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Originally published In Press as doi:10.1194/jlr.M400167-JLR200 on June 1, 2004

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Journal of Lipid Research, Vol. 45, 1429-1436, August 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

High-level expression of ABCG5 and ABCG8 attenuates diet-induced hypercholesterolemia and atherosclerosis in Ldlr–/– mice

Kenneth R. Wilund*, Liqing Yu*, Fang Xu{dagger}, Helen H. Hobbs*,{dagger},§ and Jonathan C. Cohen1,{dagger},§

* Departments of Molecular Genetics and Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75290
{dagger} McDermott Center for Human Growth and Development and Center for Human Nutrition, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75290
§ Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75290

Published, JLR Papers in Press, June 1, 2004. DOI 10.1194/jlr.M400167-JLR200

1 To whom correspondence should be addressed. e-mail: Jonathan.Cohen{at}utsouthwestern.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice expressing human ABCG5 (G5) and ABCG8 (G8) have decreased fractional absorption and increased biliary secretion of cholesterol, but their plasma cholesterol levels are unchanged (males) or modestly reduced (females). To determine whether increased expression of G5 and G8 can ameliorate hypercholesterolemia in mice lacking LDL receptors (LDLRs), we examined the effects of G5G8 transgene expression on cholesterol metabolism and atherosclerosis in Ldlr–/– mice. In chow-fed Ldlr–/– mice, the G5G8 transgene reduced fractional absorption of dietary cholesterol by 50% and increased biliary cholesterol levels by 60% but did not affect plasma cholesterol levels. On a Western diet (21% fat, 0.2% cholesterol), G5G8Tg; Ldlr–/– mice had a 30% reduction in the level of hepatic cholesterol and 45% lower plasma cholesterol levels than the Ldlr–/– mice. After 6 months on the Western diet, the atherosclerotic lesion area in the aortic root and arch was ~70% lower in the G5G8Tg;Ldlr–/– than in the Ldlr–/– mice and was correlated with the plasma cholesterol levels.

These results demonstrate that increased expression of G5 and G8 attenuates diet-induced hypercholesterolemia in Ldlr–/– mice, resulting in a significant reduction in plasma levels of cholesterol and aortic atherosclerotic lesion area.

Abbreviations: ABC, ATP binding cassette; FPLC, fast-protein liquid chromatography; SREBP, sterol-regulatory element binding protein

Supplementary key words cholesterol absorption • cholesterol secretion • plant sterol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the ATP binding cassette (ABC) proteins ABCG5 (G5) and ABCG8 (G8) cause sitosterolemia, a rare autosomal recessive disorder characterized by accumulation of cholesterol and other neutral sterols in the circulation and tissues (1, 2). Metabolic studies have demonstrated that the accumulation of sterols in these patients is due to both increased absorption and decreased biliary secretion of neutral sterols (3, 4). Deletion of G5 and G8 expression in mice by gene targeting produces a phenotype that recapitulates the cardinal features of sitosterolemia. Plasma and tissue levels of plant sterols are significantly elevated in these animals, reflecting both the increased fractional absorption and decreased biliary secretion of these sterols in the absence of G5 and G8 (5). Thus, G5 and G8 prevent the accumulation of neutral sterols in the body by limiting their absorption in the gut and promoting their excretion into bile.

In contrast to other Mendelian forms of hypercholesterolemia, sitosterolemic patients are exquisitely sensitive to dietary cholesterol (6, 7). Although large changes in dietary cholesterol content are associated with relatively modest changes in plasma cholesterol levels in normal individuals, diets containing high levels of cholesterol can elicit severe hypercholesterolemia in sitosterolemic individuals and restriction of dietary cholesterol often normalizes plasma levels of cholesterol (6, 7). The elevated plasma sterol levels are associated with severe atherosclerosis in sitosterolemic individuals, some of whom succumb to myocardial infarction in early childhood (8, 9). These findings indicate that ABCG5 and ABCG8 protect the vasculature by buffering the circulation against changes in dietary cholesterol intake. Increasing the expression of G5 and G8 should therefore ameliorate the hypercholesterolemic effects of dietary cholesterol and prevent atherosclerosis.

