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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1189-1194.)
© 1995 American Heart Association, Inc.

Articles

Genetic Differences of Lipid Metabolism in Macrophages From C57BL/6J and C3H/HeN Mice

Itsuko Ishii; Yasuhiko Ito; Nobuhiro Morisaki; Yasushi Saito; Seiyu Hirose

From the Department of Biochemistry, Faculty of Pharmaceutical Science (I.I., Y.I., S.H.), and the Second Department of Internal Medicine, School of Medicine (N.M.), Chiba University, and the Department of Laboratory Medicine, Yamagata University (Y.S.), Japan.

Correspondence to Itsuko Ishii, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku 1-33, Yayoi-cho, Chiba, 263, Japan.

	Abstract

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Abstract Cholesterol metabolism in macrophages from atherosclerosis-prone C57BL/6J mice was compared with that in macrophages from atherosclerosis-resistant C3H/HeN mice. Plasma total cholesterol levels of both types of mice were significantly increased, but HDL cholesterol level was increased only in C3H/HeN mice when a high-cholesterol diet (1% cholesterol) was fed for 5 weeks. After incubation of macrophages from male and female mice on the high-cholesterol diet with ß-VLDL for 24 hours, cholesterol content in macrophages from C57BL/6J was approximately 1.5- to 2.0-fold higher than in those from C3H/HeN mice. [³H]Cholesterol oleate–ß-VLDL incorporation into macrophages from C57BL/6J mice on the high-cholesterol diet was greater than incorporation into those from C3H/HeN mice. The release of [³H]cholesterol from macrophages from C57BL/6J mice on the high-cholesterol diet was one seventh that from macrophages from C57BL/6J mice on the basal diet or that from macrophages from C3H/HeN mice on the basal or high-cholesterol diet. Acid cholesterol esterase activity was almost the same in macrophages from any group. Acyl CoA:cholesterol acyltransferase activity in macrophages from C57BL/6J mice on the high-cholesterol diet increased compared with that from macrophages from C57BL/6J mice on the normal diet. Neutral cholesterol esterase activity in macrophages from C57BL/6J mice was about half of that in macrophages from C3H/HeN mice independent of the type of diet. There were no sex differences in these metabolisms. Considered with our previous data, these results suggested that a high-cholesterol diet may cause metabolic changes to accumulate cholesterol ester in macrophages from C57BL/6J mice in accordance with genetic abnormalities.

Key Words: high-cholesterol diet • neutral cholesterol esterase • C57BL/6J mouse • macrophage • acyl CoA:cholesterol acyltransferase

	Introduction

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C57BL/6J mice on a high-cholesterol diet are known to be very susceptible to atherosclerosis.^{1 2 3 4 5} However, C3H/HeN mice on a high-cholesterol diet for 1 year were resistant to the formation of atherosclerotic lesions.^{3 4 5} After C57BL/6J mice had consumed a high-cholesterol diet, HDL-C levels were decreased at 4 weeks⁴ and atheroscelotic lesions were observed at 7 weeks, and these lesions continued to grow until all mice had large atheromatous plaques in the aorta and coronary arteries.⁵ Foam cells apparently derived from macrophages in typical fatty lesions were observed overlying acellular areas containing cellular debris in C57BL/6J mice.^{1 2} However, the HDL-C level of C3H/HeN mice did not change with a high-cholesterol diet and they did not develop atheromatous lesions.⁴ Nevertheless, it is uncertain whether the difference of the response of plasma HDL levels is sufficient to explain the difference in atherogenicity between the two strains of mice. This prompted us to clarify the genetic difference of lipid metabolism at the cellular level.

We have reported the mechanism of foam cell formation by macrophages by use of ß-VLDL.^{6 7} That is, macrophages incorporate ß-VLDL⁸ and accumulate cholesterol ester as lipid droplets in cells. Cholesterol metabolism regulating cholesterol ester accumulation in macrophages acts as follows.⁹ First, cholesterol ester in ß-VLDL is hydrolyzed by acid cholesterol esterase in lysosomes.^{10 11} Then the product, free cholesterol, is reesterified by ACAT and stored as cholesterol ester in intracellular lipid droplets.^{12 13} After that, the reesterified cholesterol is hydrolyzed by neutral cholesterol esterase,^{14 15 16} and finally free cholesterol is released from the cells. It is hypothesized that the imbalance of incorporated cholesterol and released cholesterol induces cholesterol ester accumulation and thereby foam cell formation. Each enzyme activity affects this imbalance. To further elucidate the genetic difference between C57BL/6J and C3H/HeN mice at the cellular level, we also investigated ß-VLDL–C metabolism in macrophages.

