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

The FASEB Journal • FJ Express Full-Length Article

Regulation of hepatic cholesterol synthesis bya novel protein (SPF) that acceleratescholesterol biosynthesis

Norihito Shibata,*,† Kou-ichi Jishage,‡ Makoto Arita,* Miho Watanabe,‡ Yosuke Kawase,‡

Kiyotaka Nishikawa,§ Yasuhiro Natori,§ Hiroyasu Inoue,� Hitoshi Shimano,¶

Nobuhiro Yamada,¶ Masafumi Tsujimoto,† and Hiroyuki Arai*,1

*Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo,Tokyo, Japan; †Laboratory of Cellular Biochemistry, Riken, Saitama, Japan; ‡Pharmacology andPathology 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 ClinicalMedicine, University of Tsukuba, Ibaraki, Japan

ABSTRACT Supernatant protein factor (SPF) is anovel cholesterol biosynthesis-accelerating protein ex-pressed in liver and small intestine. Here, we report onthe physiological role of SPF by using Spf-deficientmice. Although plasma cholesterol levels were similarin chow-fed Spf �/� and wild-type (WT) mice, fastingsignificantly decreased plasma cholesterol levels inSpf �/� mice but not in WT mice. While fasting reducedhepatic cholesterol synthesis rate in WT mice, a morepronounced reduction was observed in Spf �/� mice.The expression of cholesterogenic enzymes was dra-matically suppressed by fasting both in WT and Spf �/�

mice. In contrast, hepatic SPF expression of WT micewas up-regulated by fasting in peroxisome proliferator-activated receptor � (PPAR-�)-dependent manner.These results indicate that in WT mice, the decrease ofhepatic cholesterol synthesis under fasting conditions isat least in part compensated by SPF up-regulation.Fibrates, which function as a PPAR-� agonist and arewidely used as hypotriglycemic drugs, reduced hepaticcholesterol synthesis and plasma cholesterol levels byapproximately one-half in Spf �/� mice but not in WTmice. These findings suggest that co-administration offibrates and an SPF inhibitor may reduce not onlyplasma triglyceride but also cholesterol levels, indicat-ing that SPF is a promising hypocholesterolemic drugtarget.—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 novelprotein (SPF) that accelerates cholesterol biosynthesisFASEB J. 20, E2231–E2239 (2006)

Key Words: squalene monooxygenase � peroxisome proliferator-activated receptor � � fasting � fibrate

Prolonged food deprivation induces dramaticchanges in mammalian lipid metabolism. Under

these conditions, fatty acid metabolism in the liverchanges to allow sufficient energy production (1).Starvation decreases the synthesis of fatty acids andincreases the oxidation of fatty acids released fromadipose tissue.

Cholesterol is required for a wide range of cellularfunctions in mammalian cells. It is a major lipid com-ponent of the plasma membrane, functions as a pre-cursor molecule for bile acids, and is necessary forcovalent modifications of embryonic signaling proteins(2, 3). Therefore, mammals are forced to continuouslyadjust their sterol content by regulating their hepaticcholesterol metabolism (4). However, much is notknown about the changes in hepatic cholesterol metab-olism under fasting conditions. Previous studies havedemonstrated that fasting causes a dramatic reductionin the mRNA levels of hepatic cholesterogenic enzymes(5, 6). However, hepatic cholesterol synthesis is de-creased only �50% (7), and plasma cholesterol levelsare hardly affected by fasting (5, 8). This suggests thepresence of a compensatory mechanism to maintaincholesterol levels even under extreme conditions suchas fasting.

In 1957, Bloch and colleagues identified a solublefactor from rat liver cytosol termed “supernatant pro-tein factor (SPF)”, which promotes the activity ofsqualene monooxygenase, a rate-limiting enzyme in thelate stages of cholesterol biosynthesis (9, 10). Half acentury after its discovery, we succeeded in purifyingthis protein and isolating its cDNA (11). RecombinantSPF produced in E. coli enhances microsomal squalenemonooxygenase activity and also promotes intermem-

1 Correspondence: Department of Health Chemistry, Grad-uate School of Pharmaceutical Sciences, University of Tokyo,7–3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail:[email protected]

doi: 10.1096/fj.06-6368fje

E22310892-6638/06/0020-2231 © FASEB

brane transfer of squalene in vitro, which led to thehypothesis that SPF facilitates the access of a hydropho-bic substrate (squalene) to a specific enzyme site (11,12). SPF is abundantly expressed in the liver andintestine of rats, mice, and humans, but is undetectablein cultured cells, including hepatoma cell lines. Thesedata suggest that SPF is not necessarily a prerequisitefor cholesterol biosynthesis but rather plays a role incertain specific organs in mammals (11). By use of theSPF-deficient mouse model, we demonstrate here thatSPF plays a role in hepatic cholesterol synthesis underfasting conditions. Moreover, we show that fibrates,which are widely used hypotriglycemic drugs, also re-duce plasma cholesterol levels in Spf �/� mice, impli-cating SPF as a promising hypocholesterolemic drugtarget.

