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Published as doi: 10.1096/fj.06-6368fje.
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(The FASEB Journal. 2006;20:2642-2644.)
© 2006 FASEB

Regulation of hepatic cholesterol synthesis by a novel protein (SPF) that accelerates cholesterol biosynthesis

Norihito Shibata*,{dagger}, Kou-ichi Jishage{ddagger}, Makoto Arita*, Miho Watanabe{ddagger}, Yosuke Kawase{ddagger}, Kiyotaka Nishikawa§, Yasuhiro Natori§, Hiroyasu Inoue||, Hitoshi Shimano, Nobuhiro Yamada, Masafumi Tsujimoto{dagger} and Hiroyuki Arai*,1

* Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan;

{dagger} Laboratory of Cellular Biochemistry, Riken, Saitama, Japan;

{ddagger} Pharmacology and Pathology Research Center, Chugai Research Institute For Medical Science, Inc., Shizuoka, Japan;

§ Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, Tokyo, Japan;

|| Department of Food Science and Nutrition, Faculty of Human Life and Environment, Nara Women’s University, Nara, Japan; and

Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan

1Correspondence: Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7–3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: harai{at}mol.f.u-tokyo.ac.jp

ABSTRACT

Supernatant protein factor (SPF) is a novel cholesterol biosynthesis-accelerating protein expressed in liver and small intestine. Here, we report on the physiological role of SPF by using Spf-deficient mice. Although plasma cholesterol levels were similar in chow-fed Spf–/– and wild-type (WT) mice, fasting significantly decreased plasma cholesterol levels in Spf–/– mice but not in WT mice. While fasting reduced hepatic cholesterol synthesis rate in WT mice, a more pronounced reduction was observed in Spf–/– mice. The expression of cholesterogenic enzymes was dramatically suppressed by fasting both in WT and Spf–/– mice. In contrast, hepatic SPF expression of WT mice was up-regulated by fasting in peroxisome proliferator-activated receptor {alpha} (PPAR-{alpha})-dependent manner. These results indicate that in WT mice, the decrease of hepatic cholesterol synthesis under fasting conditions is at least in part compensated by SPF up-regulation. Fibrates, which function as a PPAR-{alpha} agonist and are widely used as hypotriglycemic drugs, reduced hepatic cholesterol synthesis and plasma cholesterol levels by approximately one-half in Spf–/– mice but not in WT mice. These findings suggest that co-administration of fibrates and an SPF inhibitor may reduce not only plasma triglyceride but also cholesterol levels, indicating that SPF is a promising hypocholesterolemic drug target.—Shibata, N., Jishage, K.-i., Arita, M., Watanabe, M., Kawase, Y., Nishikawa, K., Natori, Y., Inoue, H., Shimano, H., Yamada, N., Tsujimoto, M., Arai, H. Regulation of hepatic cholesterol synthesis by a novel protein (SPF) that accelerates cholesterol biosynthesis


Key Words: squalene monooxygenase • peroxisome proliferator-activated receptor {alpha} • fasting • fibrate

PROLONGED FOOD DEPRIVATION induces dramatic changes in mammalian lipid metabolism. Under these conditions, fatty acid metabolism in the liver changes to allow sufficient energy production (1) . Starvation decreases the synthesis of fatty acids and increases the oxidation of fatty acids released from adipose tissue.

Cholesterol is required for a wide range of cellular functions in mammalian cells. It is a major lipid component of the plasma membrane, functions as a precursor molecule for bile acids, and is necessary for covalent modifications of embryonic signaling proteins (2 , 3) . Therefore, mammals are forced to continuously adjust their sterol content by regulating their hepatic cholesterol metabolism (4) . However, much is not known about the changes in hepatic cholesterol metabolism under fasting conditions. Previous studies have demonstrated that fasting causes a dramatic reduction in the mRNA levels of hepatic cholesterogenic enzymes (5 , 6) . However, hepatic cholesterol synthesis is decreased only ~50% (7) , and plasma cholesterol levels are hardly affected by fasting (5 , 8) . This suggests the presence of a compensatory mechanism to maintain cholesterol levels even under extreme conditions such as fasting.

