 μM) was used as a positive control. d Western blotting analyses of SREBP-2 and HMGCR in HepG2 cells transfected with SREBP-2 shRNA virus (LV-SREBP-2 virus) or with negative control virus followed by FSH (10 ng/mL) treatment. e Assay for the nascent transcription of SREBP-2 in the HepG2 cell line by run-on assay. f (Left) The SREBP-2 reporter construct containing IRE1 and IRE2 sites (SREBP-2/luc) and constructs with IRE motif mutants (muIRE1 or muIRE2) were made. (Right) HepG2 cells were transfected with these constructs for 24 h and were subsequently treated with FSH (10 ng/mL), the relative luciferase activity was assayed. g Live imaging of hepatic SREBP-2-luciferase activity in OVX + E2 mice injected with Ad-SREBP-2/luc via tail vein and then solvent (N.S.) or FSH (30 IU/kg) (n = 3 per group). Live imaging of luciferase activity was normalized to coinfected RSVβ-gal reporter activity in the liver. Bar graph is shown as the mean ± SEM. h HepG2 cells were incubated with FSH (10 ng/ml) for 12 h and then ChIP-qPCR analysis was performed using anti-FoxO1 antibody. The data were normalized to the corresponding IgG. i Western blotting results showing the protein level of FoxO1 in the cytoplasm and the nuclei in OVX mouse model (n = 9 per group). For all panels in this figure, data are represented as the mean ± SEM for no less than three independent experiments. N represents nuclear protein; P represents cytoplasmic protein. For b, generalized estimating equation (GEE) is used for statistical analysis. For a, c, e–h, One-way ANOVA or Student’s t-test is used for statistical analysis. *P < 0.05 and **P < 0.01; ns, not significant

Fig. 8

FSH activates hepatic cholesterol biosynthesis via the Gi2α/β-arrestin2/Akt pathway. a Representative immunoblotting of phosphorylated (p-) Akt, ERK, or JNK levels in response to FSH stimulation (10 ng/mL). b Representative immunoblotting of p-Akt, SREBP-2 and HMGCR in HepG2 cells treated with MK2206 (10 µM), a p-Akt specific inhibitor, prior to FSH (10 ng/mL) treatment. c Representative immunoblotting of p-Akt, HMGCR and SREBP-2 in HepG2 cells treated with LY294002 (10 µM), a PI3K inhibitor, prior to FSH (10 ng/mL) treatment. d The luciferase activity of HepG2 cells transfected with the hHMGCR/luc vector for 24 h and then treated with LY294002 (10 µM) followed by FSH (10 ng/mL) treatment. e qPCR of Gα proteins in HepG2 cells transfected with different siRNAs that suppress the corresponding Gα subunits. f siRNAs were used to suppress different Gα isoforms in HepG2 cells, the effect of FSH on HMGCR mRNA levels was subsequently analyzed. Negative siRNA was used as a negative control. g After the knockdown of Gi2α by siRNA in HepG2 cells, the protein levels of SREBP-2 and HMGCR were detected by Western blotting following treatment with FSH (10 ng/mL). Representative immunoblotting of Akt phosphorylation and HMGCR expression in ARRB1 (h) or ARRB2 (i) gene-knockdown HepG2 cell lines. For all panels in this figure, data are shown as the mean ± SEM for no less than three independent experiments. N represents nuclear protein; P represents cytoplasmic protein. One-way ANOVA or Student’s t-test is used for statistical analysis. **P < 0.01; ns, not significant

Fig. 9

The schematic model depicts the regulation process of de novo cholesterol biosynthesis by FSH in the liver. FSH couples with FSHR on the hepatocyte surface and then activates Gi2α, which promotes the process of Akt phosphorylation by PI3K. Moreover, ARRB2 (β-arrestin2) is involved in the regulation of Akt phosphorylation by FSH. The phosphorylated Akt decreases the transfer of FoxO1 from the cytoplasm into the nucleus, which reduces its inhibition of SREBP-2 transcription. As a gatekeeper of cholesterol biosynthesis, mature SREBP2 promotes the transcription and expression of HMGCR, the rate-limiting enzyme in cholesterol biosynthesis. In summary, elevated FSH induces hepatic de novo cholesterol biosynthesis