[go: up one dir, main page]
Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2010 Jul 29.
Published in final edited form as: Lipids. 2009 Apr 17;44(6):511–519. doi: 10.1007/s11745-009-3299-1

StAR overexpression decreases serum and tissue lipids in apolipoprotein E-deficient mice

Yanxia Ning #, Leyuan Xu #, Shunlin Ren ‡, William M Pandak ‡, Sifeng Chen #,*, Lianhua Yin #,*
PMCID: PMC2911774  NIHMSID: NIHMS219386  PMID: 19373502

Abstract

Cholesterol metabolism as initiated by mitochondrial CYP27A1 is a ubiquitous pathway capable of synthesizing multiple key regulatory oxysterols involved in lipids homeostasis. Previously we have shown that the regulation of its activities within hepatocytes is highly controlled by the rate of mitochondrial cholesterol delivery. In the present study, we hypothesized that increasing expression of the mitochondrial cholesterol delivery protein, StAR, is able to lower lipids accumulation in liver, aortic wall, as well as in serum in a well-documented animal model, apoE−/− mice. ApoE−/− mice, characterized by increased serum, liver, and endothelial cholesterol and triglyceride levels by 3 months of age, were infected with recombinant CMV-StAR adenovirus to increase StAR protein expression. Six days following infection, serum total cholesterol and triglycerides had decreased 19 % (P<0.01) and 30% (P<0.01), respectively, with a compensatory 40% (P<0.01) increase in serum HDL-cholesterol in increased StAR expressing mice as compared to controls (no or control virus). Histologic and biochemical analysis of the liver demonstrated not only a dramatic decrease in cholesterol (↓25%; P<0.01), but an even more marked decrease in triglyceride (↓56%; P<0.01) content. En bloc Sudan IV staining of the aorta revealed a >80% (P<0.01) decrease in neutral lipid staining. This study demonstrates for the first time a possible therapeutic role of the CYP27A1–initiated pathway in the treatment of dyslipidemias.

Keywords: lipids, cholesterol, lipoproteins, steroidogenic acute regulatory protein

Introduction

Dyslipidemias, as characterized by abnormal serum lipoprotein profiles, are a risk factor for atherosclerosis (1). Although current strategies have led to significant advances in their treatment, there is great interest to explore new therapeutic strategies to treat and prevent what remains a leading cause of morbidity and mortality throughout the world.

Steroidogenic acute regulatory protein (StAR/StarD1), an intracellular cholesterol transport protein, was first identified in steroidogenic tissues. This protein is a member of a family that contains a StAR homologue domain that has been found capable of binding and transporting sterols within cells. This family includes the phosphatidylcholine transfer protein (PC-TP/StarD2) (2), MLN64 (StarD3) (3, 4), and the newly discovered StarD4, StarD5, and StarD6 (5–8). All of these proteins have a similar structural lipid-binding domain referred to as the StAR-related lipid transfer (START) domain. StAR, first detected in the adrenal, was shown to facilitate cholesterol delivery to the inner mitochondrial cholesterol side-chain cleavage system in the initiation of steroidogenesis (9, 10). StAR was initially considered to be confined to steroidogenic tissues such as adrenal gland, ovary, and testicle (7, 11). However, StAR has since been found in other tissues/cells inclusive of the liver, brain, endothelium, and macrophages (12–15).

In the liver, StAR has been shown to facilitate cholesterol delivery to the inner membrane of the mitochondria where cholesterol can be oxidized by sterol 27-hydroxylase (CYP27A1) to an important regulatory oxysterol, 27-hydroxycholesterol, before ultimately being metabolized to bile acids (16). Most recently, we have shown that in addition to its metabolism of cholesterol to 27-hydroxycholesterol and subsequently to bile acids, CYP27A1 is also capable of metabolizing cholesterol to 25-hydroxycholesterol with subsequent conversion to its sulfated form (17). Like 27-hydroxycholesterol, both 25-hydroxycholesterol and sulfated 25-hydroxycholesterol are regulators of intracellular lipid metabolism (17–19). Both 27- and 25-hydroxycholesterol are known to down-regulate cholesterol synthesis while stimulating cell cholesterol secretion (18, 19). Sulfated 25-hydroxycholesterol has recently been shown to decrease fatty acid synthase as well as activate a nuclear receptor responsible for fatty acid mobilization/secretion (20).

It is possible that increased metabolism of cholesterol as initiated through the CYP27A1 could result in a coordinated lipids lowering response. Increasing StAR expression would be expected to not only increase cholesterol catabolism to bile acids as we have previously shown, but produce key regulatory oxysterols of intracellular lipid homeostasis (12, 16, 21) In support of this supposition, our recent in vitro findings in THP-1 macrophages showed that up-regulation of StAR induced a coordinated mobilization and reduction of cell cholesterol (14). Therefore, it seems reasonable to hypothesize that the StAR/CYP27A1-initiated metabolism of cholesterol is not only a pathway of cholesterol catabolism, but an important intracellular pathway capable of directing intracellular lipid homeostasis.

