FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c

Abstract

Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

FIGURE 1.

FoxO1 reduces hepatic expression of SREBP-1c in transgenic mice. A, effect of constitutively active FoxO1 (CA-FoxO1) on hepatic SREBP-1c gene expression in response to fasting and refeeding. Wild-type (WT, open bars) and transgenic mice expressing constitutively active FoxO1 (CA-FoxO1, solid bars) in the liver were fasted for 18 h and sacrificed 6 h after refeeding or continued fasting. Total RNA was prepared and mRNA levels of SREBP-1c were analyzed by qRT-PCR and results were expressed relative to levels in fasting WT mice (left panel). The right panel shows the relative abundance of SREBP-1c mRNA following refeeding versus fasting levels (refed-fasting) for each genotype. *, signifies p < 0.05, CA-FoxO1 versus WT. B, effect of liver-specific FoxO knock-out on hepatic SREBP-1c gene expression. Alleles for FoxO1, FoxO3a, and FoxO4 were disrupted in the liver using Cre-lox technology (FoxO KO, black bar), and Cre-negative littermates where floxed alleles were not disrupted served as controls (WT, open bar). Left panel, total hepatic RNA was prepared after an 18-h fast and SREBP-1c transcripts were quantified by qRT-PCR. Results were expressed relative to SREBP-1c mRNA levels in fasting or wild-type mice. *, signifies p < 0.05, CA-FoxO1 versus WT. Right panel, total cell lysates were prepared from FoxO knock-out mice and littermates and analyzed by Western blotting with the high-mobility group protein-1 (HMGB1) as loading control.

FIGURE 2.

CA-FoxO1 inhibits SREBP-1c expression in rat hepatocytes. A, effect of CA-FoxO1 on mRNA levels of SREBP-1c and fatty acid synthase. Rat hepatocytes were infected with adenovirus expressing GFP alone (Ad-GFP) or a constitutively active form of FoxO1 (Ad-CAFoxO1) and then treated with 100 nm insulin for 24 h. Total RNA was prepared and the abundance of transcripts for SREBP-1c, fatty acid (FA) synthase, and IGFBP-1 was analyzed by qRT-PCR. IGFBP-1 was included as a positive control for CA-FoxO1 action. Data for RT-PCR are the mean ± S.E. of three to six independent experiments. B, Ad-CAFoxO1 increased nuclear content of FoxO1. Rat hepatocytes were infected with Ad-GFP or Ad-CAFoxO1. Nuclear extracts were analyzed for FoxO1 protein by Western blot analysis. C, Ad-CAFoxO1-reduced SREBP-1c protein. Rat hepatocytes were treated in the same manner as described in B, and cytoplasmic and nuclear proteins were prepared for Western blot analysis of SREBP-1 protein levels. Levels of both the full-length precursor and N-terminal fragment of SREBP-1c (nuclear SREBP-1c) were measured in the cytoplasmic and nuclear fractions, respectively. Western blots are representative of three independent experiments and the numbers above the bands are average levels of relative expression of the proteins quantified by densitometry.

FIGURE 3.

FoxO1 reduces the stimulation of the SREBP-1c promoter activity by insulin. A, CA-FoxO1 reduces activation of the SREBP-1c promoter by insulin. Rat hepatocytes were co-transfected with the SREBP-1c reporter plasmid pSREBP-1c (−1516/+40)-luc and either the CA-FoxO1 expression plasmid or empty vector 24 h prior to treatment with (solid bars) or without (open bars) insulin (100 nm). Cell lysates were prepared 24 h later for analysis of luciferase activity. Cells were transfected with Renilla luciferase to control for transfection efficiency. B, nuclear content of endogenous FoxO1 but not CA-FoxO1 is regulated by insulin. Hepatocytes were co-transfected with the CA-FoxO1 expression plasmid or empty vector 24 h before treatment with or without insulin (100 nm), and nuclear extracts were prepared 12 h later for analysis of FoxO1 protein by Western blot. C, SREBP-1c promoter response to insulin is enhanced by suppression of endogenous FoxO1. Hepatocytes were co-transfected overnight with pSREBP-1c (−1516/+40)-luc and either scrambled or FoxO1-specific small RNA oligos (RNAi-Sc and RNAi-FoxO1, respectively) and then treated with or without insulin for 24 h. Cell extracts were assayed for luciferase activities. D, RNAi-FoxO1 reduced FoxO1 protein expression. Hepatocytes were treated in the same manner as that for C. Whole cell lysates were prepared and analyzed by Western blotting. A representative blot of three independent experiments is shown. *, signifies p < 0.05 insulin-treated cells, RNAi-Sc versus RNAi-FoxO1. Data in panels A and C are presented as the mean ± S.E. from three hepatocyte preparations each with triplicate treatments. Numbers above the bands in B and D are average relative levels of proteins quantified by densitometry.

FIGURE 4.