MATERIALS AND METHODS

Generation of SPF-null mice

The SPF gene was isolated from the 129/Sv mouse genomicDNA � FIX II library (Stratagene, La Jolla, CA, USA) usingmouse cDNA as a probe. A replacement-type targeting vectorwas constructed; the long arm containing a 9.7 kb SmaI/SacIIfragment spanning exons 1 and 2, a 1.3 kb SalI/BamHIfragment of the PGK-neo cassette, and the short arm contain-ing a 1.0 kb BamHI/SalI fragment in intron 5 were insertedinto 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 linear-ized targeting vector. G418/gancyclovir-resistant clones werescreened by polymerase chain reaction (PCR) using forwardand reverse primers, 5�-TCG CCT TCT ATC GCC TTC TTGACG-3� and 5�-AGA AGA CAA TTT GGG GAG CTA GC-3�,respectively. Targeted embryonic stem clones were injectedinto C57BL/6J (CLEA Japan, Tokyo, Japan) blastocysts yield-ing two lines of chimeric mice, which transmitted the dis-rupted allele through the germ line.

Animal studies

Seven- to eight-week-old male C57BL/6J mice, Ppar-��/�

mice (129S4/SvJae-Pparatm1Gonz/J; The Jackson Laboratory,ME, USA) and Spf �/� mice were housed in colony cagesunder a 12-h light/dark cycle and maintained on standardchow (NMF diet from Oriental Yeast Co., Tokyo, Japan) thatcontained 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 werefed with CR-LPF diet supplemented with fenofibrate at theindicated dose for 6 d. For the cholesterol experiments, micewere fed a cholesterol-deficient diet (CR-LPF diet) thatcontained 0.029% (w/w) cholesterol or a cholesterol-supple-mented diet [CR-LPF diet supplemented with 2% cholesterol(Sigma)] for 6 d. In all experiments, mice were anesthetizedby intraperitoneal (i.p.) injection of pentobarbital sodium(Nembutal, Abbot, North Chicago, IL, USA), and blood andliver were obtained on the days indicated during the earlyphase of the dark cycle. The experiments were performed in

accordance with institutional guidelines for animal experi-ments at the University of Tokyo.

Preparation of mouse anti-SPF specificmonoclonal antibodies

Mouse anti-SPF specific monoclonal antibodies were estab-lished according to the method described by Kaempf-Rotzollet al. (14). Briefly, the coding region of mouse SPF cDNA wasinserted into the BamHI/SalI sites of the pET21a vector(Novagen, Madison, WI, USA). After the plasmid was intro-duced into E. coli strain BL21 (DE3) (Novagen), the proteinwas expressed as a His-tagged protein by induction with 1 mMisopropyl-�-D-thiogalactopyranoside. The protein was puri-fied using nickel column chromatography (Novagen) accord-ing to the manufacturer’s protocol. Three rats (WKY strains,female, 8 wk; SLC, Hamamatsu, Japan) were immunized byinjecting the protein into the hind foot pads using Freundcomplex adjuvant. At 3-week intervals after the initial injec-tion, the rats were injected twice with the purified proteinmixed with Freund complex adjuvant. One week after the lastbooster injection, the two enlarged medial iliac lymph nodesfrom each rat were used for cell fusion with mouse myelomacells, line PAI. Several monoclonal antibody (mAb)-produc-ing hybridoma cell lines were established and selected afterchecking the produced antibodies by ELISA, Western blotanalysis screening of mouse liver cytosolic fraction. Only theclones highly positive in those screening methods were se-lected. 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 SETbuffer (0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH7.4) and centrifuged at 100,000 g for 1 h at 4°C. The resultantsupernatants were used as the hepatic cytosolic fraction. Thehepatic microsomal fraction was prepared as described pre-viously (15). Each sample (20 �g) was separated by SDS-PAGEand transferred to a polyvinylidine difluoride membrane. Themembranes were incubated with anti-SPF, anti-�-tocopheroltransfer protein (�-TTP), anti-HMG-coenzyme A reductase,or anticalnexin antibody (Ab). Mouse anti-�-TTP specificmonoclonal antibodies were previously established in ourlaboratory (16). The anti-HMG-coenzyme A reductase mAb(15) was a kind gift from Dr. Tatsuhiko Kodama. Theanticalnexin mAb was purchased from Transduction Labora-tories (Lexington, KY, USA).

SPF activity assay

Conversion of [14C] squalene to [14C] squalene 2,3-oxide wasused to measure SPF activity from individual liver, as de-scribed previously (10, 11). Briefly, [14C] squalene (20,000dpm/40 nmol, American Radiolabeled Chemicals, St. Louis,MO, USA) and 50 �g of Tween 80 in 50 �l of acetone weremixed, 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, 0.3mM Amo-1618, an inhibitor of lanosterol synthase (Calbio-chem, San Diego, CA, USA), 0.01 mM flavin adenine dinu-cleotide (FAD) (Sigma), 0.1 mg phosphatidylglycerol (AvantiPolar Lipids, Inc., Alabaster, AL, USA), 1 mM NADPH(Sigma), and 1.28 mg of hepatic microsomal and cytosolicprotein. Mixtures were incubated at 37°C for 30 min, andproducts were saponified by 500 �l of 10% KOH in methanol.Lipids were extracted with petroleum ether, evaporated un-der nitrogen, and subjected to thin-layer chromatography on