Wild-type mice are remarkably resistant to diet-induced hypercholesterolemia (10), and overexpression of human G5 and G8 confers protection against hepatic cholesterol accumulation in mice fed a high (2%) cholesterol diet, but causes either no change (males) or only a modest reduction (females) in plasma cholesterol levels (11). Significant diet-induced changes in plasma cholesterol levels can be achieved in genetically modified mice that express no LDL receptor (LDLR); these animals develop moderate hypercholesterolemia that is greatly exacerbated by cholesterol feeding (12). Ldlr–/– mice have been used to examine the effects of overexpression of other genes that play critical roles in cholesterol metabolism. For example, high-level hepatic expression of the nuclear form of sterol-regulatory element binding protein-1a (SREBP1a), a transcription factor that activates several genes controlling cholesterol and triglyceride synthesis, results in a marked increase in hepatic cholesterol and triglyceride synthesis, and yet no change in plasma lipid levels. However, when the SREBP1a transgene was expressed in Ldlr–/– mice, the mice became extremely hypercholesterolemic (13). Plasma cholesterol and triglyceride levels were several-fold higher in doubly mutant (TgBP-1a;LDLR–/–) mice than in mice lacking the LDLR. Recently, Wu et al. (14) reported that expression of a G5G8 transgene had little effect on plasma cholesterol levels or atherosclerosis in Ldlr–/– mice consuming a Western diet. However, in that study, the G5G8 transgene was only expressed in the liver, and the level of expression, as assessed by changes in biliary cholesterol secretion and fecal cholesterol excretion, was low. To determine whether overexpression of G5 and G8 in liver and intestine can protect against dietary hypercholesterolemia and atherosclerosis, we examined the effects of a human G5G8 transgene on diet-induced hypercholesterolemia in mice lacking the LDLR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and diets
Feeding studies were performed in four groups of mice: 1) wild-type (Ldlr+/+), 2) Ldlr–/–, 3) G5G8Tg, and 4) G5G8Tg;Ldlr–/–. Breedings were performed to ensure similar genetic backgrounds (~75% C57BL/6J) in each group. G5G8Tg;Ldlr–/– animals were generated by crossing male G5G8Tg mice on a mixed genetic background (50% C57BL/6J and 50% SJLF1) (11) with female Ldlr–/– mice, also of a mixed genetic background (C57BL/6J and 129S1/SvImJ). The G5G8Tg;Ldlr+/– F1 males were backcrossed to Ldlr–/– females, producing G5G8Tg;Ldlr–/– mice. The F2 males were backcrossed to female Ldlr –/– mice on a pure C57BL/6J background. The F3 mice from this cross were ~75% C57BL/6J. Male G5G8Tg;Ldlr–/– and Ldlr–/– mice were used for the feeding studies. Ldlr+/+ animals on a comparable genetic background were generated by crossing male G5G8Tg mice with Ldlr+/+ females (B6129SF2/J mice from Jackson Labs). The F1 G5G8Tg males (50% C57BL/6J) were backcrossed to C57BL/6J females. Male F2 mice that expressed the G5G8Tg and their littermates that did not express the transgene (both ~75% C57BL/6J), were used as controls for the Ldlr–/– animals.

All mice were housed in plastic cages in a temperature-controlled room (22°C) with 12 h light/12 h dark cycles (daylight cycle was 6:00 AM to 6:00 PM). Animals were fed ad-libitum with either standard rodent chow (Diet 7001; Harlan Teklad, Madison, WI) containing 0.02% cholesterol (w/w) and 4% total lipid (w/w), or a Western diet (chow diet supplemented with cholesterol and fat to a final concentration of 0.2% (w/w) cholesterol, and 21% (w/w) total fat. All mice were between 8 and 10 weeks of age at the start of the study and were fed the indicated diets for 8 weeks, except for the atherosclerosis studies, in which the mice were fed the Western diet for 6 months.

Female SREBP2Tg;Ldlr–/– mice were crossed with male G5G8Tg; Ldlr–/– mice. Male F2 mice expressing the SREBP2 transgene (SREBP2Tg;Ldlr–/–), the G5G8 transgene (G5G8Tg;Ldlr–/–), both transgenes (SREBP2Tg;G5G8Tg;Ldlr–/–), or neither transgene (Ldlr–/–) were used in the study.