In 1957, Bloch and colleagues identified a soluble factor from rat liver cytosol termed "supernatant protein factor (SPF)", which promotes the activity of squalene monooxygenase, a rate-limiting enzyme in the late stages of cholesterol biosynthesis (9 , 10) . Half a century after its discovery, we succeeded in purifying this protein and isolating its cDNA (11) . Recombinant SPF produced in E. coli enhances microsomal squalene monooxygenase activity and also promotes intermembrane transfer of squalene in vitro, which led to the hypothesis that SPF facilitates the access of a hydrophobic substrate (squalene) to a specific enzyme site (11 , 12) . SPF is abundantly expressed in the liver and intestine of rats, mice, and humans, but is undetectable in cultured cells, including hepatoma cell lines. These data suggest that SPF is not necessarily a prerequisite for cholesterol biosynthesis but rather plays a role in certain specific organs in mammals (11) . By use of the SPF-deficient mouse model, we demonstrate here that SPF plays a role in hepatic cholesterol synthesis under fasting conditions. Moreover, we show that fibrates, which are widely used hypotriglycemic drugs, also reduce plasma cholesterol levels in Spf–/– mice, implicating SPF as a promising hypocholesterolemic drug target.

MATERIALS AND METHODS

Generation of SPF-null mice
The SPF gene was isolated from the 129/Sv mouse genomic DNA {lambda} FIX II library (Stratagene, La Jolla, CA, USA) using mouse cDNA as a probe. A replacement-type targeting vector was constructed; the long arm containing a 9.7 kb SmaI/SacII fragment spanning exons 1 and 2, a 1.3 kb SalI/BamHI fragment of the PGK-neo cassette, and the short arm containing a 1.0 kb BamHI/SalI fragment in intron 5 were inserted into the SmaI/SalI sites of the vector pMCDT-A (A+T/pau) (13) . AB2.2-Prime ES cells (Lexicon Genetics, Woodlands, TX, USA) were transfected by electroporation with a linearized targeting vector. G418/gancyclovir-resistant clones were screened by polymerase chain reaction (PCR) using forward and reverse primers, 5'-TCG CCT TCT ATC GCC TTC TTG ACG-3' and 5'-AGA AGA CAA TTT GGG GAG CTA GC-3', respectively. Targeted embryonic stem clones were injected into C57BL/6J (CLEA Japan, Tokyo, Japan) blastocysts yielding two lines of chimeric mice, which transmitted the disrupted allele through the germ line.

Animal studies
Seven- to eight-week-old male C57BL/6J mice, Ppar-{alpha}–/– mice (129S4/SvJae-Pparatm1Gonz/J; The Jackson Laboratory, ME, USA) and Spf–/– mice were housed in colony cages under a 12-h light/dark cycle and maintained on standard chow (NMF diet from Oriental Yeast Co., Tokyo, Japan) that contained 0.12% (w/w) cholesterol. For fasting experiments, mice consumed standard chow ad libitum or fasted for 48 h. For the fibrate experiments, mice consumed CR-LPF diet (Oriental Yeast Co.) or CR-LPF diet supplemented with 0.2% fenofibrate (Sigma, St. Louis, MO, USA) or 0.2% ciprofibrate (Sigma) for 6 d. To test the dose-response study, mice were fed with CR-LPF diet supplemented with fenofibrate at the indicated dose for 6 d. For the cholesterol experiments, mice were fed a cholesterol-deficient diet (CR-LPF diet) that contained 0.029% (w/w) cholesterol or a cholesterol-supplemented diet [CR-LPF diet supplemented with 2% cholesterol (Sigma)] for 6 d. In all experiments, mice were anesthetized by intraperitoneal (i.p.) injection of pentobarbital sodium (Nembutal, Abbot, North Chicago, IL, USA), and blood and liver were obtained on the days indicated during the early phase of the dark cycle. The experiments were performed in accordance with institutional guidelines for animal experiments at the University of Tokyo.