E2232 Vol. 20 December 2006 SHIBATA ET AL.The FASEB Journal

silica gel plate. The plate was developed with 0.5% ethylacetate in benzene. After development, plates were exposedand 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 �gtotal RNA with oligo-dT primer by using a SuperScriptFirst-Strand Synthesis System (Invitrogen). Real-time PCRreactions were performed on an ABI PRISM 7000 SequenceDetection System (Applied Biosystems, Foster City, CA, USA)using 2 SYBR Green PCR Master Mix. Mouse 36B4 mRNAwas used as an invariant control for fasting experiment.Mouse �-actin mRNA was used as an invariant control forother experiments. PCR primers used were as follows (5� to3�): �-Actin, ATG AAG ATC AAG ATC ATT GCT CCT C andACA TCT GCT GGA AGG TGG ACA; 36B4, GCT CGA CATCAC AGA GCA GGandCCG AGG CAA CAG TTG GGT AC;SPF, TTC CGG AAG CAA AAG GAA CAT and GCC TGA CAGATA CTG TTG GAT CAC; HMG-coenzyme A synthase, CTTGCT TTG CTC GTT CTT CT and TCG GTC ACC GGT TCCTCC TTC A; HMG-coenzyme A reductase, TGG AAT TAT GAGTGC CCC AAA and CCG CGT TAT CGT CAG GAT GAT G;Squalene monooxygenase, AAG AAA GAA CAG CTG GAG TCCAA and GTC ACG AAC GAG GTC GAC ACT.

Plasma cholesterol, triglyceride, and glucose measurements

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

In vivo rates of hepatic cholesterol synthesis

The in vivo rate of hepatic cholesterol synthesis was measuredas described (17). Briefly, mice were given an i.p. injectionwith 40 mCi of [3H] water (100 mCi/mmol, MP Biomedicals,Inc, CA, USA) and were anesthetized and exsanguinated after1 h. Aliquots of liver were saponified and their content ofradiolabeled digitonin-precipitable sterols (DPS) was mea-sured (18–20). The rate of sterol synthesis was expressed asthe nmol of [3H] water incorporated into DPS per hour pergram liver. The rate of incorporation of [3H] water intosterols by the liver was also converted to an equivalent mgquantity of newly synthesized cholesterol assuming that 0.693H atoms were incorporated into the sterol molecule percarbon atom entering the biosynthetic pathway as acetyl-coenzyme A.

VLDL-cholesterol secretion

Mice were injected intravenously (i.v.) with 20 mg of TritonWR1339 (Sigma) in 100 �l of PBS. Three hours after injec-tion, plasma samples were prepared as described above.Pooled plasma samples (120 �l) were subjected to fast proteinliquid chromatography (FPLC) gel filtration on a Superose 6column (Amersham Pharmacia Biotech, Uppsala, Sweden) asdescribed previously (21). Each fraction was collected in 375�l, and the cholesterol concentrations were determined asdescribed above.

HMG-coenzyme A reductase activity assay

Mouse livers were homogenized in a buffer containing 15 mMnicotinamide, 2 mM MgCl2, and 100 mM potassium phos-phate, 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, werewashed and resuspended in the same buffer. HMG-coenzymeA reductase activities were measured essentially as describedpreviously (22).

Statistics

Statistical analyses were performed with Student’s t test set-ting the significance at P 0.05.

RESULTS

Generation of SPF-null mice

We generated mice lacking the Spf gene by targeteddeletion leading to undetectable Spf mRNA (data notshown) and protein expression in these animals (Fig.1A). As shown previously in rats (9–12), the microso-mal squalene monooxygenase activity was enhanced byadding the hepatic cytosolic fraction in WT mice,whereas the hepatic cytosolic fraction obtained fromSpf �/� mice showed substantially no effect on micro-somal squalene monooxygenase activity (Fig. 1B).These results indicated that SPF is a major cytosoliccomponent for stimulating hepatic squalene monoox-ygenase activity.

Spf �/� mice developed normally and did not displayapparent phenotypic abnormalities. Furthermore,plasma cholesterol (Fig. 1C), triglyceride concentra-tions, and plasma lipoprotein profiles (data not shown)were unaffected under normal diet conditions. How-ever, we found that the mRNAs for hepatic HMG-coenzyme A synthase and squalene monooxygenasewere up-regulated in Spf �/� mice (Fig. 1D).

Fasting decreases plasma cholesterol levels andhepatic cholesterol synthesis in Spf �/� mice

Following these experiments, the effect of various nu-tritional regimens was examined in Spf �/� mice and itwas found that the plasma cholesterol levels differedunder fasting conditions between WT and Spf �/� mice.Consistent with previous studies (5, 8), fasting for 24 or48 h lowered plasma triglyceride and glucose levels inWT mice, whereas no change was observed in plasmacholesterol levels (Fig. 2A). In contrast, fasting de-creased plasma cholesterol levels in the Spf �/� mice to�70% compared with WT mice (Fig. 2 A). Plasmatriglyceride and glucose levels were decreased to levelssimilar to those in WT mice.

To examine the rate of hepatic cholesterol synthesisin Spf �/� mice under fasting conditions, [3H] waterwas used as a precursor for the sterol biosyntheticpathway (17). Following 24 h fasting, hepatic choles-

E2233REGULATION OF HEPATIC CHOLESTEROL SYNTHESIS UNDER FASTING CONDITION

terol synthesis was reduced to approximately one-halfin WT mice, while a more pronounced reduction in thehepatic cholesterol synthesis was observed in Spf �/�

mice (Fig. 2B). The rate of hepatic VLDL-cholesterolsecretion was also examined by using Triton WR1339,which abruptly inhibits VLDL lipolysis. Consistent withthe decreased rate of hepatic cholesterol biosynthesisin Spf �/� mice, the rate of VLDL-cholesterol secretionin fasted Spf �/� mice was lower than in fasted WT mice(Fig. 2C closed squares), whereas no change was ob-served between WT and Spf �/� mice under normaldiet conditions (Fig. 2C, open squares).