Plasma, hepatic, and biliary lipid analysis
Plasma and hepatic cholesterol and triglyceride levels were measured as described (15) using a cholesterol measurement kit (catalog no. 1127771; Roche Molecular Biochemicals, Mannheim, Germany) and Infinity Trigylcerides Reagent (catalog no. 343-500P; Sigma, St. Louis, MO). Plasma lipoproteins were size fractionated using fast-protein liquid chromatography (FPLC) (16).

To measure the biliary cholesterol, bile was removed from gallbladders of mice using a 30 gauge needle after a 4 h fast. The concentrations of cholesterol, phospholipids, and bile acids were measured as previously described (17).

Fecal neutral sterol excretion
Feces were collected from individually housed mice for 3 days, dried, weighed, and ground to a powder. Aliquots (0.5 g) of the ground feces were subjected to alkaline hydrolysis at 120°C for 8 h. The samples were dried prior to the addition of 10 ml water and 10 ml ethanol. Samples were extracted in 15 ml of petroleum ether to which 1.0 mg of 5-cholestene (Sigma) was added as an internal standard. The amount of neutral sterols (cholesterol, coprostanol, and cholestanone) in the extracts was quantified by gas chromatography (18).

Fractional cholesterol absorption
Intestinal cholesterol absorption was determined by a fecal dual-isotope ratio method as previously described (18). Briefly, individually housed mice were gavaged with a mixture of 2 µCi [5,6-3H]sitostanol (American Radiolabeled Chemicals, Inc., St. Louis, MO) and 1 µCi [4-14C]cholesterol (NEN Life Science Products, Boston, MA), and feces were collected for three days. One gram of feces from each animal was extracted with chloroform-methanol (2:1, v/v), and the ratio of 14C to 3H in each sample was calculated. The percent cholesterol absorption was then determined as described (19).

Atherosclerosis analysis
Ten G5G8Tg;Ldlr–/– and 10 Ldlr–/– littermates were fed a Western diet for 6 months to examine the development of atherosclerotic lesions in the aortic root and the whole aorta (en face analysis). The mice were fasted for 4 h and anesthetized, and the heart was perfused with 50 ml of phosphate-buffered saline (PBS) using a 23 gauge needle. The heart and aortic tissue were removed from the ascending aorta to the ileal bifurcation and placed in 4% paraformaldehyde for 16 h. After fixation, the heart was dissected from the aorta, embedded in OCT (Sakura Finetechnical, Inc., Torrance, CA), and frozen at –80°C. Serial (8 µm) sections of the heart were sliced and mounted on Fisher Microprobe Plus slides (Fisher Scientific Co., Pittsburg, PA) for analyzing the lesion area in the aortic root. The slides were stained with Oil Red O for neutral lipids, and counterstained with hematoxylin to visualize the nuclei.

For the en face analysis, aortas were transferred to PBS following fixation, and stored for 16 h at 4°C. Adventitial tissue was carefully removed, and the intimal surface was exposed by a longitudinal cut through the length of the inner surface of the aorta down through the ileal bifurcation. The outer surface of the aortic arch was also cut longitudinally from the ascending arch to the left subclavian artery to allow the lumen of the aortic arch to be laid flat, and the lumen was pinned open on a black wax background. The aorta was rinsed for 5 min in 75% ethanol, stained with 0.5% Sudan IV in 35% ethanol and 50% acetone for 15 min, destained in 75% ethanol for 5 min, then rinsed with distilled water. Digital images of the aorta were captured, and the grossly discernible lesion area was quantified from the aortic arch to 5 mm distal of the left subclavian artery using Metamorph Imaging System software, version 6.0 (Universal Imaging Corporation, Downingtown, PA).