Because it has also been reported that female C57BL/6J mice were more susceptible to atherosclerosis than male mice (ie, that female mice developed more and larger-sized lesions than males⁴ ), we also compared ß-VLDL–C metabolism in male and female mouse macrophages.

	Methods

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Materials
Cholesterol [¹⁴C]oleate (2.1 GBq/mmol), [³H]cholesterol oleate (3.0 GBq/mmol), and [1-¹⁴C]oleoyl-CoA (1.5 GBq/mmol) were purchased from New England Nuclear Corp. DMEM was from Nissui Pharmaceutical Co, Ltd. Male and female C57BL/6J and C3H/HeN mice were from Japan Clea.

Treatment of Animals
Mice at 6 weeks of age were fed basal diet (Oriental Kobo) or basal diet containing 1% cholesterol (high-cholesterol diet) for 5 weeks (the composition of each diet is shown in Table 1). We designated C57BL/6J mice C57 and C3H/HeN mice C3H. The groups were C57 fed the basal diet (C57-B), C57 fed the high-cholesterol diet (C57-H), C3H fed the basal diet (C3H-B), and C3H fed the high-cholesterol diet (C3H-H).

View this table: [in this window] [in a new window]   Table 1. Diet Composition

Preparation of Macrophages
Peritoneal exudate macrophages were harvested 4 days after injection of 1 mL thioglycollate medium (DIFCO).¹⁷ Cells were plated at a density of 2x10⁶ cells/mL in DMEM supplemented with 10% FBS (DMEM/10% FBS). After 4 hours of adherence, the cells were washed and cultured overnight. The cells were then used for the experiments.

Determination of Total Cholesterol, LDL-C+VLDL-C, and HDL-C Levels in Plasma
Isolation of HDL was determined after the LDL and VLDL had been precipitated out with the precipitation reagent containing 555 µmol/L phosphotungstic acid and 25 mmol/L MgCl₂, pH 2.5.¹⁸ The precipitation reagent (100 µL) was added to 50 µL of plasma, then the mixture was incubated for 10 minutes at room temperature and centrifuged twice, for 2 minutes each time, at 14 000g. The precipitated residue contained apoB-containing lipoproteins (LDL and VLDL) and the supernatant contained HDL. The HDL fraction was determined for analysis of cholesterol within 2 hours by enzymatic method by use of a kit that detected both free and esterified cholesterol (Determiner TC 555; Kyowa Medex Co, Ltd). Total cholesterol was determined with the same kit. LDL-C plus VLDL-C was determined by subtraction of HDL-C from total cholesterol.

Preparation of Reconstituted [³H]Cholesterol Oleate Into ß-VLDL
ß-VLDL (d<1.006) was isolated from serum of cholesterol-fed rabbits by ultracentrifugation for 16 hours.⁸ Incorporation of [³H]cholesterol oleate into ß-VLDL was done essentially by the method of Brown et al¹⁹ : 1 GBq [³H]cholesterol oleate was added with 1 mL DMSO. The mixture was sonicated for 30 seconds, then 2 mL plasma density buffer (0.154 mol/L NaCl, 1 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH 7.4, 0.01% NaN₃) was added and the mixture was resonicated for 30 seconds. It was then added dropwise to 6 mL ß-VLDL (10 mg total cholesterol/mL) in 3 minutes. The solution was incubated for 8 hours at 37°C and then dialyzed against 3 L plasma density buffer for 10 hours. After the dialysis, the solution was centrifuged for 16 hours at 105 000g. The top layer was used as [³H]cholesterol oleate–incorporated ß-VLDL. The specific activity was about 1x10⁷ dpm/mg total cholesterol.

Incorporation of [³H]Cholesterol Oleate–ß-VLDL by Macrophages
Macrophages (2x10⁶ cells/well) were plated in 12-well plates and incubated for certain times in 0.75 mL of DMEM/10% FBS containing 200 µg [³H]cholesterol oleate–ß-VLDL (5x10⁶ dpm). After incubation, the cells were washed three times with DMEM/10% FBS and their radioactivity was measured with a scintillation counter. Furthermore, to determine the free [³H]cholesterol released from the cells during incubation, organic solvent (chloroform:methanol, 2:1) was added to the medium and lipids were extracted from the chloroform layer. The lipids were applied to thin-layer chromatographs.²⁰ The radioactivity in the free cholesterol fraction was then counted. The total uptake was the sums of intracellular radioactivity and free [³H]cholesterol radioactivity in the medium.⁷

Release of [³H]Cholesterol From Macrophages Loaded With [³H]Cholesterol Oleate–ß-VLDL
Macrophages (2x10⁶ cells) were plated in 12-well plates and incubated for 24 hours in 1 mL DMEM/10% FBS containing 200 µg [³H]cholesterol oleate–ß-VLDL. The cells were then washed three times with DMEM/10% FBS. These macrophages were incubated further in 2 mL DMEM/10% FBS. At the times indicated in Fig 1, 0.4 mL of the medium was removed and its radioactivity was measured with a scintillation counter.⁷