CA-FoxO1 is associated with the SREBP-1c promoter and reduces promoter occupancy by SREBP-1c, and RNA-polymerase II. A, reduced binding of SREBP-1 to the proximal SREBP-1c promoter in liver of CA-FoxO1 overexpressing transgenic mice as determined by ChIP assay. Freshly isolated nuclei from livers of wild type (WT) and transgenic (TGN) mice were cross-linked with formaldehyde, and sheared chromatin was immunoprecipitated with specific antibodies against SREBP-1c, LXRα, CBP, or IgG. The occupancy of the SREBP-1c promoter by nuclear proteins was demonstrated by the amplification with primers targeting the proximal SREBP-1c promoter. B, CA-FoxO1 reduces binding of SREBP-1c and LXRα to the proximal SREBP-1c promoter as determined by ChIP assay. Rat hepatocytes were infected with adenovirus expressing constitutively active FoxO1 (Ad-CAFoxO1) or control adenovirus (Ad-GFP). After incubation for 24 h, hepatocytes were treated with or without insulin (100 nm) for 6 h. Chromatin was prepared and immunoprecipitated with specific antibodies against SREBP-1c, LXRα, RNA polymerase, histone 3, or IgG. Occupancy of the SREBP-1c promoter by nuclear proteins was determined by amplification with primers targeting the proximal SREBP-1c promoter. C, effect of CA-FoxO1 and insulin on SREBP-1c, LXRα, and RNA polymerase II protein levels in primary hepatocyte cultures. Hepatocytes were infected with adenoviral vectors and treated with/without insulin as above before preparation of nuclear protein extracts for analysis by Western blotting. One hundred micrograms of nuclear protein was resolved by SDS-PAGE and transferred for Western blot. Representative blots from three independent experiments are shown. D, recruitment of endogenous FoxO1 and CA-FoxO1 to the SREBP-1c and IGFBP-1 promoters. Hepatocytes were infected with Ad-CA-FoxO1 or control adenovirus and treated with or without insulin as described in panel A and chromatin was precipitated using FoxO1 specific antibody or IgG. Precipitated DNA fragments were PCR amplified as described under “Experimental Procedures” using specific primers directed against the proximal IGFBP-1 or SREBP-1c promoter as indicated. Numbers above bands in A, B, and D are the average relative signal intensities, and the numbers in C represent the average relative levels of proteins, all quantified by densitometry.

FIGURE 5.

CA-FoxO1 does not alter response of SREBP-1c to the LXR agonist TO901317. A, effect of T0901317 on SREBP-1c expression in transgenic mice expressing CA-FoxO1. Wild-type (WT) and transgenic mice expressing CA-FoxO1 in the liver were treated with 40 mg/kg of LXR agonist (T090137) or vehicle (control) by gavage every 24 h for 2 days and sacrificed 4 h after the second dose in the nonfasted state. SREBP-1c (left panel) and IGFBP-1 (right panel) mRNA levels in livers were determined by qRT-PCR and expressed relative to levels in vehicle-treated WT mice. Results shown are mean ± S.E. from 4 animals in each group. B, CA-FoxO1 suppresses SREBP-1c promoter activity. Hepatocytes were co-transfected with luciferase reporter constructs containing either the wild-type (−1516/+40) SREBP-1c promoter (pSREBP-1cWT-luc) or the corresponding construct where both LXREs in the proximal SREBP-1c promoter were mutated (pSREBP-1cMutLXRE-luc), together with the CA-FoxO1 expression vector or empty vector. Cells were treated with 10 μm TO901317 or carrier alone for 24 h before cell lysates were prepared for analysis of luciferase activity. Data are presented as mean ± S.E. from three independent experiments each performed in triplicate.

FIGURE 6.

FoxO1 inhibits SREBP-1c through Sp1 and SREBP-1c. A, CA-FoxO1 inhibits full-length and truncated SREBP-1c promoter. Hepatocytes were transfected with serial deletions of the SREBP-1c promoter and empty pFlagCMV2 vector or a plasmid expressing CA-FoxO1. Thirty-six hours post-transfection, cell lysates were assayed for luciferase expression. B, CA-FoxO1 suppresses wild-type and mutant SREBP-1c promoters. Rat hepatocytes were transfected with the wild-type (WT) SREBP-1c promoter construct (pSREBP-1c (−1516/+40)-luc) or with mutant constructs in which the indicated transcription factor binding sites were disrupted, with or without plasmid expressing CA-FoxO1. Luciferase analyses were performed as described in A. The data are presented as percent inhibition of SREBP-c promoter activity by CA-FoxO1.

FIGURE 7.

Inhibitory effects of FoxO1 on Sp1 and SREBP-1c. A, CA-FoxO1 does not reduce the response of Gal4-LXR to the LXR agonist TO9. Hepatocytes were co-transfected with pGal4-luc composed of four Gal4-binding sites fused to a minimal TK promoter along with plasmid expressing the Gal4 DNA binding alone (Gal4DBD) or a chimeric protein containing the Gal4 DNA-binding domain in-frame with the ligand binding and transactivation domains of human LXRα (Gal4LXRα), plus either the CA-FoxO1 expression vector or empty vector. Luciferase activity was quantified in cell extracts following 24 h treatment with/without TO901317. Data are presented as mean ± S.E. from three independent experiments each performed in triplicate. B, CA-FoxO1 inhibits the ability of Sp1 to activate a GC box-driven promoter. Hepatocytes were co-transfected with a luciferase reporter construct driven by six GC-box elements and an Sp1-expressing plasmid, in the presence or absence of CA-FoxO1. Cell extracts were assayed for luciferase expression 36 h post-transfection. C, CA-FoxO1 reduces transactivating capacity of Gal4-SREBP-1c. Hepatocytes were co-transfected with the pGal4-luc reporter gene construct, composed of four Gal4-binding sites fused to a minimal TK promoter (inset) together with plasmid vectors expressing the Gal4 DNA binding alone (Gal4DBD) or a chimeric protein containing the Gal4 DNA-binding domain in-frame with the transactivation domain of rat SREBP-1c (amino acids 1–300) (Gal4SREBP1c), plus either the CA-FoxO1 expression vector or empty vector. Luciferase activity was quantified in cell extracts following 36 h post-transfection. Data in A–C are presented as mean ± S.E. from 3–7 independent experiments done in triplicate.