Statistics
All data are reported as means ± SEM. Differences between mean values in groups were tested for statistical significance by Student's t - test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of the G5G8 transgene ameliorates diet-induced hypercholesterolemia
Expression of the G5G8 transgene had little effect on plasma or liver cholesterol levels in chow-fed Ldlr+/+ or Ldlr–/– mice (Fig. 1). Following 8 weeks on a Western diet, plasma cholesterol levels were 45% lower (521 ± 81 mg/dl vs. 939 ± 23 mg/dl, P = 0.0002) and liver cholesterol levels were 30% lower (5.2 ± 0.5 mg/g vs. 7.2 ± 0.9 mg/g, P = 0.03) in the Ldlr–/– mice expressing the G5G8 transgene compared with their Ldlr–/– littermates (Fig. 1). FPLC analysis indicated that high-level expression of G5G8 in the Ldlr–/– animals fed a Western diet was associated with lower plasma levels of LDL cholesterol (LDL-C) and VLDL-C (Fig. 2). No significant differences were found in the plasma HDL-C levels in the G5G8Tg;Ldlr–/– and Ldlr–/– mice. Expression of the G5G8 transgene in the Ldlr–/– mice on the Western diet resulted in lower hepatic and plasma triglyceride levels (Table 1). In the liver, expression of the G5G8 transgene was also associated with lower triglyceride levels in the chow-fed Ldlr+/+ mice (Table 1).



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  		Fig. 1. Levels of plasma and hepatic cholesterol in Ldlr+/+, G5G8Tg, Ldlr–/–, and G5G8Tg;Ldlr–/– mice. Six- to eight-week-old male mice (5 per group) were fed a Western or chow diet for 2 months. After a 4 h fast, the mice were sacrificed, blood was collected from the inferior vena cava, and liver samples were snap frozen in liquid nitrogen. Plasma was isolated by centrifugation, and the plasma and liver cholesterol levels were determined as described in Experimental Procedures. Error bars indicate mean ± SEM. *P < 0.05.

 


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  		Fig. 2. Fast-protein liquid chromatography (FPLC) fractionation of plasma samples from Ldlr+/+, G5G8Tg, Ldlr–/–, and G5G8Tg;Ldlr–/– mice fed a chow or Western diet for 2 months. Fasting plasma from 5 male mice in each group was pooled and subjected to FPLC fractionation, and the cholesterol content of each fraction was assayed as described in Experimental Procedures.

 

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  		TABLE 1. Liver weights, plasma and liver triglycerides, and biliary lipid levels in 6–8-week-old mice (n = 5 in each group) consuming a chow or Western diet for 2 months

 
Cholesterol absorption is reduced in G5G8 transgenic mice
Expression of the G5G8 transgene was associated with a significant reduction in the fractional absorption of dietary cholesterol in all groups (Fig. 3). The magnitude of the decrease (~50%) was similar in the Ldlr+/+ and Ldlr–/– mice on both the chow and the Western diets. The fraction of cholesterol absorbed was consistently lower in mice consuming the Western diet, but the total amount of dietary cholesterol absorbed, calculated by multiplying the estimated cholesterol intake by the fractional absorption, was markedly higher in the mice fed the Western diet than in the chow-fed animals. Mice typically consume ~5 g chow/day (unpublished observation), and the chow diet contains 0.02% cholesterol by weight. On the chow diet, the nontransgenic animals absorbed ~35% of the cholesterol they consumed, resulting in a net cholesterol influx of ~350 µg cholesterol/day, whereas the G5G8Tg mice had a fractional absorption of cholesterol of 19% and the net absorption of dietary cholesterol was 190 µg/day. Thus, on the chow diet, expression of the transgene was associated with a reduction in dietary cholesterol influx of ~150 µg/day. On the Western diet, which contains 0.2% cholesterol, the fractional cholesterol absorption was 28% in the control mice and 17% in the mice expressing the G5G8 transgene. Thus, expression of the transgene was associated with a reduction in net cholesterol absorption from 2,800 µg cholesterol/day to 1,700 µg/day. Therefore, the transgene had a significantly greater effect on dietary cholesterol uptake in mice consuming the Western diet than in mice consuming the chow diet.



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  		Fig. 3. The fractional absorption of dietary cholesterol in Ldlr+/+, G5G8Tg, Ldlr–/–, and G5G8Tg;Ldlr–/– mice. Following 2 months on a chow or Western diet, mice were gavaged with [14C]cholesterol and [3H]sitostanol. Feces were collected for 3 days, and the ratio of the two isotopes was measured to determine the fractional absorption of cholesterol, as described in Experimental Procedures. Error bars indicate mean ± SEM. * P < 0.05.

