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* Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan;
Laboratory of Cellular Biochemistry, Riken, Saitama, Japan;
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
SPECIFIC AIMS
Supernatant protein factor (SPF) is a novel cholesterol biosynthesis-accelerating protein that promotes the activity of squalene monooxygenase, a rate-limiting enzyme in the late stages of cholesterol biosynthesis, and expresses abundantly in the liver and small intestine. Although SPF may play an important role in the tissue-specific regulation of cholesterol metabolism in vivo, no information was available on the physiological function of SPF. The aim of this study was to elucidate the physiological function of SPF by using SPF-deficient mice.
PRINCIPAL FINDINGS
1. Fasting decreases plasma cholesterol levels and hepatic cholesterol synthesis in Spf–/– mice
As shown previously in rats, the microsomal squalene monooxygenase activity was enhanced by adding the hepatic cytosolic fraction in wild-type (WT) mice, whereas the hepatic cytosolic fraction obtained from Spf –/– mice showed substantially no effect on microsomal squalene monooxygenase activity, indicating that SPF is a major cytosolic component for stimulating hepatic squalene monooxygenase activity. Spf –/– mice developed normally and did not display apparent phenotypic abnormalities. Furthermore, plasma cholesterol, triglyceride concentrations, and plasma lipoprotein profiles were unaffected under normal diet conditions.
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, fasting for 24 h or 48 h lowered plasma triglyceride and glucose levels in WT mice, whereas no change was observed in plasma cholesterol levels (Fig. 1
A). In contrast, fasting decreased plasma cholesterol levels in the Spf –/– mice to 
70% compared with WT mice (Fig. 1A
). Plasma triglyceride and glucose levels were decreased to levels similar to those in WT mice.
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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. 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. 1B
). 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. 1C
, closed squares), whereas no change was observed between WT and Spf –/– mice under normal diet conditions (Fig. 1C
, 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 SREBP-2. As expected, fasting markedly reduced the mRNA of cholesterogenic enzymes, such as HMG-coenzyme A synthase, HMG-coenzyme A reductase (Fig. 1D
), squalene synthase, squalene monooxygenase, and lanosterol synthase (data not shown) in both the WT and Spf –/– mice. In contrast to the cholesterogenic enzymes, hepatic SPF mRNA, and protein levels increased significantly under fasting conditions (Fig. 1E
).
These results indicate that the significant decrease in plasma cholesterol level of Spf –/– mice under fasting conditions (Fig. 1A
) can at least in part be attributed to the more pronounced reduction in hepatic cholesterol synthesis and VLDL-cholesterol secretion in these animals (Fig. 1B, 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.
2. SPF expression is PPAR-
-dependent
The nuclear receptor PPAR- is known to play a role in regulating the hepatic transcriptional response to fasting. 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-–/– mice and found that fasting did not affect hepatic SPF expression in Ppar-–/– mice, indicating that fasting up-regulated hepatic SPF expression through PPAR-.
Then the effect of two fibrates was examined, both of which are agonists of PPAR-. 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. No significant difference was observed in hepatic SPF protein levels on treatment with fenofibrate in Ppar-–/– mice, indicating that fibrate-induced up-regulation of SPF is mediated by PPAR-.
However, agonists of other nuclear receptors that play important roles in regulating hepatic lipid metabolism (PPAR-
, PPAR- 
, liver X receptor , and farnesol X receptor) did not exhibit any effect on hepatic SPF expression. 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, suggesting that SPF does not belong to the family of proteins that are transcriptionally regulated by SREBP-2.
3. Fibrates reduce plasma cholesterol levels in Spf–/– mice
Fibrates efficiently lower plasma triglyceride levels but have no effect on plasma cholesterol levels. Then, we explored the effect of fibrates on plasma lipid levels in Spf –/– mice. As shown previously, plasma triglyceride levels were decreased by fibrate treatment in both WT and Spf –/– mice. Interestingly, plasma cholesterol levels were also reduced significantly by fibrate treatment in Spf –/– mice but not WT mice. The reduction of plasma cholesterol levels in Spf –/– mice by fibrate treatment was dose-dependent. 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. Fibrate treatment reduced protein levels and activity of hepatic HMG-coenzyme A reductase in both WT mice and Spf –/– mice, as previously reported. 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.
CONCLUSIONS AND SIGNIFICANCE
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. Although mammals are forced to continuously adjust their sterol content by regulating their hepatic cholesterol metabolism, 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. However, plasma cholesterol levels are hardly affected by fasting. This suggests the presence of a compensatory mechanism to maintain cholesterol levels even under extreme conditions such as fasting. The results presented here showed that hepatic cholesterol synthesis and plasma cholesterol levels in fasting Spf –/– mice were much lower than in fasting WT mice (Fig. 1A, B
). Moreover, hepatic SPF expression was increased 1.5-fold by fasting in WT mice (Fig. 1E
). These results indicate that SPF plays a role in compensating hepatic cholesterol synthesis during fasting (Fig. 2
).
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Our results also suggest that SPF is a highly useful therapeutic target for hypercholesterolemia. Fibrate drugs effectively lower serum triglyceride levels by up-regulating genes involved in the cellular uptake and ßbeta;-oxidation of fatty acids, whereas plasma cholesterol levels are not affected by fibrate treatment. 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. HMG-coenzyme A reductase is a key rate-limiting enzyme in the cholesterol biosynthetic pathway, and several HMG-coenzyme A reductase inhibitors (statins) have been developed and are now in clinical use. However, at times these inhibitors induce adverse hepatic and muscular effects. Moreover, statin-fibrate combination therapy is a well-known risk factor for myopathy, rhabdomyolysis, and severe renal failure. 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, and Spf –/– mice develop without apparent phenotypic abnormalities, indicating the practical possibilities of using SPF as a drug target.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6368fje
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