Next, we examined the effect of fasting on theexpressions of hepatic SPF and various cholesterogenicenzymes. It has previously been demonstrated thatfasting dramatically reduces the mRNAs for cholestero-genic enzymes due to the fall in nuclear sterol regula-

tory element binding protein-2 (SREBP-2) (5, 6). Asexpected, fasting markedly reduced the mRNA of cho-lesterogenic enzymes, such as HMG-coenzyme A syn-thase, HMG-coenzyme A reductase (Fig. 2D), squalenesynthase, squalene monooxygenase, and lanosterol syn-thase (data not shown) in both the WT and Spf �/�

mice. The level of HMG-coenzyme A reductase mRNAwas slightly higher in fasted Spf �/� mice than in fastedWT mice (Fig. 2D). In contrast to the cholesterogenicenzymes, hepatic SPF mRNA and protein levels in-creased significantly under fasting conditions (Fig. 2E).

These results indicate that the significant decrease inplasma cholesterol level of Spf �/� mice under fastingconditions (Fig. 2A) can at least in part be attributed tothe more pronounced reduction in hepatic cholesterolsynthesis and VLDL-cholesterol secretion in these ani-mals (Fig. 2B, C). It can, therefore, be postulated that inWT mice, SPF compensates the decrease of cholesterolsynthesis under fasting conditions through an up-regu-lation of SPF expression.

SPF expression is PPAR-�-dependent

The nuclear receptor PPAR-� is known to play a role inregulating the hepatic transcriptional response to fast-ing (1, 23). As described above, hepatic SPF mRNA andprotein expressions were up-regulated under fastingconditions in WT mice. We then examined SPF expres-sion using Ppar-� �/� mice and found that fasting didnot affect hepatic SPF expression in Ppar-� �/� mice(Fig. 3A), indicating that fasting up-regulated hepaticSPF expression through PPAR-�.

Then the effect of two fibrates was examined, both ofwhich are agonists of PPAR-�. Feeding WT mice a dietsupplemented with 0.2% fenofibrate or 0.2% ciprofi-brate for 6 d significantly increased hepatic SPF mRNAand protein levels (Fig. 3B). No significant differencewas observed in hepatic SPF protein levels on treatmentwith fenofibrate in Ppar-��/� mice (Fig. 3A), indicatingthat fibrate-induced up-regulation of SPF is mediatedby PPAR-�.

However, agonists of other nuclear receptors thatplay important roles in regulating hepatic lipid metab-olism [PPAR-�, PPAR- �, liver X receptor �, and farne-sol X receptor (24)] did not exhibit any effect onhepatic SPF expression (data not shown). We alsotested the effect of dietary cholesterol on the expres-sion of hepatic SPF. WT mice were fed a cholesterol-deficient or cholesterol-supplemented diet for 6 d.Although significant reduction of mRNA for choles-terogenic enzymes such as HMG-coenzyme A synthaseand HMG-coenzyme A reductase were observed undercholesterol-supplemented diet conditions, no signifi-cant differences were observed in SPF mRNA andprotein levels between mice fed the cholesterol-defi-cient or cholesterol-supplemented diet (Fig. 3C, D). It iswell established that cholesterogenic enzymes are tran-scriptionally regulated by dietary cholesterol through amechanism dependent on the transcription factorSREBP-2 (25, 26). These data indicate that SPF does

Figure 1. Cholesterol metabolism of Spf �/� mice vs. WTmice on a standard diet. A) Immunoblot analysis of SPF inhepatic cytosolic fractions isolated from Spf �/� mice andtheir WT littermates (WT). B) In vitro squalene monooxygen-ase promoting activity of hepatic cytosolic fraction isolatedfrom WT mice and Spf �/� mice. Each value represents theamount of squalene 2,3-oxide relative to that in the absenceof hepatic cytosolic fraction. Data show mean values sem.For each group, n � 3. C) Plasma cholesterol levels of WTmice and Spf �/� mice. Data show mean values sem. Foreach group, n � 3. D) The mRNA levels of hepatic choles-terogenic enzymes of WT mice and Spf �/� mice. Each valuerepresents the amount of mRNA relative to that of the WTmice. Data show mean values sem. For each group, n � 3.*P 0.05 compared with WT mice.


not belong to the family of proteins that are transcrip-tionally regulated by SREBP-2.

Fibrates reduce plasma cholesterol levels inSpf �/� 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 plasmalipid levels in Spf �/� mice. As show previously, plasmatriglyceride levels were decreased by fibrate treatmentin both WT and Spf �/� mice (Fig. 4A). Interestingly,plasma cholesterol levels were also reduced signifi-cantly by fibrate treatment in Spf �/� mice but not WTmice (Fig. 4A). The reduction of plasma cholesterollevels in Spf �/� mice by fibrate treatment was dose-dependent (Fig. 4B). We also examined the effect offibrates on hepatic cholesterol synthesis and found thatthe rates of hepatic cholesterol synthesis were reducedby fibrate treatment in Spf �/� mice, whereas no signif-icant decrease was observed in WT mice (Fig. 4C).Fibrate treatment reduced protein levels and activity ofhepatic HMG-coenzyme A reductase in both WT miceand Spf �/� mice (Fig. 4D, E), as previously reported(30). It can be speculated that the reduction of thisenzyme after fibrate treatment is a cause for the de-crease in hepatic cholesterol synthesis in Spf �/� mice.

In WT mice receiving fibrate treatment, cholesterolsynthesis is adequately maintained, probably becausethe decrease in HMG-coenzyme A reductase is compen-sated by SPF up-regulation.