 
Cholesterol excretion is increased in G5G8 transgenic mice
Expression of the G5G8 transgene significantly increased biliary cholesterol concentrations and fecal cholesterol excretion rates in all groups (Fig. 4). Cholesterol feeding increased the sterol content of the bile and of the stool, reflecting the increase in intestinal uptake and biliary secretion of dietary cholesterol. Biliary bile acid levels were significantly reduced in the G5G8Tg mice, compared with the Ldlr+/+ mice, on both chow and Western diets (Table 1). This result contrasts with previous studies from our laboratory, which failed to detect any effect of the G5G8 transgene on biliary bile acid secretion (11), and may be due to differences in the genetic background of the mice used in the two studies. Biliary phospholipids were not significantly different between the wild-type and G5G8Tg mice but were significantly higher in the G5G8Tg;Ldlr–/– compared with the Ldlr–/– mice, on both the chow and Western diets (Table 1).



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  		Fig. 4. Biliary cholesterol, phospholipids, bile acid concentrations, and fecal neutral sterol levels in Ldlr+/+, G5G8Tg, Ldlr–/–, and G5G8Tg;Ldlr–/– mice. Upper panel: Following 2 months on a chow or Western diet, mice were fasted for 4 h, gallbladder bile was collected using a 30 gauge needle, and biliary cholesterol, phospholipids, and bile acids were determined as described in Experimental Procedures. Lower panel: Feces were collected for 3 days, dried, weighed, and ground to a powder, and fecal neutral sterols were measured as described in Experimental Procedures. Error bars indicate mean ± SEM. * P < 0.05.

 
Expression of the G5G8 transgene confers protection against diet-induced atherosclerosis
To determine whether the reductions in plasma cholesterol levels observed with expression of the G5G8 transgene translated into a reduction in aortic atherosclerosis, we examined the effect of G5G8 expression on atherosclerotic lesion area in the cholesterol-fed wild-type and Ldlr–/– mice. Atherosclerotic lesions were only observed in the Ldlr–/– and G5G8Tg;Ldlr–/– mice fed the Western diet. In these animals, expression of the G5G8 transgene was associated with a significant reduction in lesion development (Fig. 5). The lesion area was directly related to the plasma cholesterol levels in both groups of animals (r = 0.782; P < 0.001). Mice with similar plasma cholesterol levels, irrespective of transgene expression (Fig. 6) had similar lesion areas measured en face and in the aortic root.




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  		Fig 5. A: Comparison of atherosclerotic lesion area in aortic arch in Ldlr–/– and G5G8Tg;Ldlr–/– mice fed a Western diet for 6 months. Mice were sacrificed, the aortas were cleaned and fixed, and the lumen was exposed as described in Experimental Procedures. Lesions in the lumen were stained for neutral lipids with Sudan IV, and the lesion area in the aortic arch to 5 mm distal of the left subclavian artery was quantified as described in Experimental Procedures. * P < 0.05. B: Relationship between plasma cholesterol levels and aortic lesion area.

 



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  		Fig. 6. Neutral lipid staining of lesions in aortic arch (A) and aortic root (B) in Ldlr–/– and G5G8Tg;Ldlr–/– mice with similar plasma cholesterol levels. Mice were fed a Western diet for 6 months before sacrifice. The lumen of the aortic arch was stained with Sudan IV, and the lesion area in the arch was quantified as described in Experimental Procedures. B: Cross sections of hearts from mice were sliced and stained with Oil Red O as described in Experimental Procedures.