Preparation of mouse anti-SPF specific monoclonal antibodies
Mouse anti-SPF specific monoclonal antibodies were established according to the method described by Kaempf-Rotzoll et al. (14) . Briefly, the coding region of mouse SPF cDNA was inserted into the BamHI/SalI sites of the pET21a vector (Novagen, Madison, WI, USA). After the plasmid was introduced into E. coli strain BL21 (DE3) (Novagen), the protein was expressed as a His-tagged protein by induction with 1 mM isopropyl-ß-D-thiogalactopyranoside. The protein was purified using nickel column chromatography (Novagen) according to the manufacturer’s protocol. Three rats (WKY strains, female, 8 wk; SLC, Hamamatsu, Japan) were immunized by injecting the protein into the hind foot pads using Freund complex adjuvant. At 3-week intervals after the initial injection, the rats were injected twice with the purified protein mixed with Freund complex adjuvant. One week after the last booster injection, the two enlarged medial iliac lymph nodes from each rat were used for cell fusion with mouse myeloma cells, line PAI. Several monoclonal antibody (mAb)-producing hybridoma cell lines were established and selected after checking the produced antibodies by ELISA, Western blot analysis screening of mouse liver cytosolic fraction. Only the clones highly positive in those screening methods were selected. In this study, the mAb from clone H3–7B (rat IgG2a) was used for Western blot analysis.

Western blot analysis
Mouse livers were homogenized in 2.5 volumes (w/v) of SET buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) and centrifuged at 100,000 g for 1 h at 4°C. The resultant supernatants were used as the hepatic cytosolic fraction. The hepatic microsomal fraction was prepared as described previously (15) . Each sample (20 µg) was separated by SDS-PAGE and transferred to a polyvinylidine difluoride membrane. The membranes were incubated with anti-SPF, anti-{alpha}-tocopherol transfer protein ({alpha}-TTP), anti-HMG-coenzyme A reductase, or anticalnexin antibody (Ab). Mouse anti-{alpha}-TTP specific monoclonal antibodies were previously established in our laboratory (16) . The anti-HMG-coenzyme A reductase mAb (15) was a kind gift from Dr. Tatsuhiko Kodama. The anticalnexin mAb was purchased from Transduction Laboratories (Lexington, KY, USA).

SPF activity assay
Conversion of [14C] squalene to [14C] squalene 2,3-oxide was used to measure SPF activity from individual liver, as described previously (10 , 11) . Briefly, [14C] squalene (20,000 dpm/40 nmol, American Radiolabeled Chemicals, St. Louis, MO, USA) and 50 µg of Tween 80 in 50 µl of acetone were mixed, and the solvents were evaporated under nitrogen. Along with substrate and detergent, the reactions contained, in a vol of 1 ml, 0.1 M Tris-HCl (pH 7.3), 1 mM EDTA, 0.3 mM Amo-1618, an inhibitor of lanosterol synthase (Calbiochem, San Diego, CA, USA), 0.01 mM flavin adenine dinucleotide (FAD) (Sigma), 0.1 mg phosphatidylglycerol (Avanti Polar Lipids, Inc., Alabaster, AL, USA), 1 mM NADPH (Sigma), and 1.28 mg of hepatic microsomal and cytosolic protein. Mixtures were incubated at 37°C for 30 min, and products were saponified by 500 µl of 10% KOH in methanol. Lipids were extracted with petroleum ether, evaporated under nitrogen, and subjected to thin-layer chromatography on silica gel plate. The plate was developed with 0.5% ethyl acetate in benzene. After development, plates were exposed and analyzed using a bioimage analyzer (Fuji Photo Film Co., Tokyo, Japan).

Quantitative real-time PCR
Total RNA was prepared from liver with Isogen (Nippongene, Osaka, Japan). First-strand cDNA was synthesized from 1 µg total RNA with oligo-dT primer by using a SuperScript First-Strand Synthesis System (Invitrogen). Real-time PCR reactions were performed on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using 2 x SYBR Green PCR Master Mix. Mouse 36B4 mRNA was used as an invariant control for fasting experiment. Mouse ß-actin mRNA was used as an invariant control for other experiments. PCR primers used were as follows (5' to 3'): ß-Actin, ATG AAG ATC AAG ATC ATT GCT CCT C and ACA TCT GCT GGA AGG TGG ACA; 36B4, GCT CGA CAT CAC AGA GCA GGandCCG AGG CAA CAG TTG GGT AC; SPF, TTC CGG AAG CAA AAG GAA CAT and GCC TGA CAG ATA CTG TTG GAT CAC; HMG-coenzyme A synthase, CTT GCT TTG CTC GTT CTT CT and TCG GTC ACC GGT TCC TCC TTC A; HMG-coenzyme A reductase, TGG AAT TAT GAG TGC CCC AAA and CCG CGT TAT CGT CAG GAT GAT G; Squalene monooxygenase, AAG AAA GAA CAG CTG GAG TCC AA and GTC ACG AAC GAG GTC GAC ACT.