DISCUSSION

To determine how SPF affects cholesterol metabolismin vivo, we generated mice lacking the Spf gene andfound that this gene is closely involved in hepaticcholesterol synthesis during fasting. Mammals havedeveloped a metabolic response system that allowsthem to survive prolonged energy deprivation. Onemajor response to fasting is the reduction of plasmatriglyceride and glucose levels as triglycerides and glu-cose are consumed to maintain energy production (1,8, 31). However, it has been reported that fasting causesa dramatic reduction, by as much as 80%, in the mRNAlevels of cholesterogenic enzymes such HMG-coenzymeA synthase, HMG-coenzyme A reductase, and squalenesynthase due to a fall in SREBP-2 (5, 6), while hepaticcholesterol synthesis is decreased only by �50% (7),and plasma cholesterol levels are hardly affected (5, 8).The results presented here showed that hepatic choles-terol synthesis in fasting Spf �/� mice were much lowerthan in fasting WT mice (Fig. 2B). Moreover, hepatic

Figure 2. Plasma cholesterol levels and hepaticcholesterol metabolism in Spf �/� mice vs. wild-type (WT) mice under fasting conditions. A)Plasma lipids and glucose levels of WT miceSpf �/� mice in nonfasted and fasted animals.Plasma cholesterol, triglyceride, and glucoselevels collected after 0, 24, 48 h of fasting. 0 hsamples were obtained before fasting. Datashow 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 fastedconditions (Fasted). The rates of hepatic sterolsynthesis are expressed as the nmol of [3H]water incorporated into [3H] DPS per hour pergram liver. Data show mean values sem. Foreach group, n � 3. *P 0.05 compared to thefasted WT mice. C) Lipoprotein distribution ofcholesterol after injection of Triton WR1339 inWT mice (WT) and Spf�/� mice under non-fasted (Fed, open square) or 24 h fasted condi-tions (Fasted, closed square). The approximateelution positions of VLDL and HDL are indi-cated. D) The mRNA levels of hepatic choles-terogenic enzymes of WT mice and Spf�/� miceunder nonfasted (Fed) or 24 h fasted conditions(Fasted). E) Hepatic expression of SPF mRNAand protein in WT mice under nonfasted (Fed)or 24 h fasted (Fasted) conditions. Each valuerepresents the amount of mRNA relative to thatof the fed WT mice. Data show mean values sem. For each group, n � 3. *P 0.05 com-pared with the fed mice.


SPF expression was increased �1.5-fold by fasting inWT mice (Fig. 2E). These results indicate that SPF playsa role in compensating hepatic cholesterol synthesisduring fasting (Fig. 5). It has recently been reportedthat protein kinase A (PKA) phosphorylates SPF andincreases SPF activity by more than 2-fold in vitro (32,33). Since it is well known that PKA activity is enhancedin the liver under fasting conditions (34, 35), it can bespeculated that SPF activity is much more elevated byincreasing both SPF protein expression and its phos-phorylation under fasting conditions. Studies address-ing this point are now in progress in our laboratory.

As has been demonstrated previously (5, 8), we alsoobserved that plasma cholesterol levels were not de-creased even by 48 h fasting in WT mice (Fig. 2A). Incontrast, fasting decreased plasma cholesterol levels inthe Spf �/� mice to �70% compared to WT mice (Fig.2A). Steady-state plasma cholesterol of WT mice isalmost exclusively located in the high-density lipopro-tein (HDL) fraction (36). The concentration of HDL-cholesterol in plasma is known to be determined by anumber of factors, including apolipoprotein A-I(apoA-I) synthesis, ATP-binding cassette transporter A1(ABCA1)-mediated cholesterol secretion from periph-eral tissues, lipoprotein lipase (LPL)-mediated lipolysisof chylomicrons and VLDL, and the hepatic scavengerreceptor class B type I (SR-BI)-mediated HDL-choles-terol uptake by the liver. Haas et al. showed that fastingfor 48 h in rats significantly increases the synthesis ofhepatic and intestinal apoA-I (37). In addition, Kok etal. reported that fasting in mice increases the expres-sion of ABCA1 and decreases the expression of hepaticSR-BI (38). Hepatic VLDL production also affectsplasma HDL levels (39), which is confirmed by ourstudies demonstrating the marked reduction of HDL-cholesterol levels by LPL inhibitor Triton WR1339treatment (Fig. 2C). We found in this study that hepaticVLDL cholesterol production was significantly reducedunder fasting conditions in Spf �/� mice, which mayresult in the reduction of the VLDL lipolysis-derivedHDL cholesterol level. Thus, SPF can be considered asa new factor for determining steady-state plasma cho-lesterol level during fasting. Cholesterol is an indispens-able substance in mammals, making it likely that mul-tiple overlapping strategies are necessary to maintaincertain levels of hepatic cholesterol supply even underextreme conditions such as fasting.