Apolipoprotein-E mediates the clearance of serum lipids such as cholesterol and triglyceride (22). Apolipoprotein E–deficient (apoE−/−) mice develop hypercholesterolemia as a result of an accumulation of chylomicron remnants, very low-density lipoproteins (VLDL), and intermediate-density lipoproteins (IDL) (23). Mice homozygous for the inactivated apoE gene spontaneously develop hypercholesterolemia and aortic lipid accumulation (24, 25). The lipid lesions which develop in the aortas of mice fed a standard rodent chow diet are similar to the early lipid accumulations found in humans (23, 26, 27). In addition, apoE−/− mice are also known to develop significant hepatic lipid (i.e. cholesterol/triglyceride) accumulation. Therefore, apoE−/− mice provide not only a practical model for the study of early developmental atherosclerosis (28), but an excellent model for the outlined study.

The described trial was simply designed as “an in vivo proof of concept” that increasing CYP27A1-initiated cholesterol metabolism as facilitated by the mitochondria-directed cholesterol transport protein, StAR, could represent a novel approach to reduce serum and tissue cholesterol and triglyceride levels. As shown, increased StAR expression led to a coordinated response in serum triglycerides and HDL cholesterol. Furthermore, hepatic cholesterol was significantly reduced. Of equal importance, neutral lipid levels in the serum, liver, and aorta were also dramatically reduced in this classic animal model of dyslipidemia.

Experimental Procedure

Materials

TRIzol regent and SuperScript TMIII First–Strand Synthesis System for RT-PCR were purchased from Invitrogen (Carlsbad, CA). Taq DNA Polymerase, dNTP mix and PageRuler Prestained Protein Ladder were purchased from Fermentas MBI (San Diego, CA). Primary antibody against StAR and GAPDH were purchased from Abcam Ltd (Cambridge Science Park, Cambridge, UK) and Kangcheng Bio-Tech (Shanghai, China), respectively. Second antibody against rabbit and mouse IgG were obtained from Kirkegaard & Perry Laboratories (Guildford, UK). SuperSignal West Pico Chemiluminescent Substrate was obtained from Pierce Biotechnology, Inc (Rockford, IL). All other reagents were from Sigma-Aldrich Chemical Co (St. Louis, MO) unless otherwise indicated.

Experimental Animals

C57BL/6J mice and homozygous apoE-deficient (apoE−/−) mice (same inherited background as the C57BL/6J mice) were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center (Beijing, China). The animals were allowed to acclimatize for 1 week while being maintained at a room temperature of 22 ± 2 °C on a 12 h light/dark cycle with free access to standard rodent chow food and water (Standard sustain feed, from Institute of Laboratory Animal Science, Shanghai, China). Housing facilities and all experimental protocols were approved by the Animals Care and Use Committee of Fudan University Shanghai Medical College which adopts the guideline for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Ninety-six C57BL/6J and apoE−/− mice of both sexes at 1, 3 and 5 months old were used to determine serum level of lipids. An additional 27 6-month-old male apoE−/− mice were used to investigate the effect of StAR overexpression on serum, liver, and aortic lipid levels. Prior to and post infusion, the animals dietary intake was not significantly different from either control group. Before sacrificing, the mice were fasted for 12 hours. The mice were then anesthetized (60 mg pentobarbital/kg body weight, intravenously) and euthanized. Blood was collected from the retro-orbital plexus and aliquots of plasma were stored at −70 °C until determination of serum parameters as described below. The liver was excised, weighed, snap frozen in liquid nitrogen, and stored at −70 °C until further analysis. The aorta and liver were harvested for gross and histological examination.

Preparation of Recombinant Adenoviruses

The recombinant adenoviruses encoding StAR were prepared as previously described (29). The virus was then amplified by infecting confluent monolayers of human embryonic kidney 293 cells as previously described (30). Aliquots of the amplified virus were stored at −70 °C until used. The control adenoviruses expressing the enhanced green fluorescence protein were purchased from the Vector Gene Technology Company Ltd (Beijing, China). The infection and particle titers of the viruses were determined using plaque and optical density assays, respectively.

Intravenous Infusion of Adenovirus

For the adenovirus infections, the 27 6-month-old apoE−/− mice fed a standard rodent chow diet were equally and randomly divided into 3 groups. Eighteen of mice were tail vein injected with 1×1011 virus particles of Ad-CMV-StAR or Ad-CMV-EGFP. In 9 non-transduced (control) mice the virus was replaced with the same volume of normal saline (NS). Six days after infection, the animals were briefly anesthetized and euthanized after fasting for 12 hours. Blood was collected for determination of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lipid levels. Organs were harvested for histology and biochemical analysis. The histological specimens were fixed in 10% formalin. Additional tissue (liver) was quickly frozen in liquid nitrogen and stored at −70 °C for further analysis.