Plasma cholesterol, triglyceride, and glucose measurements
Blood was obtained from the tail vein and collected into a tube containing 2 mM EDTA, 0.2% NaN3, 0.77% gentamicin, 1 mM PMSF, and 1 mM benzamidine (final concentrations). Total cholesterol, triglyceride, and glucose concentrations were determined using enzymatic assay kits (Wako, Osaka, Japan).

In vivo rates of hepatic cholesterol synthesis
The in vivo rate of hepatic cholesterol synthesis was measured as described (17) . Briefly, mice were given an i.p. injection with 40 mCi of [3H] water (100 mCi/mmol, MP Biomedicals, Inc, CA, USA) and were anesthetized and exsanguinated after 1 h. Aliquots of liver were saponified and their content of radiolabeled digitonin-precipitable sterols (DPS) was measured (18 19 20) . The rate of sterol synthesis was expressed as the nmol of [3H] water incorporated into DPS per hour per gram liver. The rate of incorporation of [3H] water into sterols by the liver was also converted to an equivalent mg quantity of newly synthesized cholesterol assuming that 0.69 3H atoms were incorporated into the sterol molecule per carbon atom entering the biosynthetic pathway as acetyl-coenzyme A.

VLDL-cholesterol secretion
Mice were injected intravenously (i.v.) with 20 mg of Triton WR1339 (Sigma) in 100 µl of PBS. Three hours after injection, plasma samples were prepared as described above. Pooled plasma samples (120 µl) were subjected to fast protein liquid chromatography (FPLC) gel filtration on a Superose 6 column (Amersham Pharmacia Biotech, Uppsala, Sweden) as described previously (21) . Each fraction was collected in 375 µl, and the cholesterol concentrations were determined as described above.

HMG-coenzyme A reductase activity assay
Mouse livers were homogenized in a buffer containing 15 mM nicotinamide, 2 mM MgCl2, and 100 mM potassium phosphate, pH 7.4, and centrifuged at 10,000 g for 20 min at 4°C. The supernatants were centrifuged at 100,000 g for 1 h at 4°C. The resultant pellets, comprising a microsomal fraction, were washed and resuspended in the same buffer. HMG-coenzyme A reductase activities were measured essentially as described previously (22) .

Statistics
Statistical analyses were performed with Student’s t test setting the significance at P < 0.05.

RESULTS

Generation of SPF-null mice
We generated mice lacking the Spf gene by targeted deletion leading to undetectable Spf mRNA (data not shown) and protein expression in these animals (Fig. 1 A). As shown previously in rats (9 10 11 12) , the microsomal squalene monooxygenase activity was enhanced by adding the hepatic cytosolic fraction in WT mice, whereas the hepatic cytosolic fraction obtained from Spf–/– mice showed substantially no effect on microsomal squalene monooxygenase activity (Fig. 1B ). These results indicated that SPF is a major cytosolic component for stimulating hepatic squalene monooxygenase activity.


Figure 1
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  		Figure 1. Cholesterol metabolism of Spf–/– mice vs. WT mice on a standard diet. A) Immunoblot analysis of SPF in hepatic cytosolic fractions isolated from Spf–/– mice and their WT littermates (WT). B) In vitro squalene monooxygenase promoting activity of hepatic cytosolic fraction isolated from WT mice and Spf–/– mice. Each value represents the amount of squalene 2,3-oxide relative to that in the absence of hepatic cytosolic fraction. Data show mean values ± SEM. For each group, n = 3. C) Plasma cholesterol levels of WT mice and Spf–/– mice. Data show mean values ± SEM. For each group, n = 3. D) The mRNA levels of hepatic cholesterogenic enzymes of WT mice and Spf–/– mice. Each value represents the amount of mRNA relative to that of the WT mice. Data show mean values ± SEM. For each group, n = 3. *P < 0.05 compared with WT mice.