View larger version (20K): [in this window] [in a new window]   Figure 1. Bar graphs show cholesterol ester content in macrophages incubated with ß-VLDL. Monolayers of macrophages were incubated with ß-VLDL (1 mg total cholesterol/mL) at 37°C for 24 hours. Open columns indicate incubation without ß-VLDL; solid columns indicate incubation with ß-VLDL. Values are mean±SD for triplicate cultures, and these experiments were repeated three times. ***P<.01.

Assay of Acid and Neutral Cholesterol Esterase Activities⁵
Macrophages (2x10⁷ cells) were washed three times with PBS and suspended in 1 mL 10 mmol/L Tris-HCl (pH 7.4) containing 0.25 mol/L sucrose. The cells were then sonicated twice for 15 seconds and used as the enzyme solution. The reaction mixture contained, in addition to the enzyme solution, 0.5 mmol/L cholesterol oleate, 0.37 MBq cholesterol [¹⁴C]oleate, 0.5 mmol/L phosphatidic acid, and 100 mmol/L Tris-HCl, pH 7.4, for neutral cholesterol esterase or 0.5 mmol/L phosphatidylcholine and sodium acetate buffer, pH 4.0, for acid cholesterol esterase in a total volume of 200 µL. The incubation was carried out at 37°C for 1 hour. The [¹⁴C]oleate released was extracted by a modification of the method of Belfrage and Vaughan.²¹ In brief, the reaction was stopped with 3.25 mL chloroform/methanol/heptane (1.42:1.25:1.00) and then 1 mL 0.1N NaOH was added. The radioactivity in the water phase was measured.

Assay of ACAT Activity
ACAT activity of the above enzyme solution was assayed by the method of Gillies et al²⁰ with [1-¹⁴C]oleoyl-CoA without exogenous cholesterol.

Analyses
Intracellular cholesterol content was measured as follows⁷ : the washed cells in each well were treated with 1 mL hexane/isopropanol (2:1), and the organic solvent was evaporated. The pellet was dissolved in 100 mL methanol, and the total and free cholesterol contents in the methanol solution were assayed enzymatically by use of Determiner TC 555 and Determiner FC 555 kits. The cholesterol ester content was taken as the difference between the total and free cholesterol contents.

Protein concentration was determined with a kit by use of Bradford's method (Bio-Rad, Protein Assay) .

Significance was analyzed by Student's t test.

	Results

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Effect of a High-Cholesterol Diet on Total Cholesterol Levels in the Plasma of Mice
Plasma lipid levels are shown in Table 2. The total cholesterol level of female C57-B mice was less than the levels of the other three groups. HDL-C levels of C57-B mice of both sexes were less than those of C3H-B mice of both sexes. Moreover, the level in male C57-B mice was higher than that in female C57-B mice. The LDL-C+VLDL-C level in male C57-B mice was higher than the levels in the other three groups. The ratio of HDL-C to LDL-C+VLDL-C was in the order male C3H-B>female C3H-B>female C57-B>male C57-B.

View this table: [in this window] [in a new window]   Table 2. Cholesterol Levels in C57BL/6J and C3H/HeN Mice

After consumption of a high-cholesterol diet, total cholesterol and LDL-C+VLDL-C levels were increased in all groups. However, HDL-C levels in C57-H mice of both sexes did not change statistically, whereas those in C3H-H mice of both sexes increased. The ratios of HDL-C to LDL-C+VLDL-C were much less in all groups on the high-cholesterol diet than in those on the basal diet. Nevertheless, the ratio in C57-H mice was about half or less than that in C3H-H mice.

Cholesterol Metabolism in Male Mouse Macrophages
First, to clarify the effect of the high-cholesterol diet on cholesterol ester accumulation in macrophages from male mice, we measured the cholesterol ester content after incubation with or without ß-VLDL (Fig 1). The cholesterol content was very low without ß-VLDL, and there were hardly any differences among the four groups. However, after incubation with ß-VLDL, the cholesterol ester content of macrophages of all the groups increased greatly. That in C57-B macrophages was almost the same as in C3H-B and C3H-H macrophages, but that in C57-H macrophages was twofold higher than in C57-B macrophages.

Next, we investigated the incorporation of ß-VLDL into macrophages in male mice by using [³H]cholesterol oleate–ß-VLDL (Fig 2). The incorporation of [³H]cholesterol oleate–ß-VLDL into C57-H macrophages was increased 1.3-fold compared with C57-B macrophages at 12 and 36 hours. The levels of incorporation into C57-B, C3H-B, and C3H-H macrophages were quite similar.

