doi: 10.1101/gad.891301.
SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation
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- PMID:11358865
- PMCID: PMC313801
- DOI: 10.1101/gad.891301
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SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation
M Matsuda et al. Genes Dev..
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Abstract
In liver, the synthesis of cholesterol and fatty acids increases in response to cholesterol deprivation and insulin elevation, respectively. This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors (statins) and high calorie diets (insulin). In nonhepatic cells, lipid synthesis is controlled by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose active domains are released proteolytically to enter the nucleus and activate genes involved in the synthesis and uptake of cholesterol and fatty acids. SCAP (SREBP cleavage-activating protein) is a sterol-regulated escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of cleavage in the Golgi. Here, we produced a conditional deficiency of SCAP in mouse liver by genomic recombination mediated by inducible Cre recombinase. SCAP-deficient mice showed an 80% reduction in basal rates of cholesterol and fatty acid synthesis in liver, owing to decreases in mRNAs encoding multiple biosynthetic enzymes. Moreover, these mRNAs failed to increase normally in response to cholesterol deprivation produced by a cholesterol synthesis inhibitor and to insulin elevation produced by a fasting-refeeding protocol. These data provide in vivo evidence that SCAP and the SREBPs are required for hepatic lipid synthesis under basal and adaptive conditions.
Figures
Generation and characterization of a floxedSCAP allele. (A) Schematic of the sequence-replacement gene-targeting strategy. The map of the wild-type allele spans the SCAP promoter region (the sequence upstream of exon 1), exon 1, intron 1, and exon 2. The targeting vector containing theneo gene driven by thepgk-neo-pA cassette (phosphoglyceride kinase promoter followed by the 3′-untranslated region of the bovine growth hormone gene containing the polyadenylation signal) (Soriano et al. 1991) is flanked on each side by one copy of theloxP sequence as denoted by the solid arrows (Kuhn et al. 1995). The transcriptional direction of theneo gene is shown by the arrow in its box. The entire cassette was inserted 3 kb upstream of theSCAP gene. The downstreamloxP sequence was inserted within intron 1. Excision of the sequences between theloxP sites by the Cre recombinase deletes 3 kb of upstream sequence and exon 1, which includes the initiator methionine and residues encoding the first transmembrane domain of SCAP. The location of the probe used for Southern analysis (2-kbEcoRI–HindIII fragment) is denoted by a solid box. (B) Representative Southern blot analysis ofSpeI-digested genomic DNA from the livers of wild-type;MX1-Cre transgenic mice andSCAPf/f;MX1-Cre transgenic mice that were treated with three intraperitoneal injections of pIpC (250 μg/injection) as described in Materials and Methods. Genomic DNA from liver was prepared 14 d after the last injection. The positions of migration of the fragments derived from wild-type, targeted, and disrupted alleles are indicated.
Expression of mRNAs and proteins in livers of wild-type;MX1-Cre andSCAPf/f;MX1-Cre. The mice used in these experiments are described in Table 1. (A,B) Immunoblot analysis of SCAP, SREBP-1, and SREBP-2 in hepatic membranes and nuclear extracts. After treatment of mice with or without pIpC, the livers from the four groups in Table 1 were separately pooled, and aliquots of the membrane fraction (50 μg protein) (A andtop gel inB) and nuclear extract fraction (30 μg) (bottom gel inB) were subjected to 6% SDS-PAGE for membranes and 8% SDS-PAGE for nuclear extracts. Immunoblot analysis was performed with 5 μg/mL of rabbit anti-hamster SCAP IgG, rabbit anti-mouse SREBP-1 IgG, or rabbit anti-mouse SREBP-2 IgG as the primary antibody and 0.25 μg/mL horseradish peroxidase-coupled donkey anti-rabbit IgG as the secondary antibody. Filters were exposed to Reflection NEF496 film 15–30 sec at room temperature. (*) A non-specific band. (P and N) The precursor and cleaved nuclear forms of SREBPs, respectively. (C) Amounts of mRNAs for SREBP-1a, SREBP-1c, and SREBP-2, as measured by RNase protection assay. The cRNA probe for SREBP-1 generates protected fragments of 262 bp for SREBP-1a and 168 bp for SREBP-1c. Total RNA isolated from livers of mice (Table 1) was pooled, and 15-μg aliquots were subjected to the RNase protection assay as described in Materials and Methods. After RNase digestion, the protected fragments were separated by gel electrophoresis and exposed to film for 16 h at −80°C, and then quantified. The intensity of each band relative to lane1 is denoted above the band. (D) Amounts of various mRNAs in livers, as measured by Northern blot hybridization. Total RNA isolated from livers of mice (Table 1) was pooled, and 20-μg aliquots were subjected to electrophoresis and blot hybridization with the indicated32P-labeled cDNA probe. Filters were exposed to film with intensifying screens at −80°C for 8–24 h, and then quantified. The intensity of each band relative to lane1 is denoted below the band.