Spf–/– mice developed normally and did not display apparent phenotypic abnormalities. Furthermore, plasma cholesterol (Fig. 1C ), triglyceride concentrations, and plasma lipoprotein profiles (data not shown) were unaffected under normal diet conditions. However, we found that the mRNAs for hepatic HMG-coenzyme A synthase and squalene monooxygenase were up-regulated in Spf–/– mice (Fig. 1D ).

Fasting decreases plasma cholesterol levels and hepatic cholesterol synthesis in Spf –/– mice
Following these experiments, the effect of various nutritional regimens was examined in Spf–/– mice and it was found that the plasma cholesterol levels differed under fasting conditions between WT and Spf–/– mice. Consistent with previous studies (5 , 8) , fasting for 24 or 48 h lowered plasma triglyceride and glucose levels in WT mice, whereas no change was observed in plasma cholesterol levels (Fig. 2 A). In contrast, fasting decreased plasma cholesterol levels in the Spf–/– mice to ~70% compared with WT mice (Fig. 2A ). Plasma triglyceride and glucose levels were decreased to levels similar to those in WT mice.


Figure 2
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  		Figure 2. Plasma cholesterol levels and hepatic cholesterol metabolism in Spf–/– mice vs. wild-type (WT) mice under fasting conditions. A) Plasma lipids and glucose levels of WT mice Spf–/– mice in nonfasted and fasted animals. Plasma cholesterol, triglyceride, and glucose levels collected after 0, 24, 48 h of fasting. 0 h samples were obtained before fasting. Data show mean values ± SEM. For each group, n = 5. *P < 0.05 compared with the nonfasted mice. B) Sterol synthesis in WT mice (WT) and Spf–/– mice under nonfasted (Fed) or 24 h fasted conditions (Fasted). The rates of hepatic sterol synthesis are expressed as the nmol of [3H] water incorporated into [3H] DPS per hour per gram liver. Data show mean values ± SEM. For each group, n = 3. *P < 0.05 compared to the fasted WT mice. C) Lipoprotein distribution of cholesterol after injection of Triton WR1339 in WT mice (WT) and Spf–/– mice under nonfasted (Fed, open square) or 24 h fasted conditions (Fasted, closed square). The approximate elution positions of VLDL and HDL are indicated. D) The mRNA levels of hepatic cholesterogenic enzymes of WT mice and Spf–/– mice under nonfasted (Fed) or 24 h fasted conditions (Fasted). E) Hepatic expression of SPF mRNA and protein in WT mice under nonfasted (Fed) or 24 h fasted (Fasted) conditions. Each value represents the amount of mRNA relative to that of the fed WT mice. Data show mean values ± SEM. For each group, n = 3. *P < 0.05 compared with the fed mice.

To examine the rate of hepatic cholesterol synthesis in Spf–/– mice under fasting conditions, [3H] water was used as a precursor for the sterol biosynthetic pathway (17) . Following 24 h fasting, hepatic cholesterol synthesis was reduced to approximately one-half in WT mice, while a more pronounced reduction in the hepatic cholesterol synthesis was observed in Spf–/– mice (Fig. 2B ). The rate of hepatic VLDL-cholesterol secretion was also examined by using Triton WR1339, which abruptly inhibits VLDL lipolysis. Consistent with the decreased rate of hepatic cholesterol biosynthesis in Spf–/– mice, the rate of VLDL-cholesterol secretion in fasted Spf–/– mice was lower than in fasted WT mice (Fig. 2C closed squares), whereas no change was observed between WT and Spf–/– mice under normal diet conditions (Fig. 2C , open squares).

Next, we examined the effect of fasting on the expressions of hepatic SPF and various cholesterogenic enzymes. It has previously been demonstrated that fasting dramatically reduces the mRNAs for cholesterogenic enzymes due to the fall in nuclear sterol regulatory element binding protein-2 (SREBP-2) (5 , 6) . As expected, fasting markedly reduced the mRNA of cholesterogenic enzymes, such as HMG-coenzyme A synthase, HMG-coenzyme A reductase (Fig. 2D ), squalene synthase, squalene monooxygenase, and lanosterol synthase (data not shown) in both the WT and Spf–/– mice. The level of HMG-coenzyme A reductase mRNA was slightly higher in fasted Spf–/– mice than in fasted WT mice (Fig. 2D ). In contrast to the cholesterogenic enzymes, hepatic SPF mRNA and protein levels increased significantly under fasting conditions (Fig. 2E ).