Both plasma cholesterol level and the rate of hepaticcholesterol synthesis were not affected by fibrate treat-ment in WT mice but reduced appreciably in Spf �/�

mice. When exploring the effect of fibrates on hepaticcholesterogenic enzyme expressions, we found thatfibrate treatment reduced the protein levels of hepaticHMG-coenzyme A reductase in both WT and Spf �/�

mice. In sharp contrast, fibrate treatment increasedhepatic SPF levels in WT mice. According to the recentreport (40), SPF stimulates not only squalene monoox-ygenase activity but also HMG-coenzyme A reductaseactivity. Increase of these activities may explain theapparently unaltered hepatic cholesterol biosynthesis

Figure 3. Effect of fibrate or dietary cholesterol level on hepaticSPF expression. A) Hepatic expression of SPF protein inPpar-a �/� mice fed CR-LPF diet without (Control) or withsupplementation of 0.2% fenofibrate (Feno) or fasted for 24 h(Fasted). B) Hepatic expression of SPF mRNA and protein inWT mice fed CR-LPF diet [cholesterol-deficient diet thatcontained 0.029% (w/w) cholesterol) without (Control) orwith supplementation of 0.2% fenofibrate (Feno) or ciprofi-brate (Cipro)]. Each value for the fenofibrate-treated andciprofibrate-treated group represents the amount of mRNArelative to that of the control group. Data show mean values sem. For each group, n � 3. *P 0.05 compared to controlgroup. C, D) WT mice (WT) and Spf �/� mice were fed witha cholesterol-deficient diet [cluster of differentiation (CD),open bar] or a cholesterol-supplemented diet (CS, closedbar). Hepatic expressions of cholesterogenic enzymes (C)and SPF (D). Each value represents the amount of mRNArelative to that of the WT mice fed with a cholesterol-deficientdiet. Data show mean values sem. Each group n � 3.


despite significant reduction of HMG-coenzyme A re-ductase in fibrate-treated WT mice. The results re-ported here show that fibrates have an impact not onlyon the activities and/or expression levels of cholesterolsynthetic enzymes but also on the SPF levels at least inmice, revealing SPF to be one of crucial factors forfibrate-mediated regulation of cholesterol metabolism.

Our results also suggest that SPF is a highly usefultherapeutic target for hypercholesterolemia. An impor-tant class of PPAR-� ligands is fibrate drugs, whicheffectively lower serum triglyceride levels by up-regulat-ing genes involved in the cellular uptake and �-oxida-tion of fatty acids (27, 28). We demonstrated that theadministration of fibrate to Spf �/� mice resulted in asignificant decrease of plasma cholesterol concentra-tions, suggesting that co-administration of fibrates andan SPF inhibitor may reduce not only plasma triglycer-ide but also cholesterol levels (Fig. 4A). HMG-coen-zyme A reductase is a key rate-limiting enzyme in thecholesterol biosynthetic pathway (4), and several HMG-coenzyme A reductase inhibitors (statins) have beendeveloped and are now in clinical use (41). However, attimes these inhibitors induce adverse hepatic and mus-cular effects (42). Moreover, statin-fibrate combinationtherapy is a well-known risk factor for myopathy, rhab-domyolysis, and severe renal failure (43–45). There-fore, research is currently focusing on the developmentof new target reagents that can suppress cholesterolbiosynthesis. SPF is expressed selectively in the liver and

Figure 4. Cholesterol metabolism in Spf �/� mice vs. WTmice treated with fibrate. A) Plasma cholesterol and triglyc-eride concentrations in WT mice and Spf �/� mice fedCR-LPF diet without (Control) or with supplementation of0.2% fenofibrate. Data show mean values sem. For eachgroup, n � 3. *P 0.05 compared to control group. B) Plasmacholesterol concentrations in WT mice and Spf �/� mice fedCR-LPF diet with supplementation of fenofibrate at increas-ing doses. Data show mean values sem. For each group, n �3 *P 0.05 compared to control group. C) Sterol synthesis inWT mice and Spf �/� mice fed CR-LPF diet without (Control)

Figure 5. Hepatic cholesterol synthesis regulation modelunder fasting conditions. Hepatic cholesterol synthesis isgoverned by two mechanisms under fasting conditions; therepression of biosynthesis enzymes through the inhibition ofthe transcription factor SREBP-2 (25, 46) and the inductionof SPF through the activation of the nuclear receptor PPAR-�.See text for details.

or with supplementation of 0.2% fenofibrate. The rates ofhepatic 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 ofHMG-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 supplementa-tion of 0.2% fenofibrate (Feno). Data show mean values sem. For each group, n � 3. *P 0.05 compared to controlgroup.


intestine (11), and Spf �/� mice develop without appar-ent phenotypic abnormalities. Future studies must elu-cidate the practical possibilities of using SPF as a drugtarget.

We thank T. Kodama for the anti-HMG-coenzyme A reduc-tase mAb. This work was supported by grants from the JapanSociety for the Promotion of Science. N.S. was a researchfellow of the Special Postdoctoral Researchers Program,RIKEN.

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


The FASEB Journal • FJ Express Summary

Regulation of hepatic cholesterol synthesis bya novel protein (SPF) that acceleratescholesterol biosynthesis

Norihito Shibata,*,† Kou-ichi Jishage,‡ Makoto Arita,* Miho Watanabe,‡ Yosuke Kawase,‡

Kiyotaka Nishikawa,§ Yasuhiro Natori,§ Hiroyasu Inoue,� Hitoshi Shimano,¶

Nobuhiro Yamada,¶ Masafumi Tsujimoto,† and Hiroyuki Arai*,1

*Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo,Tokyo, Japan; †Laboratory of Cellular Biochemistry, Riken, Saitama, Japan; ‡Pharmacology andPathology 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 ClinicalMedicine, University of Tsukuba, Ibaraki, Japan

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6368fje

SPECIFIC AIMS

Supernatant protein factor (SPF) is a novel cholesterolbiosynthesis-accelerating protein that promotes the activ-ity of squalene monooxygenase, a rate-limiting enzyme inthe late stages of cholesterol biosynthesis, and expressesabundantly in the liver and small intestine. Although SPFmay play an important role in the tissue-specific regula-tion of cholesterol metabolism in vivo, no information wasavailable on the physiological function of SPF. The aim ofthis study was to elucidate the physiological function ofSPF by using SPF-deficient mice.