Determination of Blood Parameters

The levels of triglyceride (TG), total cholesterol (T-CHO), and HDL cholesterol (HDL-CHO) in serum were measured by enzymatic methods (31) according to the manufacturer’s instructions using the detection kit purchased from Rongsheng Biotechnology Company Ltd (Shanghai, China). Serum ALT, AST, and ALP activities were determined by the clinical laboratory at the Longhua Hospital (Shanghai, China).

Histology

After adenoviruses infection, the StAR protein expression in heart, coronary artery and liver were detected by immunohistochemistry by ABC methods as previously described by the authors (13). A portion of liver was harvested and embedded in optimum cutting temperature compound (O.C.T compound, Sakura Finetek, Inc., Torrance, CA), mixed with sucrose (20%), and stored at −80°C. Ten-micrometer cryosections were made lengthwise through the liver lobe. All cryosections were fixed for 1 min in formaldehyde solution, stained for 10 min with Oil Red O (stains lipids red), and counterstained for 1 min with hematoxylin. En face lipid accumulation was determined by removing the aortas from the aortic arch to the ileal bifurcation. The aortas were fixed in 10% neutral buffered formalin. To increase the reliability of lipid quantification, only the aortic segment from left subclavian artery to ileal bifurcation was used for the measurement. The aortas were cut longitudinally, splayed, and pinned in a dish filled with Sudan IV stain for 10 min, destained in 80% ethanol for 5 min, and then photographed. Quantitation of neutral lipid staining was performed by measuring stained area using ImageM software (Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, China). The accumulated stained area in a given aorta was calculated as a percentage of the total surface area of the aorta.

Liver Lipid Measurement

Lipids were extracted from liver according to a published method (32). Triglyceride and cholesterol were measured using the same method described previously for blood lipid analysis (31).

Determination StAR Protein Levels in Tissues

Total proteins of heart, lung, liver, spleen, and adrenal gland from the infected mice were isolated using 1×SDS-PAGE sample buffer (50 mM Tris-Cl buffer, pH 6.8, 2% (w/v) SDS, 2% mercaptoethanol, 10% (v/v) glycerol, and 0.1% (w/v) bromophenol blue). Protein was quantitated using the bicinchoninic acid assay purchased from Pierce (Rockford, IL). Fifty micrograms of total protein were separately on 10% SDS-PAGE gels. Western blot analysis of StAR was performed as previously described by the authors (13).

Statistics

Data were reported as mean ± standard deviation (S.D) and subjected to ANOVA analysis. F-test and the Student-Neuman-Keuls post test analyses were performed on these data to analyze the variances and significances between groups. Statistical significance was defined as P < 0.05.

Results

Plasma Lipid Levels Increased in ApoE−/− Mice

The absence of apoE−/− in humans and animal models is associated with increases in serum, liver, and aortic lipid levels. To simply confirm the apoE−/− mouse model available as representative and as a viable model for examining the effects of increased StAR expression on serum, aortic, and liver lipids, serum lipids were determined at 1, 3, and 5 months of age and compared to C57BL/J6 mice. On a standard rodent chow diet (4.4% fat; 0.06% cholesterol), apoE−/− mice had a significantly higher total cholesterol (T-CHO) and a significantly lower HDL-cholesterol (HDL-CHO) at all time periods vs. age-matched C57BL/6J mice (Table. 1). Serum triglycerides were not significantly different. Of note is that a significant steady decrease in HDL-CHO was observed from month 1 to month 5 in apoE−/− mice. The results indicated that the apoE−/− mice available for study to be a representative and would serve as a viable apoE−/− model. Certain assumptions were made, i.e. that there would be significant liver and aortic lipid accumulation.

Table 1.

Increased plasma lipid levels in apoE−/− micea

Mice Total cholesterol Triglyceride HDL-cholesterol
Female Male Female Male Female Male
Wild type
 1 M 1.98±0.14 2.32±0.25 1.96±0.83 1.60±0.54 1.29±0.02 1.73±0.05
 3M 2.08±0.14 2.22±0.21 1.18±0.59 1.26±0.19 1.52±0.17 1.74±0.21
 5M 1.76±0.34 2.31±0.11 0.94±0.41 0.97±0.95 1.15±0.16 1.97±0.43
 apoE−/−
 1M 7.55±3.42* 7.53±3.26* 0.84±0.53* 1.31±0.52 0.63±0.33* 0.95±0.20*
 3M 9.58±1.53* 7.81±3.88* 1.08±0.64 1.50±0.46 0.56±0.15* 0.78±0.41*
 5M 9.98±0.97 * 8.96±0.67* 1.02±0.25* 1.54±1.29 0.36±0.15* 0.53±0.31*
Open in a new tab
a

Values are mmol/L.

ApoE−/−: apolipoprotein E-deficient; Values are presented as means±S.D; n =6.

Statistical analyses were performed by one-way ANOVA within each age group.

*

P <0.01 versus wild type C57BL/6J mice at the same age.