Synthesis of cholesterol and fatty acids in liver, adrenal gland, and small intestine in wild-type mice andSCAPf/f; MX1-Cre transgenic mice. Mice (five females per group; 7–9 wk of age) were injected three times intraperitoneally with pIpC (250 μg/injection) as described in Materials and Methods. Fourteen days after the last injection, each mouse was injected intraperitoneally with3H-labeled water (50 mCi in 0.25 mL of isotonic saline), and 1 h later the indicated tissue was removed for measurement of its content of3H-labeled fatty acids and digitonin-precipitable sterols as described in Materials and Methods. Bars, mean ±sem of the values from five mice.
Rates of lipid synthesis and secretion by primary hepatocytes from wild-type mice (□, ○) andSCAPf/f;MX1-Cre transgenic mice (▪, ●). Male mice (6 weeks of age) were injected intraperitoneally with pIpC four times (300 μg/injection) as described in Materials and Methods. Fourteen days after the last injection, primary hepatocytes were prepared as described in Materials and Methods. After a 2-h attachment period, the hepatocytes were incubated with 0.5 mM sodium14C-labeled acetate (50 dpm/pmole) in DMEM supplemented with 5% human lipoprotein-deficient serum. At the indicated time, the medium was removed, the monolayers were washed, and the cells were harvested. The content of14C-labeled cholesterol and fatty acids in cells and medium was quantified as described in Material and Methods. Each value is the average of duplicate incubations. Similar results were obtained in three other experiments.
Expression of mRNAs and proteins in livers of wild-type mice andSCAPf/f;MX1-Cre subjected to fasting and refeeding. The mice used in these experiments are described in Table 2. (A–C) Immunoblot analysis of SCAP, SREBP-1, and SREBP-2 in hepatic membranes and nuclear extracts. After treatment with pIpC, the wild-type andSCAPf/f;MXI-Cre mice were each divided into three groups. The nonfasted group was maintained ad lib, the fasted group was fasted for 24 h, and the refed group was fasted for 24 h and then refed for 12 h prior to study. The treatments were staggered so that all animals could be killed at the same time. Livers from each group were separately pooled, and aliquots of the membrane pellet (50 μg protein) for SCAP, SREBP-1, and SREBP-2 and aliquots of the nuclear extract (30 μg) for SREBPs were subjected to SDS-PAGE and immunoblot analysis as described in the legend to Fig. 2. Filters were exposed to film for 15–30 sec at room temperature. (D) Amounts of various mRNAs in livers, as measured by Northern blot hybridization. Aliquots of pooled total RNA (20 μg) isolated from livers of the indicated mice were subjected to electrophoresis and blot hybridization with the indicated32P-labeled cDNA probe. Filters were exposed to film with intensifying screens at −80°C for 8–24 h, and then quantified. The intensity of each band relative to lane1 is shown below the band.
Expression of mRNAs and proteins in wild-type;MX1-Cre mice andSCAPf/f;MX1-Cre mice fed a diet supplemented with 0.2% lovastatin plus 2% colestipol (L/C) for 10 d (A–C) or 0.2% lovastatin (L) alone for 4 d (D). Mice (9–10 wk of age) were injected intraperitoneally with pIpC four times (300 μg/injection). The dietary treatments were begun immediately after the fourth injection. (A,B) Immunoblot analysis. Livers from five mice in each group (2 males, 3 females) were pooled, and aliquots of the membrane fraction (50 μg protein for SCAP and SREBPs) and nuclear extracts (30 μg) were subjected to SDS-PAGE and immunoblot analysis as described in the legend to Fig. 2. Filters were exposed to film for 15–30 sec at room temperature. (*) A nonspecific band. (P and N) The precursor and cleaved nuclear forms of SREBPs. (C,D) Northern blot analysis. Livers from five mice in each group (2 males, 3 females) were pooled for preparation of total RNA, and 20-μg aliquots were subjected to electrophoresis and blot hybridization with the indicated32P-labeled cDNA probes as described in Materials and Methods. Filters were exposed to film with intensifying screens at −80°C for 0.5–12 h, and then quantified. The intensity of each band relative to lane1 is shown below the band.
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