Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation

Abstract

Stearoyl-coenzyme A desaturase (SCD) is the rate-limiting enzyme necessary for the biosynthesis of monounsaturated fatty acids. In this study, we investigated the regulation of mouse SCD1 by liver X receptor (LXR) and its role in plasma lipoprotein metabolism upon LXR activation. In vivo, the SCD1 gene remained induced upon LXR activation in the absence of sterol regulatory element-binding protein 1c (SREBP-1c), a known transcriptional regulator of SCD1. Serial deletion and point mutation analyses in reporter gene assays, as well as a gel mobility shift assay, identified an LXR response element in the mouse SCD1 promoter. In addition, SCD1 deficiency prevented the hypertriglyceridemic effect and reduced hepatic triglyceride accumulation associated with LXR activation despite induced hepatic expression of SREBP-1c protein and several SREBP1c and LXR target genes involved in lipoprotein metabolism. Unlike wild-type mice, SCD1-deficient mice failed to elevate the hepatic triglyceride monounsaturated acid (MUFA)/saturated fatty acid (SFA) ratio despite induction of the SCD2 gene. Together, these findings suggest that SCD1 plays a pivotal role in the regulation of hepatic and plasma triglyceride accumulation, possibly by modulating the MUFA-to-SFA ratio. In addition, SCD1 deficiency also increased plasma high-density lipoprotein cholesterol levels induced by LXR activation.

FIG. 1.

In vivo regulation of SCD1 independent of SREBP-1c. Wild-type and SREBP-1c^−/− mice (four to six mice/group) were fed a chow diet supplemented with either vehicle (DMSO) or 0.025% T0901317 for 2 days before sacrifice. (A) Nuclear and membrane proteins were isolated from the liver and subjected to immunoblot analysis with anti-SREBP1, which detects both SREBP-1a and -1c isoforms. pSREBP-1, precursor SREBP-1 protein. (B) Measurement of hepatic gene expression by real-time quantitative PCR and Northern blot analysis. (C) FPLC fractionation of plasma triglycerides. (D) Measurement of liver TG content. (E) The MUFA-to-SFA ratio was measured for hepatic triglycerides and total hepatic lipids. a, P < 0.05 compared with the control group; b, P < 0.05 compared with wild-type mice fed an LXR diet.

FIG. 2.

Mapping of LXR response element in the mouse SCD1 gene promoter. (A) Deletion analysis of the SCD1 promoter using a luciferase reporter gene assay. The schematic illustrations represent the serially deleted SCD1 luciferase reporter constructs. (B) Sequence comparison of the putative LXRE-DR4 in the promoter region of the SCD1 gene with the LXRE-DR4 in LXR target genes reported previously. (C) Mutational analysis of the SCD1 promoter pGL3/−1537+155mSCD1. The asterisk indicates the mutation made in the LXRE of the SCD1 gene promoter. (D) Mutational analysis of pGL3p/−1537−563mSCD1. Control cells were treated with 0.1% DMSO in the absence of LXR-RXR receptors. Results are expressed as the relative luciferase units (RLU) of induction (n-fold) over the control value for each construct and represent the means ± SE of three independent transfection experiments of duplicates.

FIG. 3.

LXR-RXR complex specifically binds to the mouse SCD1 DR4 element. Electrophoretic mobility shift assays were performed using ³²P-radiolabeled double-stranded oligonucleotides corresponding to either the LXR response element of the mouse SCD1 promoter (SCD1 WT LXRE) or the LXR response element of the mouse SREBP-1c promoter (SREBP-1c LXRE). Competitive assays were performed using a 50-fold molar excess of unlabeled oligonucleotides corresponding to the LXR response element of the mouse SREBP-1c promoter (A), the wild-type LXR response element of the SCD1 promoter (B), or the mutant LXR response element of the SCD1 promoter (C) as indicated. The asterisks indicate the specific base pairs mutated in the LXR response element of the SCD1 promoter.

FIG. 4.

Hepatic response to T0901317 treatment in wild-type and SCD1^−/− mice. Mice (four mice/group) were fed a chow diet supplemented with either vehicle or 0.025% T0901317 for 2 days. The total content of triglyceride (A) and the fatty acid composition in hepatic triglyceride fractions (B) in the liver of wild-type and SCD1^−/− mice were determined. (C) MUFA-to-SFA ratios in hepatic triglycerides. (D) Aliquots of microsome fractions (100 μg) from the liver of each mouse were subjected to SCD enzyme activity analysis. (E) SCD2 mRNA expression levels were determined by real-time quantitative PCR and normalized to those of cyclophilin. Each value represents the mean ± SE. a, P < 0.01 compared with the control group; b, P < 0.05 compared with the control group; c, P < 0.01 compared with wild-type mice fed an LXR diet.

FIG. 5.

Plasma lipid levels after LXR agonist T0901317 treatment for 2 days. Wild-type and SCD1^−/− mice were fed a chow diet supplemented with either vehicle or 0.025% T0901317 for 2 days and fasted for 4 h before sacrifice. Plasma triglyceride (A) and cholesterol (B) levels were measured. FPLC fractionation of triglycerides (C) and cholesterol (D). Each data point in the lipoprotein profile represents the total triglyceride or cholesterol mass per fraction. The lipoprotein peaks for VLDL and HDL are indicated.

FIG. 6.

Analysis of SREBP-1 protein, hepatic gene expressions, and the PXR pathway. Mice (four mice/group) were fed a chow diet supplemented with either vehicle or 0.025% T0901317 for 2 days. (A) Nuclear and membrane proteins were isolated from the liver and subjected to immunoblot analysis with antibody against mouse SREBP1 (upper panels). Ponceau S staining of the membrane used for immunodetection served as a loading control (lower panel). pSREBP-1, precursor SREBP-1 protein. (B) Measurement of various hepatic genes involved in lipid regulation by real-time quantitative PCR. (C) Measurement of PXR target genes under T0901317 treatment. (D) Measurement of hepatic gene expressions upon PCN treatment in wild-type mice. The expression levels of various genes were normalized to those of cyclophilin, with mean expression of the control wild-type vehicle group normalized to 1. Each value represents the mean ± standard deviation. a, P < 0.01 compared with the control group; b, P < 0.001 compared with wild-type mice fed an LXR diet.