Recombinant StAR Gene Was Successfully Expressed in ApoE−/− Mice

To determine if StAR protein was being overexpressed following mouse infection with Ad-CMV-StAR, 6 days following infection selected organs were screened for StAR protein expression by immunohistochemistry and Western blotting. After infection, StAR expression was higher in the mice infused with adenovirus encoding CMV-StAR than the other two groups in heart, coronary artery and liver tissue. The increases of StAR in the heart and coronary arteries were marginal (Fig. 1a). StAR protein was detected in the adrenal gland, spleen, liver, and lung of controls (NS: normal saline; Ad-CMV-EGFP), with large amounts of StAR found in the spleen and adrenal gland. Following Ad-CMV-StAR infection, StAR protein was found in all tested tissues with the largest increases found in the liver, a known reservoir for infecting adenovirus (Fig 1b).

Figure 1. Successful gene transduction of StAR in apoE−/− mice fed with normal diet.

Figure 1

Open in a new tab

The StAR expression in paraffin imbedded slices of heart, coronary artery and liver were detected by immunohistochemistry as shown in panel a. Western blot of StAR protein levels in different major organs after StAR overexpression as shown in panel b. NS: apoE−/− mice injected with normal saline. EGFP: apoE−/− mice injected with control adenovirus encoding CMV-EGFP. StAR: apoE−/− mice injected with adenovirus encoding CMV-StAR. Bar = 10μm

Liver Transaminases

Serum ALT, AST, and ALP activity levels in mice were measured to determine if there was liver injury following adenovirus infection. No significant differences were observed among the three groups of NS, Ad-CMV-StAR and Ad-CMV-EGFP (data not shown).

Transduction of Ad-CMV-StAR Decreases Lipid Accumulation in the Serum and Liver of ApoE−/− Mice

Sera

Six days following infection with Ad-CMV-StAR, the serum T-CHO and TG were 19% (P<0.01) and 30% (P<0.01) lower in apoE−/− mice receiving Ad-CMV-StAR than in those receiving NS or Ad-CMV-EGFP, respectively. In contrast, there was a dramatic reversal in the HDL-CHO levels with HDL-CHO in their plasma about 40% higher (P<0.01) than controls (Table 2).

Table 2.

The level of plasma lipids in apolipoprotein E-deficient mice after StAR overexpression a

NS Ad-CMV-EGFP Ad-CMV-StAR
T-CHO 13.53±1.17 13.76±1.21 11.01±0.62**##
TG 1.25±0.30 1.12±0.08 0.82±0.14**#
HDL-CHO 0.73±0.08 0.67±0.13 1.00±0.19**##
Open in a new tab
a

Values are mmol/L.

Values are presented as means±S.D;

NS: apoE−/− mice injected with normal saline. Ad-CMV-EGFP: apoE−/− mice injected with control adenovirus encoding CMV-EGFP. Ad-CMV-StAR: apoE−/− mice injected with adenovirus encoding CMV-StAR.

**

P <0.01 versus NS group

##

P <0.01 versus Ad-CMV-EGFP group

#

P <0.05 versus Ad-CMV-EGFP group.

Liver tissues

Liver sections stained with Oil Red O to evaluate the hepatic accumulation of lipids showed that the lipid levels were significantly less in the livers of Ad-CMV-StAR mice as compared to NS and Ad-CMV-EGFP mice (Fig. 2a). To confirm these results, lipids were extracted from the liver tissues and measured using commercial colorimetric kits for both cholesterol and triglycerides (Rongsheng Biotechnology Company Ltd, Shanghai, China). The hepatic cholesterol and triglyceride concentrations in the Ad-CMV-StAR mice decreased 25% (P<0.01) and 56% (P<0.01) as compared to NS and Ad-CMV-EGFP mice, respectively (Fig. 2b and 2c). There were no significant differences in hepatic cholesterol and triglyceride levels between the NS and Ad-CMV-EGFP groups (Fig. 2b and 2c).

Figure 2. Transduction of Ad-CMV-StAR decreases lipid accumulation in the liver of apoE−/− mice fed with normal diet.

Figure 2

Open in a new tab

a: Oil Red O-stained liver sections (bar = 10μm) of apoE−/− mice. b: Total hepatic cholesterol. c: Hepatic triglyceride. Data are means ± S.D. ** represent P < 0.01 versus NS mice. ## represent P < 0.01versus Ad-CMV-EGFP mice. NS: apoE−/− mice injected with normal saline (n = 11). EGFP: apoE−/− mice injected with control adenovirus encoding CMV-EGFP (n = 9). StAR: apoE−/− mice injected with adenovirus encoding CMV-StAR (n = 9).

Transduction of StAR Reduces Aortic Neutral Lipid Accumulation in ApoE−/− Mice

A previous study has shown that apoE−/− mice will develop visible aortic lipid accumulation at 20 weeks on a normal mouse diet (23). We found the microscopic presence of aortic lipid accumulation by the 3rd month with accumulation grossly visible by the 5th month in apoE−/− mice fed a similar diet. Six days following Ad-CMV-StAR transduction, examination en face of aortas from Ad-CMV-StAR injected mice using ImageM software revealed significantly less neutral lipid than in NS or Ad-CMV-EGFP mice (Fig. 3a). The amount of lipid accumulation in aortas between NS and Ad-CMV-EGFP mice was found to not be different (Fig 3a). The accumulated area of lipid stained lesions had decreased from 21.03 ± 2.66 % and 19.42 ± 2.39% in NS and Ad-CMV-EGFP mice, respectively, to 3.74 ± 1.57% (Fig. 3b).