 
The G5G8 transgene fails to lower plasma cholesterol levels in SREBP2Tg;Ldlr–/– mice
To determine whether high-level expression of the G5G8 transgene would protect against an increase in endogenous synthesis of cholesterol, we crossed G5G8Tg mice into Ldlr–/– mice expressing a dominant-positive form of SREBP2. Expression of the SREBP2 transgene is associated with increased hepatic cholesterol synthesis (20). Expression of the SREBP2 transgene in the Ldlr–/– mice resulted in only a modest increase in mean biliary cholesterol levels (3.1 vs. 4.4 µmol/ml) (Fig. 7A) but a dramatic increase in the plasma cholesterol levels, from 482 ± 46 mg/dl to 733 ± 53 mg/dl (P < 0.001) (Fig. 7B). In contrast to these results, biliary cholesterol levels increased significantly when the G5G8 transgene was expressed in the SREBP2Tg;Ldlr–/– mice (4.4 vs. 29.9 µmol/ml). Despite the dramatic increase in biliary cholesterol secretion in the SREBP2Tg;Ldlr–/– mice expressing the G5G8 transgene (Fig. 7A), no reduction in plasma cholesterol levels was seen in these mice (Fig. 7B).



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  		Fig. 7. Biliary and plasma levels of cholesterol in Ldlr–/–, SREBP-2Tg;Ldlr–/–, G5G8Tg;Ldlr–/–, and SREBP-2Tg;G5G8Tg;Ldlr–/–mice. Male Ldlr–/– mice expressing the human SREBP2 transgene (SREBP-2Tg;Ldlr–/–) were crossed with female G5G8Tg;Ldlr–/– mice. F1 mice (Ldlr–/–, SREBP-2Tg;Ldlr–/–, G5G8Tg;Ldlr–/–, and SREBP-2Tg;G5G8Tg;Ldlr–/–) were weaned at 4–5 weeks and fed a 6% chow diet for 2 months. Mice were sacrificed after a 4 h fast, gallbladder bile was extracted, and blood was drawn from inferior vena cavae. Biliary (A) and plasma (B) cholesterol were assayed as described in Experimental Procedures. Error bars indicate mean ± SEM. * P < 0.05. Biliary cholesterol levels were also significantly higher in SREBP-2Tg;G5G8Tg;Ldlr–/– than in G5G8Tg;Ldlr–/– mice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding of this study is that overexpression of G5 and G8 ameliorates diet-induced hypercholesterolemia and atherosclerosis in Ldlr–/– mice. After 8 weeks on a Western diet, plasma cholesterol levels were 45% lower in the G5G8Tg;Ldlr–/– mice (521 ± 81 mg/dl) than in Ldlr–/– littermates not expressing the G5G8 transgene (939 ± 23 mg/dl). The cholesterol-lowering effect of G5G8 overexpression was specific to the VLDL and LDL fractions and was only observed in animals consuming Western diets; no reduction of plasma cholesterol was seen when the G5G8 transgene was expressed in the Ldlr–/– mice on the chow diet, or in Ldlr–/– mice in which hypercholesterolemia was exacerbated by expression of an SREBP2 transgene. Taken together, these findings indicate that expression of G5 and G8 confers protection against diet-induced hypercholesterolemia but not against hypercholesterolemia induced by stimulation of endogenous cholesterol synthesis.

In mice, the LDLR efficiently clears lipoproteins from the circulation and prevents changes in plasma lipoprotein levels (12). For this reason, we used LDLR-deficient animals to examine the effect of G5G8 expression on plasma cholesterol levels. Expression of the G5G8 transgene in chow-fed Ldlr–/– mice decreased the absorption of dietary cholesterol and increased the excretion of cholesterol into the bile, resulting in a marked increase in fecal neutral sterol excretion. Despite these changes in cholesterol trafficking, no changes in liver cholesterol levels or plasma levels of cholesterol were found. Thus, the mice compensated for the G5G8-stimulated increase in cholesterol excretion by increasing endogenous cholesterol synthesis.

Previously, we showed that expression of the G5G8 transgene in wild-type mice resulted in increased hepatic cholesterol synthesis (11). Chow-fed mice derive most of their cholesterol from de novo synthesis; dietary cholesterol contributes less than 20% to the total cholesterol pool (21). Thus, the 50% reduction in the uptake of dietary cholesterol in the G5G8 transgenic animals only accounts for ~10% of the total daily cholesterol turnover. On the Western diet, cholesterol consumption increased 10-fold and the fractional absorption of cholesterol was reduced only modestly. Therefore, in the mice fed a Western diet, the fraction of the cholesterol pool derived from dietary cholesterol is substantially increased. The calculated influx of dietary cholesterol was reduced from 2,800 µg/day in wild-type animals to 1,700 µg/day in those expressing the transgene. Thus, the reduction in dietary cholesterol absorption associated with G5G8 expression had a much greater impact in the Ldlr–/– mice consuming the Western diet and resulted in a significant reduction in liver and plasma cholesterol levels.

