Expression of SREBP-1c Requires SREBP-2-mediated Generation of a Sterol Ligand for LXR in Livers of Mice

Abstract

The synthesis of cholesterol and fatty acids (FA) in the liver is independently regulated by SREBP-2 and SREBP-1c, respectively. Here, we genetically deleted Srebf-2 from hepatocytes and confirmed that SREBP-2 regulates all genes involved in cholesterol biosynthesis, the LDL receptor, and PCSK9; a secreted protein that degrades LDL receptors in the liver. Surprisingly, we found that elimination of Srebf-2 in hepatocytes of mice also markedly reduced SREBP-1c and the expression of all genes involved in FA and triglyceride synthesis that are normally regulated by SREBP-1c. The nuclear receptor LXR is necessary for Srebf-1c transcription. The deletion of Srebf-2 and subsequent lower sterol synthesis in hepatocytes eliminated the production of an endogenous sterol ligand required for LXR activity and SREBP-1c expression. These studies demonstrate that cholesterol and FA synthesis in hepatocytes are coupled and that flux through the cholesterol biosynthetic pathway is required for the maximal SREBP-1c expression and high rates of FA synthesis.

Keywords: LXR; SREBP; cholesterol; fatty acids; human biology; medicine; mouse.

Figure 1.. Gene-targeting strategy and characterization of the floxed Srebf-2 allele.

(A) Schematic of gene-targeting strategy. Cre-mediated excision of the sequences flanked by the loxP sites deletes 660 bp of the Srebf-2 promoter and exon 1, which includes the initiator methionine and residues encoding the NH₂-terminal domain of Srebf-2. The positions of primers (P1 and P2, P3 and P4) used for PCR detection of homologous recombination are denoted by arrowheads. (B) Genotype analysis of the conditionally targeted Srebf-2 mice by PCR of tail-derived DNA. (C) Levels of proteins in the livers of WT and hepatocyte-Srebf-2^-/- mice. Nuclear and membrane protein was made from each mouse liver described in Table 1 and equal aliquots from each were pooled (total, 30 µg) and subjected to SDS-PAGE and immunoblot analysis was carried out for the indicated protein as described in ‘Materials and methods.’ The precursor and nuclear form of SREBPs were denoted as P and N, respectively. DOI: http://dx.doi.org/10.7554/eLife.25015.002

Figure 2.. Levels of mRNAs in livers of WT and hepatocyte-Srebf-2^-/- mice.

Total RNA from livers of each mouse liver described in Table 1 was subjected to real-time RT-PCR as described in ‘Materials and methods.’ Apo B was used as the invariant control. Values represent the amount of mRNA relative to those in the wild-type mice, which are arbitrarily assigned a value of 1. (A) Genes involved in cholesterol homeostasis. (B) Genes involved in FA homeostasis. DOI: http://dx.doi.org/10.7554/eLife.25015.004

Figure 3.. In vivo sterol and FA synthesis rates in livers of WT and hepatocyte-Srebf-2^-/- mice.

Six 4-month-old male WT and hepatocyte-Srebf-2^-/- mice were injected intraperitoneally with 50 mCi ³H-labeled water and rates of hepatic sterol and FA synthesis were determined as described in ‘Materials and methods'. DOI: http://dx.doi.org/10.7554/eLife.25015.005

Figure 4.. Levels of mRNAs and proteins in the livers of WT and hepatocyte-Srebf-2^-/-mice fed chow diet supplemented with an LXR agonist.

Mice 7–11 weeks of age were fed ad libitum chow or chow supplemented with 25 mg/kg of a LXR agonist (T901317) for three weeks prior to study. (A) Liver membrane and nuclear extract protein was made from each mouse and equal aliquots were pooled (total, 30 µg) and subjected to SDS-PAGE and immunoblot analysis as described in ‘Materials and methods.’ The precursor and nuclear form of SREBPs are denoted as P and N, respectively. (B) Total RNA from each mouse liver was subjected to real-time RT-PCR as described in ‘Materials and methods.’ Apo B was used as the invariant control. Values represent the amount of mRNA relative to those in the WT mice, which are arbitrarily assigned a value of 1. The following figure supplements are available for Figure 4. DOI: http://dx.doi.org/10.7554/eLife.25015.006

Figure 4 —figure supplement 1.. Liver mRNAs levels of WT and hepatocyte-Scap^-/- mice fed chow diet supplemented with an LXR agonist.

DOI: http://dx.doi.org/10.7554/eLife.25015.007

Figure 5.. Liver lipid concentrations, mRNA, and protein levels in WT and hepatocyte-Srebf-2^-/- mice fed chow or chow supplemented with cholesterol.

Mice 7–11 weeks of age were fed chow (n = 6–7) or chow supplemented with 0.2% cholesterol (n = 6–7) for six weeks prior to study. (A) Liver cholesterol and TG concentrations were measured as described in ‘Materials and methods.’ (B) Equal aliquots of nuclear and membrane protein from each mouse liver were pooled (total, 30 µg) and subjected to SDS-PAGE and immunoblot analysis for the indicated protein as described in ‘Materials and methods.’ The precursor and nuclear form of SREBPs were denoted as P and N, respectively. (C) Total RNA from the livers of each mouse was subjected to real-time RT-PCR as described in ‘Materials and methods.’ Apo B was used as the invariant control. Values represent the amount of mRNA relative to those in WT mice, which are arbitrarily assigned a value of 1. * denotes a level of statistical significance of p<0.05 (Student’s t test) between WT and hepatic-Srebf-2^-/-mice, ND denotes no significant difference between the indicated groups. DOI: http://dx.doi.org/10.7554/eLife.25015.008

Figure 6.. In vivo VLDL secretion and LDL clearance in WT and hepatocyte-Srebf-2^-/- mice.

(A) Eleven male mice (8 weeks of age) of each genotype were subjected to i.v. injection of ¹²⁵I-labeled LDL (15 µg of protein, 496 cpm/ng protein). Blood was obtained at 30 s (time 0) and 10, 30, 60, 120, and 240 min for the quantification of plasma content of ¹²⁵I-labeled apoB. Data were plotted as the percentage of 0 time value. (B) Five male mice (8 wks of age) of each genotype were fasted for 4 hr prior to the study. Each mouse was injected i.v. with 10% triton-saline solution at 500 mg/kg. Plasma TG accumulation of each mouse at 0, 0.5, 1, and 2 hr after the triton injection were measured. (C) Plasma TG secretion rate during a detergent block of lipolysis was calculated for each mouse from the linear regression analysis of the time vs. TG concentration. DOI: http://dx.doi.org/10.7554/eLife.25015.011