Figure 3. Effect of transduction of StAR on aortic lipid accumulation in apoE−/− mice.

Figure 3

Open in a new tab

a: En face staining of Sudan IV-stained aortas of mice fed with normal diet. Arrows indicate neutral lipid stained plaques. b: Quantification of neutral lipid staining presented as percentage of total en face aorta surface area of mice fed with normal diet (see “Methods”). ** represent P < 0.01 versus NS. ## represent P< 0.01 versus Ad-CMV-EGFP. NS, EGFP and StAR represent apoE−/− mice injected with normal saline, control adenovirus encoding CMV-EGFP, and adenovirus encoding CMV-StAR, respectively.

Discussion

Hyperlipidemia is an important risk factor for atherosclerosis. The treatment response to current medications is frequently inadequate. Therefore, there remains great interest to develop new therapeutic strategies. Based upon recent observations, it was hypothesized that facilitating mitochondria-directed cholesterol transport could elicit in an in vivo model a sequence of metabolic events resulting in both serum and tissue cholesterol lowering. The current study was designed to explore this hypothesis by increasing expression of StAR, a mitochondrial directed cholesterol transport protein, in a well-described animal model of dyslipidemia.

Cholesterol metabolism as initiated via mitochondrial CYP27A1 is subsequently metabolized to key regulatory oxysterols of cholesterol homeostasis; and within the liver, continued catabolism to bile acids. For proper function, inner mitochondrial cholesterol levels must be tightly controlled, and it has been suggested that the key function of CYP27A1 is to initiate the metabolism of cholesterol to a metabolite that can be more easily mobilized out of the mitochondria. As CYP27A1 has been detected in all examined peripheral tissues as well as the liver, others have suggested that the conversion of cholesterol to 27-hydroxycholesterol as important under conditions of cholesterol excess. Under these conditions it has been proposed that the 27-hydroxylation can facilitate a process of reverse cholesterol transport of cholesterol from peripheral tissues to the liver with 27-hydroxycholesterol’s subsequent elimination as bile acids (16, 33–36). In support of this hypothesis, CTX (Cerebrotendinous xanthomatosis), characterized by the inability of cells to 27-hydroxylate sterols, is associated in humans with early dementia, premature atherosclerosis, and cataracts due to abnormal tissue sterol accumulation (37, 38). As a function of these observations, CYP27A1 was overexpressed in transgenic mice, but interpreted as having little appreciable effect on lipid metabolism (39). We also found that following overexpression of CYP27A1 in hepatocyte cultures only a modest increase in the rates of bile acid synthesis was observed as compared to the 8 to 10-fold increase consistently seen following CYP7A1 overexpression (40). This suggested that inner mitochondrial substrate availability could be the rate-determining step in the CYP27A1-initiated pathway.

As described, StAR has been shown to not only facilitate cholesterol delivery to the inner mitochondrial cholesterol side-chain cleavage system in the initiation of steroidogenesis, but that increasing its expression would markedly increase steroidogenesis (9, 10). Applying this concept to hepatocytes, we demonstrated that increased StAR expression markedly increased the rates of bile acid synthesis (BAS) in both in vitro and in vivo models (16, 21). Our inability to up-regulate bile acid synthesis in CYP27A1−/− mouse hepatocytes demonstrated that StAR’s effects were mediated through the CYP27A1-initiated pathway (17).

Recently, Ren et al. demonstrated for the first time that CYP27A1 also metabolizes cholesterol to 25-hydroxycholesterol. 25-hydroxycholesterol, in a manner like 27-hydroxycholesterol, can down-regulate cholesterol synthesis, stimulate cholesterol secretion and stimulate fatty acid synthesis (17, 18). Sulfated 25-hydroxycholesterol, a newly uncovered and subsequent metabolite of 25-hydroxycholesterol, like its precursor, also down-regulates cholesterol synthesis (17–19). However, in direct contrast to 25-hydroxycholesterol and 27-hydroxycholesterol, sulfated 25-hydroxycholesterol down-regulates fatty acid synthetase (19). Furthermore, it is capable of activating PPARγ, a nuclear receptor known to play a role in the redistribution body fat as well as increase tissue insulin responsiveness (41, 42). All three oxysterols are increased following increased StAR expression as evidenced by their increased cytosolic and nuclear levels (16, 18, 21, 30). Furthermore, in addition to their intestinal lipid solubilization properties, bile acids, an end product of cholesterol/27-hydroxycholesterol metabolism, are also known regulators of key steps of lipid homeostasis (43–45).