These results indicate that the significant decrease in plasma cholesterol level of Spf–/– mice under fasting conditions (Fig. 2A ) can at least in part be attributed to the more pronounced reduction in hepatic cholesterol synthesis and VLDL-cholesterol secretion in these animals (Fig. 2B, C ). It can, therefore, be postulated that in WT mice, SPF compensates the decrease of cholesterol synthesis under fasting conditions through an up-regulation of SPF expression.

SPF expression is PPAR-{alpha}-dependent
The nuclear receptor PPAR-{alpha} is known to play a role in regulating the hepatic transcriptional response to fasting (1 , 23) . As described above, hepatic SPF mRNA and protein expressions were up-regulated under fasting conditions in WT mice. We then examined SPF expression using Ppar-{alpha} –/– mice and found that fasting did not affect hepatic SPF expression in Ppar-{alpha} –/– mice (Fig. 3 A), indicating that fasting up-regulated hepatic SPF expression through PPAR-{alpha}.


Figure 3
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  		Figure 3. Effect of fibrate or dietary cholesterol level on hepatic SPF expression. A) Hepatic expression of SPF protein in Ppar-a–/– mice fed CR-LPF diet without (Control) or with supplementation of 0.2% fenofibrate (Feno) or fasted for 24 h (Fasted). B) Hepatic expression of SPF mRNA and protein in WT mice fed CR-LPF diet [cholesterol-deficient diet that contained 0.029% (w/w) cholesterol) without (Control) or with supplementation of 0.2% fenofibrate (Feno) or ciprofibrate (Cipro)]. Each value for the fenofibrate-treated and ciprofibrate-treated group represents the amount of mRNA relative to that of the control group. Data show mean values ± SEM. For each group, n = 3. *P < 0.05 compared to control group. C, D) WT mice (WT) and Spf –/– mice were fed with a cholesterol-deficient diet [cluster of differentiation (CD), open bar] or a cholesterol-supplemented diet (CS, closed bar). Hepatic expressions of cholesterogenic enzymes (C) and SPF (D). Each value represents the amount of mRNA relative to that of the WT mice fed with a cholesterol-deficient diet. Data show mean values ± SEM. Each group n = 3.

Then the effect of two fibrates was examined, both of which are agonists of PPAR-{alpha}. Feeding WT mice a diet supplemented with 0.2% fenofibrate or 0.2% ciprofibrate for 6 d significantly increased hepatic SPF mRNA and protein levels (Fig. 3B ). No significant difference was observed in hepatic SPF protein levels on treatment with fenofibrate in Ppar-{alpha}–/– mice (Fig. 3A ), indicating that fibrate-induced up-regulation of SPF is mediated by PPAR-{alpha}.

However, agonists of other nuclear receptors that play important roles in regulating hepatic lipid metabolism [PPAR-{delta}, PPAR- {gamma}, liver X receptor {alpha}, and farnesol X receptor (24) ] did not exhibit any effect on hepatic SPF expression (data not shown). We also tested the effect of dietary cholesterol on the expression of hepatic SPF. WT mice were fed a cholesterol-deficient or cholesterol-supplemented diet for 6 d. Although significant reduction of mRNA for cholesterogenic enzymes such as HMG-coenzyme A synthase and HMG-coenzyme A reductase were observed under cholesterol-supplemented diet conditions, no significant differences were observed in SPF mRNA and protein levels between mice fed the cholesterol-deficient or cholesterol-supplemented diet (Fig. 3C, D ). It is well established that cholesterogenic enzymes are transcriptionally regulated by dietary cholesterol through a mechanism dependent on the transcription factor SREBP-2 (25 , 26) . These data indicate that SPF does not belong to the family of proteins that are transcriptionally regulated by SREBP-2.