PRINCIPAL FINDINGS

1. Fasting decreases plasma cholesterol levels andhepatic cholesterol synthesis in Spf �/� mice

As shown previously in rats, the microsomal squalenemonooxygenase activity was enhanced by adding thehepatic cytosolic fraction in wild-type (WT) mice, whereasthe hepatic cytosolic fraction obtained from Spf �/� miceshowed substantially no effect on microsomal squalenemonooxygenase activity, indicating that SPF is a majorcytosolic component for stimulating hepatic squalenemonooxygenase activity. Spf �/� mice developed normallyand did not display apparent phenotypic abnormalities.Furthermore, plasma cholesterol, triglyceride concentra-tions, and plasma lipoprotein profiles were unaffectedunder normal diet conditions.

Following these experiments, the effect of variousnutritional regimens was examined in Spf �/� mice andit was found that the plasma cholesterol levels differedunder fasting conditions between WT and Spf �/� mice.

Consistent with previous studies, fasting for 24 h or 48 hlowered plasma triglyceride and glucose levels in WTmice, whereas no change was observed in plasmacholesterol levels (Fig. 1A). In contrast, fasting de-creased plasma cholesterol levels in the Spf �/� mice to�70% compared with WT mice (Fig. 1A). Plasmatriglyceride and glucose levels were decreased to levelssimilar to those in WT mice.

To examine the rate of hepatic cholesterol synthesisin Spf �/� mice under fasting conditions, [3H] waterwas used as a precursor for the sterol biosyntheticpathway. Following 24 h fasting, hepatic cholesterolsynthesis was reduced to approximately one-half in WTmice, while a more pronounced reduction in thehepatic cholesterol synthesis was observed in Spf �/�

mice (Fig. 1B). The rate of hepatic VLDL-cholesterolsecretion was also examined by using Triton WR1339,which abruptly inhibits VLDL lipolysis. Consistent withthe decreased rate of hepatic cholesterol biosynthesisin Spf �/� mice, the rate of VLDL-cholesterol secretionin fasted Spf �/� mice was lower than in fasted WT mice(Fig. 1C, closed squares), whereas no change wasobserved between WT and Spf �/� mice under normaldiet conditions (Fig. 1C, open squares).

Next, we examined the effect of fasting on theexpressions of hepatic SPF and various cholesterogenicenzymes. It has previously been demonstrated thatfasting dramatically reduces the mRNAs for cholestero-

1 Correspondence: Department of Health Chemistry, Grad-uate School of Pharmaceutical Sciences, University of Tokyo,7–3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail:[email protected]

doi: 10.1096/fj.06-6368fje

2642 0892-6638/06/0020-2642 © FASEB

genic enzymes due to the fall in nuclear SREBP-2. Asexpected, fasting markedly reduced the mRNA of cho-lesterogenic enzymes, such as HMG-coenzyme A syn-thase, HMG-coenzyme A reductase (Fig. 1D), squalenesynthase, squalene monooxygenase, and lanosterol syn-thase (data not shown) in both the WT and Spf �/�

mice. In contrast to the cholesterogenic enzymes, he-patic SPF mRNA, and protein levels increased signifi-cantly under fasting conditions (Fig. 1E).

These results indicate that the significant decrease inplasma cholesterol level of Spf �/� mice under fastingconditions (Fig. 1A) can at least in part be attributed tothe more pronounced reduction in hepatic cholesterolsynthesis and VLDL-cholesterol secretion in these ani-mals (Fig. 1B, C). It can, therefore, be postulated that inWT mice, SPF compensates the decrease of cholesterolsynthesis under fasting conditions through an up-regu-lation of SPF expression.

2. SPF expression is PPAR-�-dependent

The nuclear receptor PPAR-� is known to play a role inregulating the hepatic transcriptional response to fast-ing. As described above, hepatic SPF mRNA and pro-tein expressions were up-regulated under fasting con-ditions in WT mice. We then examined SPF expressionusing Ppar-��/� mice and found that fasting did not

affect hepatic SPF expression in Ppar-��/� mice, indi-cating that fasting up-regulated hepatic SPF expressionthrough PPAR-�.

Then the effect of two fibrates was examined, both ofwhich are agonists of PPAR-�. Feeding WT mice a dietsupplemented with 0.2% fenofibrate or 0.2% ciprofi-brate for 6 d significantly increased hepatic SPF mRNAand protein levels. No significant difference was ob-served in hepatic SPF protein levels on treatment withfenofibrate in Ppar-��/� mice, indicating that fibrate-induced up-regulation of SPF is mediated by PPAR-�.

However, agonists of other nuclear receptors thatplay important roles in regulating hepatic lipid metab-olism (PPAR-�, PPAR- �, liver X receptor �, and farne-sol X receptor) did not exhibit any effect on hepaticSPF expression. We also tested the effect of dietarycholesterol on the expression of hepatic SPF. WT micewere fed a cholesterol-deficient or cholesterol-supple-mented diet for 6 d. Although significant reduction ofmRNA for cholesterogenic enzymes, such as HMG-coen-zyme A synthase and HMG-coenzyme A reductase, wereobserved under cholesterol-supplemented diet condi-tions, no significant differences were observed in SPFmRNA and protein levels between mice fed the choles-terol-deficient or cholesterol-supplemented diet, suggest-ing that SPF does not belong to the family of proteins thatare transcriptionally regulated by SREBP-2.