Based on these observations, we hypothesized that increased cholesterol metabolism as initiated by the CYP27A1 pathway and stimulated by increased StAR expression would result, not only in increased cholesterol elimination as bile acids, but elicit a homeostatic response as a function of increased levels of at least three known regulatory oxysterols. More specifically, these oxysterols, working in a coordinate fashion, should lead to decreased cholesterol synthesis and increased cholesterol export; responses that would be expected to decrease serum and tissue cholesterol levels. Although not originally considered, a metabolic response in fatty acid metabolism should have been entertained given how tightly cholesterol and fatty acid metabolism are interwoven.

The apoE−/− mouse was chosen as the animal model to explore this hypothesis as it is a well studied model of atherogenesis that on a rodent chow diet will develop serum and tissue dyslipidemia as it ages. The model was first confirmed with serum lipid analysis. As predicted, based upon prior in vivo studies in rats and mice, 6 month old mice tolerated the level of increased StAR expression for 6 days without any detectable adverse effects or dietary differences from controls. Increased StAR expression led to dramatic normalization of both serum and tissue cholesterol levels in apoE−/− mice. Serum and tissue triglyceride levels demonstrated an even greater response. These responses demonstrate for the first time a possible therapeutic role for the CYP27A1-initiated pathway in the treatment of dyslipidemias.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (30871021) of the People’s Republic of China (L.H), and the United States National Institutes of Health R01HL078898 (S.R.).