Fibrates reduce plasma cholesterol levels in Spf–/– mice
Fibrates efficiently lower plasma triglyceride levels (27 , 28) but have no effect on plasma cholesterol levels (28 , 29) . Here, we explored the effect of fibrates on plasma lipid levels in Spf–/– mice. As show previously, plasma triglyceride levels were decreased by fibrate treatment in both WT and Spf–/– mice (Fig. 4 A). Interestingly, plasma cholesterol levels were also reduced significantly by fibrate treatment in Spf–/– mice but not WT mice (Fig. 4A ). The reduction of plasma cholesterol levels in Spf–/– mice by fibrate treatment was dose-dependent (Fig. 4B ). We also examined the effect of fibrates on hepatic cholesterol synthesis and found that the rates of hepatic cholesterol synthesis were reduced by fibrate treatment in Spf–/– mice, whereas no significant decrease was observed in WT mice (Fig. 4C ). Fibrate treatment reduced protein levels and activity of hepatic HMG-coenzyme A reductase in both WT mice and Spf–/– mice (Fig. 4D, E ), as previously reported (30) . It can be speculated that the reduction of this enzyme after fibrate treatment is a cause for the decrease in hepatic cholesterol synthesis in Spf–/– mice. In WT mice receiving fibrate treatment, cholesterol synthesis is adequately maintained, probably because the decrease in HMG-coenzyme A reductase is compensated by SPF up-regulation.


Figure 4
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  		Figure 4. Cholesterol metabolism in Spf–/– mice vs. WT mice treated with fibrate. A) Plasma cholesterol and triglyceride concentrations in WT mice and Spf –/– mice fed CR-LPF diet without (Control) or with supplementation of 0.2% fenofibrate. Data show mean values ± SEM. For each group, n = 3. *P < 0.05 compared to control group. B) Plasma cholesterol concentrations in WT mice and Spf –/– mice fed CR-LPF diet with supplementation of fenofibrate at increasing doses. Data show mean values ± SEM. For each group, n = 3 *P < 0.05 compared to control group. C) Sterol synthesis in WT mice and Spf–/– mice fed CR-LPF diet without (Control) or with supplementation of 0.2% fenofibrate. The rates of hepatic sterol synthesis are expressed as the nmol of [3H] water incorporated into [3H] DPS per hour per gram liver. Data show mean values ± SEM. For each group, n = 3. *P < 0.05 compared to control group. D, E) Hepatic expression of HMG-coenzyme A reductase protein (D) and hepatic HMG-coenzyme A reductase activity (E) in WT mice and Spf–/– mice fed CR-LPF diet without (Control) or with supplementation of 0.2% fenofibrate (Feno). Data show mean values ± SEM. For each group, n = 3. *P < 0.05 compared to control group.

DISCUSSION

To determine how SPF affects cholesterol metabolism in vivo, we generated mice lacking the Spf gene and found that this gene is closely involved in hepatic cholesterol synthesis during fasting. Mammals have developed a metabolic response system that allows them to survive prolonged energy deprivation. One major response to fasting is the reduction of plasma triglyceride and glucose levels as triglycerides and glucose are consumed to maintain energy production (1 , 8 , 31) . However, it has been reported that fasting causes a dramatic reduction, by as much as 80%, in the mRNA levels of cholesterogenic enzymes such HMG-coenzyme A synthase, HMG-coenzyme A reductase, and squalene synthase due to a fall in SREBP-2 (5 , 6) , while hepatic cholesterol synthesis is decreased only by ~50% (7) , and plasma cholesterol levels are hardly affected (5 , 8) . The results presented here showed that hepatic cholesterol synthesis in fasting Spf–/– mice were much lower than in fasting WT mice (Fig. 2B ). Moreover, hepatic SPF expression was increased ~1.5-fold by fasting in WT mice (Fig. 2E ). These results indicate that SPF plays a role in compensating hepatic cholesterol synthesis during fasting (Fig. 5 ). It has recently been reported that protein kinase A (PKA) phosphorylates SPF and increases SPF activity by more than 2-fold in vitro (32 , 33) . Since it is well known that PKA activity is enhanced in the liver under fasting conditions (34 , 35) , it can be speculated that SPF activity is much more elevated by increasing both SPF protein expression and its phosphorylation under fasting conditions. Studies addressing this point are now in progress in our laboratory.


Figure 5
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  		Figure 5. Hepatic cholesterol synthesis regulation model under fasting conditions. Hepatic cholesterol synthesis is governed by two mechanisms under fasting conditions; the repression of biosynthesis enzymes through the inhibition of the transcription factor SREBP-2 (25 , 46) and the induction of SPF through the activation of the nuclear receptor PPAR-{alpha}. See text for details.