Figure 1. Plasma cholesterol levels and hepaticcholesterol metabolism in Spf �/� mice vs. WTmice under fasting conditions. A) Plasma lipidsand glucose levels of WT mice and Spf �/� micein nonfasted and fasted animals. Plasma choles-terol, triglyceride, and glucose levels collectedafter 0, 24, 48 h of fasting. Samples (0 h) wereobtained before fasting. Data show mean val-ues � sem. For each group, n � 5. *P � 0.05compared to the nonfasted mice. B) Sterolsynthesis in WT mice (WT) and Spf �/� miceunder nonfasted (Fed) or 24 h fasted conditions(Fasted). The rates of hepatic sterol synthesisare expressed as the nmol of [3H] water incor-porated 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 WTmice. C) Lipoprotein distribution of cholesterolafter 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 posi-tions of VLDL and HDL are indicated. D) ThemRNA levels of hepatic cholesterogenic en-zymes of WT mice and Spf �/� mice undernonfasted (Fed) or 24 h fasted conditions(Fasted). E) Hepatic expression of SPF mRNAand protein in WT mice (wild-type) undernonfasted (Fed) or 24 h fasted (Fasted) condi-tions. Each value represents the amount ofmRNA relative to that of the fed WT mice. Datashow mean values � sem. For each group, n �3. *P � 0.05 compared to the fed mice.

2643REGULATION OF HEPATIC CHOLESTEROL SYNTHESIS UNDER FASTING CONDITION

3. Fibrates reduce plasma cholesterol levels inSpf �/� mice

Fibrates efficiently lower plasma triglyceride levels buthave no effect on plasma cholesterol levels. Then, weexplored the effect of fibrates on plasma lipid levels inSpf �/� mice. As shown previously, plasma triglyceridelevels were decreased by fibrate treatment in both WTand Spf �/� mice. Interestingly, plasma cholesterollevels were also reduced significantly by fibrate treat-ment in Spf �/� mice but not WT mice. The reductionof plasma cholesterol levels in Spf �/� mice by fibratetreatment was dose-dependent. We also examined theeffect of fibrates on hepatic cholesterol synthesis andfound that the rates of hepatic cholesterol synthesiswere reduced by fibrate treatment in Spf �/� mice,whereas no significant decrease was observed in WTmice. Fibrate treatment reduced protein levels andactivity of hepatic HMG-coenzyme A reductase in bothWT mice and Spf �/� mice, as previously reported. Itcan be speculated that the reduction of this enzymeafter fibrate treatment is a cause for the decrease inhepatic cholesterol synthesis in Spf �/� mice. In WTmice receiving fibrate treatment, cholesterol synthesisis adequately maintained, probably because the de-crease in HMG-coenzyme A reductase is compensatedby SPF up-regulation.

CONCLUSIONS AND SIGNIFICANCE

To determine how SPF affects cholesterol metabolismin vivo, we generated mice lacking the Spf gene andfound that this gene is closely involved in hepaticcholesterol synthesis during fasting. Although mam-mals are forced to continuously adjust their sterolcontent by regulating their hepatic cholesterol metab-olism, much is not known about the changes in hepaticcholesterol metabolism under fasting conditions. Pre-vious studies have demonstrated that fasting causes adramatic reduction in the mRNA levels of hepaticcholesterogenic enzymes. However, plasma cholesterollevels are hardly affected by fasting. This suggests thepresence of a compensatory mechanism to maintaincholesterol levels even under extreme conditions suchas fasting. The results presented here showed thathepatic cholesterol synthesis and plasma cholesterollevels in fasting Spf �/� mice were much lower than infasting WT mice (Fig. 1A, B). Moreover, hepatic SPF

expression was increased �1.5-fold by fasting in WTmice (Fig. 1E). These results indicate that SPF plays arole in compensating hepatic cholesterol synthesis dur-ing fasting (Fig. 2).

Our results also suggest that SPF is a highly usefultherapeutic target for hypercholesterolemia. Fibratedrugs effectively lower serum triglyceride levels byup-regulating genes involved in the cellular uptake and-oxidation of fatty acids, whereas plasma cholesterollevels are not affected by fibrate treatment. We demon-strated that the administration of fibrate to Spf �/�

mice resulted in a significant decrease of plasma cho-lesterol concentrations, suggesting that co-administra-tion of fibrates and an SPF inhibitor may reduce notonly plasma triglyceride but also cholesterol levels.HMG-coenzyme A reductase is a key rate-limiting en-zyme in the cholesterol biosynthetic pathway, and sev-eral HMG-coenzyme A reductase inhibitors (statins)have been developed and are now in clinical use.However, at times these inhibitors induce adverse he-patic and muscular effects. Moreover, statin-fibratecombination therapy is a well-known risk factor formyopathy, rhabdomyolysis, and severe renal failure.Therefore, research is currently focusing on the devel-opment of new target reagents that can suppress cho-lesterol biosynthesis. SPF is expressed selectively in theliver and intestine, and Spf �/� mice develop withoutapparent phenotypic abnormalities, indicating the practi-cal possibilities of using SPF as a drug target.

Figure 2. Hepatic cholesterol synthesis regulation modelunder fasting conditions. Hepatic cholesterol synthesis isgoverned by two mechanisms under fasting conditions; therepression of biosynthesis enzymes through the inhibition ofthe transcription factor SREBP-2 and the induction of SPFthrough the activation of the nuclear receptor PPAR-�. Seetext for details.

2644 Vol. 20 December 2006 SHIBATA ET AL.The FASEB Journal

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