Plasma cholesterol levels were also significantly higher in the Ldlr–/– mice expressing the SREBP2 transgene to augment hepatic cholesterol synthesis (773 vs. 482 mg/dl). Expression of the G5G8 transgene in the SREBP2Tg;Ldlr–/– mice resulted in a dramatic increase in biliary cholesterol excretion, but no change in the plasma cholesterol levels (Fig. 7). Thus, increased biliary cholesterol secretion mediated by the G5G8 transgene did not mitigate the effects of increased hepatic cholesterol synthesis on plasma cholesterol levels in the SREBP2Tg;Ldlr–/– mice. These data are consistent with the G5G8-mediated reduction in plasma cholesterol levels in the Ldlr–/– animals on the Western diet being due predominantly to the reduction in cholesterol absorption. We cannot rule out the possibility that increased efflux of cholesterol from the circulation contributes to the lower plasma cholesterol levels in the G5G8Tg;Ldlr–/– mice. Most of excess circulating cholesterol in Ldlr–/– mice is transported in apolipoprotein B-containing lipoproteins, which are normally cleared from the circulation by the LDLR. In animals lacking the LDLR, apolipoprotein B-containing lipoproteins are cleared by low-affinity pathways (12). Therefore, plasma LDL levels in these animals are determined primarily by the rate of input of cholesterol into the circulation. Further studies will be required to determine whether increased expression of G5G8 reduces secretion of apolipoprotein B-containing lipoproteins from the liver.

Expression of the G5G8 transgene ameliorated the development of atherosclerotic lesions in Ldlr–/– mice on the Western diet. The reduction in lesion area was directly related to the reduction in plasma cholesterol levels. Plasma cholesterol levels are positively correlated with aortic lesion area in many mouse models, but the distribution of cholesterol within different-sized lipoprotein particles also influences lesion formation (22). In mice expressing apolipoprotein B-100 only, deletion of the Ldlr resulted in the accumulation of small LDL-sized lipoproteins and severe atherosclerosis, whereas deletion of Apoe led to the accumulation of large VLDL-sized particles and more modest atherosclerosis (22). In the present study, expression of the G5G8 transgene did not influence the size distribution of plasma lipoproteins. Thus, the atheroprotective effect of the G5G8 transgene in Ldlr–/– animals is directly related to its ability to reduce plasma cholesterol levels, and is not related to changes in lipoprotein size distribution.

The results of this study differ from those of Wu et al. (14), who reported that overexpression of a human G5G8 transgene did not affect plasma cholesterol levels or atherosclerosis in Ldlr–/– mice fed either chow or 4% cholesterol diets. The different outcomes of these two studies probably reflect differences in the expression of the G5G8 transgenes used. The G5G8 transgene developed by Wu et al. was expressed only in the liver (14), whereas the G5G8 transgene used in the present study was expressed both in liver and intestine. Thus, the G5G8-mediated reduction in plasma cholesterol levels in our study may be due to intestinal expression of the transgene, which reduced cholesterol absorption. Comparison of biliary cholesterol concentrations also suggests that the G5G8 transgene described by Wu et al. was expressed at a lower level than was the G5G8 transgene used in the present study (14). Accordingly, it is possible that the reduced plasma cholesterol levels in our study are due to high expression levels of the G5G8 transgene. Further studies will be required to determine the metabolic mechanism by which increased expression of G5 and G8 lowers plasma cholesterol levels.


   ACKNOWLEDGMENTS
 
The authors thank Yinyan Ma and James Jennings for excellent technical assistance. This work was supported by National Institutes of Health Grant HL-20948, the Perot Family Fund, and the Donald W. Reynolds Clinical Cardiovascular Research Center in Dallas. H.H.H. is an investigator in the Howard Hughes Medical Institute.

Manuscript received April 29, 2004 and in revised form May 27, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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