As has been demonstrated previously (5 , 8) , we also observed that plasma cholesterol levels were not decreased even by 48 h fasting in WT mice (Fig. 2A ). In contrast, fasting decreased plasma cholesterol levels in the Spf–/– mice to ~70% compared to WT mice (Fig. 2A ). Steady-state plasma cholesterol of WT mice is almost exclusively located in the high-density lipoprotein (HDL) fraction (36) . The concentration of HDL-cholesterol in plasma is known to be determined by a number of factors, including apolipoprotein A-I (apoA-I) synthesis, ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol secretion from peripheral tissues, lipoprotein lipase (LPL)-mediated lipolysis of chylomicrons and VLDL, and the hepatic scavenger receptor class B type I (SR-BI)-mediated HDL-cholesterol uptake by the liver. Haas et al. showed that fasting for 48 h in rats significantly increases the synthesis of hepatic and intestinal apoA-I (37) . In addition, Kok et al. reported that fasting in mice increases the expression of ABCA1 and decreases the expression of hepatic SR-BI (38) . Hepatic VLDL production also affects plasma HDL levels (39) , which is confirmed by our studies demonstrating the marked reduction of HDL-cholesterol levels by LPL inhibitor Triton WR1339 treatment (Fig. 2C ). We found in this study that hepatic VLDL cholesterol production was significantly reduced under fasting conditions in Spf–/– mice, which may result in the reduction of the VLDL lipolysis-derived HDL cholesterol level. Thus, SPF can be considered as a new factor for determining steady-state plasma cholesterol level during fasting. Cholesterol is an indispensable substance in mammals, making it likely that multiple overlapping strategies are necessary to maintain certain levels of hepatic cholesterol supply even under extreme conditions such as fasting.

Both plasma cholesterol level and the rate of hepatic cholesterol synthesis were not affected by fibrate treatment in WT mice but reduced appreciably in Spf–/– mice. When exploring the effect of fibrates on hepatic cholesterogenic enzyme expressions, we found that fibrate treatment reduced the protein levels of hepatic HMG-coenzyme A reductase in both WT and Spf–/– mice. In sharp contrast, fibrate treatment increased hepatic SPF levels in WT mice. According to the recent report (40) , SPF stimulates not only squalene monooxygenase activity but also HMG-coenzyme A reductase activity. Increase of these activities may explain the apparently unaltered hepatic cholesterol biosynthesis despite significant reduction of HMG-coenzyme A reductase in fibrate-treated WT mice. The results reported here show that fibrates have an impact not only on the activities and/or expression levels of cholesterol synthetic enzymes but also on the SPF levels at least in mice, revealing SPF to be one of crucial factors for fibrate-mediated regulation of cholesterol metabolism.

Our results also suggest that SPF is a highly useful therapeutic target for hypercholesterolemia. An important class of PPAR-{alpha} ligands is fibrate drugs, which effectively lower serum triglyceride levels by up-regulating genes involved in the cellular uptake and ß-oxidation of fatty acids (27 , 28) . We demonstrated that the administration of fibrate to Spf–/– mice resulted in a significant decrease of plasma cholesterol concentrations, suggesting that co-administration of fibrates and an SPF inhibitor may reduce not only plasma triglyceride but also cholesterol levels (Fig. 4A ). HMG-coenzyme A reductase is a key rate-limiting enzyme in the cholesterol biosynthetic pathway (4) , and several HMG-coenzyme A reductase inhibitors (statins) have been developed and are now in clinical use (41) . However, at times these inhibitors induce adverse hepatic and muscular effects (42) . Moreover, statin-fibrate combination therapy is a well-known risk factor for myopathy, rhabdomyolysis, and severe renal failure (43 44 45) . Therefore, research is currently focusing on the development of new target reagents that can suppress cholesterol biosynthesis. SPF is expressed selectively in the liver and intestine (11) , and Spf–/– mice develop without apparent phenotypic abnormalities. Future studies must elucidate the practical possibilities of using SPF as a drug target.

ACKNOWLEDGMENTS

We thank T. Kodama for the anti-HMG-coenzyme A reductase mAb. This work was supported by grants from the Japan Society for the Promotion of Science. N.S. was a research fellow of the Special Postdoctoral Researchers Program, RIKEN.

Received for publication May 1, 2006. Accepted for publication July 5, 2006.

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