References

  • 1.Ansell BJ. Hyperlipidaemia and cardiovascular disease. Curr Opin Lipidol. 2008;19:433–434. doi: 10.1097/MOL.0b013e328308b63d. [DOI] [PubMed] [Google Scholar]
  • 2.Cohen DE, Green RM, Wu MK, Beier DR. Cloning, tissue-specific expression, gene structure and chromosomal localization of human phosphatidylcholine transfer protein. Biochim Biophys Acta. 1999;1447:265–270. doi: 10.1016/s0167-4781(99)00163-3. [DOI] [PubMed] [Google Scholar]
  • 3.Alpy F, Stoeckel ME, Dierich A, Escola JM, Wendling C, Chenard MP, Vanier MT, Gruenberg J, Tomasetto C, Rio MC. The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein. J Biol Chem. 2001;276:4261–4269. doi: 10.1074/jbc.M006279200. [DOI] [PubMed] [Google Scholar]
  • 4.Watari H, Arakane F, Moog-Lutz C, Kallen CB, Tomasetto C, Gerton GL, Rio MC, Baker ME, Strauss JF., III MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis. Proc Natl Acad Sci U S A. 1997;94:8462–8467. doi: 10.1073/pnas.94.16.8462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Romanowski MJ, Soccio RE, Breslow JL, Burley SK. Crystal structure of the Mus musculus cholesterol-regulated START protein 4 (StarD4) containing a StAR-related lipid transfer domain. Proc Natl Acad Sci U S A. 2002;99:6949–6954. doi: 10.1073/pnas.052140699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Tsujishita Y, Hurley JH. Structure and lipid transport mechanism of a StAR-related domain. Nat Struct Biol. 2000;7:408–414. doi: 10.1038/75192. [DOI] [PubMed] [Google Scholar]
  • 7.Maxfield FR, Wustner D. Intracellular cholesterol transport. J Clin Invest. 2002;110:891–898. doi: 10.1172/JCI16500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Soccio RE, Adams RM, Maxwell KN, Breslow JL. Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress. J Biol Chem. 2005;280:19410–19418. doi: 10.1074/jbc.M501778200. [DOI] [PubMed] [Google Scholar]
  • 9.Christenson LK, Strauss JF., III Steroidogenic acute regulatory protein (StAR) and the intramitochondrial translocation of cholesterol. Biochim Biophys Acta. 2000;1529:175–187. doi: 10.1016/s1388-1981(00)00147-5. [DOI] [PubMed] [Google Scholar]
  • 10.Stocco DM. Intramitochondrial cholesterol transfer. Biochim Biophys Acta. 2000;1486:184–197. doi: 10.1016/s1388-1981(00)00056-1. [DOI] [PubMed] [Google Scholar]
  • 11.Jefcoate C. High-flux mitochondrial cholesterol trafficking, a specialized function of the adrenal cortex. J Clin Invest. 2002;110:881–890. doi: 10.1172/JCI16771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hall EA, Ren S, Hylemon PB, Rodriguez-Agudo D, Redford K, Marques D, Kang D, Gil G, Pandak WM. Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells. Biochim Biophys Acta. 2005;1733:111–119. doi: 10.1016/j.bbalip.2005.01.004. [DOI] [PubMed] [Google Scholar]
  • 13.Ning Y, Chen S, Li X, Ma Y, Zhao F, Yin L. Cholesterol, LDL, and 25-hydroxycholesterol regulate expression of the steroidogenic acute regulatory protein in microvascular endothelial cell line (bEnd.3) Biochem Biophys Res Commun. 2006;342:1249–1256. doi: 10.1016/j.bbrc.2006.02.093. [DOI] [PubMed] [Google Scholar]
  • 14.Ning Y, Bai Q, Lu H, Li X, Pandak WM, Zhao F, Chen S, Ren S, Yin L. Overexpression of mitochondrial cholesterol delivery protein, StAR, decreases intracellular lipids and inflammatory factors secretion in macrophages. Atherosclerosis. 2008 doi: 10.1016/j.atherosclerosis.2008.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ma Y, Ren S, Pandak WM, Li X, Ning Y, Lu C, Zhao F, Yin L. The effects of inflammatory cytokines on steroidogenic acute regulatory protein expression in macrophages. Inflamm Res. 2007;56:495–501. doi: 10.1007/s00011-007-6133-3. [DOI] [PubMed] [Google Scholar]
  • 16.Pandak WM, Ren S, Marques D, Hall E, Redford K, Mallonee D, Bohdan P, Heuman D, Gil G, Hylemon P. Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes. J Biol Chem. 2002;277:48158–48164. doi: 10.1074/jbc.M205244200. [DOI] [PubMed] [Google Scholar]
  • 17.Li X, Pandak WM, Erickson SK, Ma Y, Yin L, Hylemon P, Ren S. Biosynthesis of the regulatory oxysterol, 5-cholesten-3beta,25-diol 3-sulfate, in hepatocytes. J Lipid Res. 2007;48:2587–2596. doi: 10.1194/jlr.M700301-JLR200. [DOI] [PubMed] [Google Scholar]
  • 18.Ren S, Hylemon P, Zhang ZP, Rodriguez-Agudo D, Marques D, Li X, Zhou H, Gil G, Pandak WM. Identification of a novel sulfonated oxysterol, 5-cholesten-3beta,25-diol 3-sulfonate, in hepatocyte nuclei and mitochondria. J Lipid Res. 2006;47:1081–1090. doi: 10.1194/jlr.M600019-JLR200. [DOI] [PubMed] [Google Scholar]
  • 19.Ren S, Li X, Rodriguez-Agudo D, Gil G, Hylemon P, Pandak WM. Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes. Biochem Biophys Res Commun. 2007;360:802–808. doi: 10.1016/j.bbrc.2007.06.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Ma Y, Xu L, Rodriguez-Agudo D, Li X, Heuman DM, Hylemon PB, Pandak WM, Ren S. 25-Hydroxycholesterol-3-sulfate regulates macrophage lipid metabolism via the LXR/SREBP-1 signaling pathway. Am J Physiol Endocrinol Metab. 2008;295:E1369–E1379. doi: 10.1152/ajpendo.90555.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ren S, Hylemon PB, Marques D, Gurley E, Bodhan P, Hall E, Redford K, Gil G, Pandak WM. Overexpression of cholesterol transporter StAR increases in vivo rates of bile acid synthesis in the rat and mouse. Hepatology. 2004;40:910–917. doi: 10.1002/hep.20382. [DOI] [PubMed] [Google Scholar]
  • 22.Harris JD, Schepelmann S, Athanasopoulos T, Graham IR, Stannard AK, Mohri Z, Hill V, Hassall DG, Owen JS, Dickson G. Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E. Gene Ther. 2002;9:21–29. doi: 10.1038/sj.gt.3301615. [DOI] [PubMed] [Google Scholar]
  • 23.Plump AS, Smith JD, Hayek T, alto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992;71:343–353. doi: 10.1016/0092-8674(92)90362-g. [DOI] [PubMed] [Google Scholar]
  • 24.Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994;14:133–140. doi: 10.1161/01.atv.14.1.133. [DOI] [PubMed] [Google Scholar]
  • 25.d’Uscio LV, Baker TA, Mantilla CB, Smith L, Weiler D, Sieck GC, Katusic ZS. Mechanism of endothelial dysfunction in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2001;21:1017–1022. doi: 10.1161/01.atv.21.6.1017. [DOI] [PubMed] [Google Scholar]
  • 26.Tamminen M, Mottino G, Qiao JH, Breslow JL, Frank JS. Ultrastructure of early lipid accumulation in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 1999;19:847–853. doi: 10.1161/01.atv.19.4.847. [DOI] [PubMed] [Google Scholar]
  • 27.Cyrus T, Witztum JL, Rader DJ, Tangirala R, Fazio S, Linton MF, Funk CD. Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice. J Clin Invest. 1999;103:1597–1604. doi: 10.1172/JCI5897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992;258:468–471. doi: 10.1126/science.1411543. [DOI] [PubMed] [Google Scholar]
  • 29.Pandak WM, Bohdan P, Franklund C, Mallonee DH, Eggertsen G, Bjorkhem I, Gil G, Vlahcevic ZR, Hylemon PB. Expression of sterol 12alpha-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo. Gastroenterology. 2001;120:1801–1809. doi: 10.1053/gast.2001.24833. [DOI] [PubMed] [Google Scholar]
  • 30.Ren S, Hylemon P, Marques D, Hall E, Redford K, Gil G, Pandak WM. Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis. J Lipid Res. 2004;45:2123–2131. doi: 10.1194/jlr.M400233-JLR200. [DOI] [PubMed] [Google Scholar]
  • 31.Belcher JD, Egan JO, Bridgman G, Baker R, Flack JM. A micro-enzymatic method to measure cholesterol and triglyceride in lipoprotein subfractions separated by density gradient ultracentrifugation from 200 microliters of plasma or serum. J Lipid Res. 1991;32:359–370. [PubMed] [Google Scholar]
  • 32.Carlson SE, Goldfarb S. A sensitive enzymatic method for determination of free and esterified tissue cholesterol. Clin Chim Acta. 1977;79:575–582. doi: 10.1016/0009-8981(77)90178-4. [DOI] [PubMed] [Google Scholar]
  • 33.Babiker A, Diczfalusy U. Transport of side-chain oxidized oxysterols in the human circulation. Biochim Biophys Acta. 1998;1392:333–339. doi: 10.1016/s0005-2760(98)00047-2. [DOI] [PubMed] [Google Scholar]
  • 34.Babiker A, Andersson O, Lund E, Xiu RJ, Deeb S, Reshef A, Leitersdorf E, Diczfalusy U, Bjorkhem I. Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport. J Biol Chem. 1997;272:26253–26261. doi: 10.1074/jbc.272.42.26253. [DOI] [PubMed] [Google Scholar]
  • 35.Lund E, Andersson O, Zhang J, Babiker A, Ahlborg G, Diczfalusy U, Einarsson K, Sjovall J, Bjorkhem I. Importance of a novel oxidative mechanism for elimination of intracellular cholesterol in humans. Arterioscler Thromb Vasc Biol. 1996;16:208–212. doi: 10.1161/01.atv.16.2.208. [DOI] [PubMed] [Google Scholar]
  • 36.Westman J, Kallin B, Bjorkhem I, Nilsson J, Diczfalusy U. Sterol 27-hydroxylase- and apoAI/phospholipid-mediated efflux of cholesterol from cholesterol-laden macrophages: evidence for an inverse relation between the two mechanisms. Arterioscler Thromb Vasc Biol. 1998;18:554–561. doi: 10.1161/01.atv.18.4.554. [DOI] [PubMed] [Google Scholar]
  • 37.Cali JJ, Hsieh CL, Francke U, Russell DW. Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis. J Biol Chem. 1991;266:7779–7783. [PMC free article] [PubMed] [Google Scholar]
  • 38.Oftebro H, Bjorkhem I, Skrede S, Schreiner A, Pederson JI. Cerebrotendinous xanthomatosis: a defect in mitochondrial 26-hydroxylation required for normal biosynthesis of cholic acid. J Clin Invest. 1980;65:1418–1430. doi: 10.1172/JCI109806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Meir K, Kitsberg D, Alkalay I, Szafer F, Rosen H, Shpitzen S, Avi LB, Staels B, Fievet C, Meiner V, Bjorkhem I, Leitersdorf E. Human sterol 27-hydroxylase (CYP27) overexpressor transgenic mouse model. Evidence against 27-hydroxycholesterol as a critical regulator of cholesterol homeostasis. J Biol Chem. 2002;277:34036–34041. doi: 10.1074/jbc.M201122200. [DOI] [PubMed] [Google Scholar]
  • 40.Hall E, Hylemon P, Vlahcevic Z, Mallonee D, Valerie K, Avadhani N, Pandak W. Overexpression of CYP27 in hepatic and extrahepatic cells: role in the regulation of cholesterol homeostasis. Am J Physiol Gastrointest Liver Physiol. 2001;281:G293–G301. doi: 10.1152/ajpgi.2001.281.1.G293. [DOI] [PubMed] [Google Scholar]
  • 41.Bouhlel MA, Staels B, Chinetti-Gbaguidi G. Peroxisome proliferator-activated receptors--from active regulators of macrophage biology to pharmacological targets in the treatment of cardiovascular disease. J Intern Med. 2008;263:28–42. doi: 10.1111/j.1365-2796.2007.01892.x. [DOI] [PubMed] [Google Scholar]
  • 42.Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP, Teissier E, Minnich A, Jaye M, Duverger N, Brewer HB, Fruchart JC, Clavey V, Staels B. PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med. 2001;7:53–58. doi: 10.1038/83348. [DOI] [PubMed] [Google Scholar]
  • 43.Javitt NB. 25R,26-Hydroxycholesterol revisited: synthesis, metabolism, and biologic roles. J Lipid Res. 2002;43:665–670. [PubMed] [Google Scholar]
  • 44.Lefebvre P, Cariou B, Lien F, Kuipers F, Staels B. Role of bile acids and bile Acid receptors in metabolic regulation. Physiol Rev. 2009;89:147–191. doi: 10.1152/physrev.00010.2008. [DOI] [PubMed] [Google Scholar]
  • 45.Li T, Chen W, Chiang JY. PXR induces CYP27A1 and regulates cholesterol metabolism in the intestine. J Lipid Res. 2007;48:373–384. doi: 10.1194/jlr.M600282-JLR200. [DOI] [PubMed] [Google Scholar]

RESOURCES