View larger version (19K): [in this window] [in a new window]   Figure 2. Graphs show incorporation of [3H]cholesterol oleate–ß-VLDL by macrophages. Macrophages were incubated with [3H]cholesterol oleate–ß-VLDL (200 µg total cholesterol/mL) at 37°C for the indicated times. indicates macrophages from C3H/HeN mice fed basal diet; , macrophages from C3H/HeN mice fed high-cholesterol diet; , macrophages from C57BL/6J mice fed basal diet; and , macrophages from C57BL/6J mice fed high-cholesterol diet. The radioactivity in the cells was determined as described in "Methods." Values are mean±SD for triplicate cultures, and these experiments were repeated three times. *P<.1, **P<.05.

Next, we examined the release of [³H]cholesterol into the medium from macrophages loaded with [³H]cholesterol oleate–ß-VLDL. Fig 3 shows the ratio of radioactivity released into the medium to total incorporated radioactivity in macrophages as a percentage. The amount increased in a time-dependent manner in all macrophages. However, the amount from the C57-H macrophages was remarkably low; it was about one sixth the amounts from the macrophages of the other three groups at 12 hours. The above results indicated that C57-H macrophages accumulate cholesterol ester by taking up a large amount of ß-VLDL and hardly releasing free cholesterol from the cells.

View larger version (19K): [in this window] [in a new window]   Figure 3. Graphs show ratio of released [3H]cholesterol to incorporated [3H]cholesterol oleate–ß-VLDL, expressed as a percentage. The cells were incubated with [3H]cholesterol oleate–ß-VLDL (200 µg total cholesterol/mL) at 37°C for 24 hours. indicates C3H-B macrophages; , C3H-H macrophages; , C57-B macrophages; , C57-H macrophages. After incubation, the cells were washed three times with PBS and then incubated in medium. Samples of medium were taken at the indicated times and their radioactivities were measured as described in "Methods." Values are mean±SD for triplicate cultures, and these experiments were repeated three times. ***P<.01.

We then measured the enzyme activities involved in intracellular cholesterol metabolism (Figs 4, 5, and 6). It is known that there are three important enzymes involved in lipid accumulation in macrophages; the first is acid cholesterol esterase (Fig 4), the second ACAT (Fig 5), and the third neutral cholesterol esterase (Fig 6). Acid cholesterol esterase activities in male mice (Fig 4) did not change among the four groups of macrophages. The ACAT activity of C57-B macrophages from male mice (Fig 5) was about one third that of C3H-B macrophages. However, the activity of C57-H macrophages increased by more than threefold compared with that of C57-B macrophages. The activity of C3H-H macrophages was not changed. The neutral cholesterol esterase activities of C57-B and C57-H macrophages in male mice (Fig 6) were about half those of C3H-B and C3H-H macrophages. The enzyme activity was not significantly changed by high-cholesterol diet in the two strains of mice. These results thus strongly suggested the involvement of genetic regulation in these enzyme activities and their response to a high-cholesterol diet.

View larger version (21K): [in this window] [in a new window]   Figure 4. Bar graphs show acid cholesterol esterase activity in macrophages. The macrophages were sonicated twice for 15 seconds each time, and the solution was used as the enzyme source. Enzyme activities were assayed as described in "Methods." Values are mean±SD for triplicate cultures, and these experiments were repeated three times.

View larger version (21K): [in this window] [in a new window]   Figure 5. Bar graphs show ACAT activity in macrophages. The macrophages were sonicated twice for 15 seconds each time, and the solution was used as the enzyme source. Enzyme activities were assayed as described in "Methods." Values are mean±SD for triplicate cultures, and these experiments were repeated three times. ***P<.01.

View larger version (21K): [in this window] [in a new window]   Figure 6. Bar graphs show neutral cholesterol esterase activity in macrophages. The macrophages were sonicated twice for 15 seconds each time, and the solution was used as the enzyme source. Enzyme activities were assayed as described in "Methods." Values are mean±SD for triplicate cultures, and these experiments were repeated three times. **P<.05, ***P<.01.

Comparison of Cholesterol Metabolism Between Male and Female Mice
ß-VLDL–C metabolism in macrophages was almost the same in male and female mice in both strains. Cholesterol ester content in female C57-H macrophages incubated with ß-VLDL was highest among all the groups of macrophages and 1.6-fold higher than that in C57-B macrophages (Fig 1). The release of [³H]cholesterol from female C57-H macrophages was the lowest, being only one sixth those of other macrophages (Fig 3). Absolute and relative enzyme activities in male and female mouse macrophages were almost the same (Figs 3, 4, and 5).

However, a slight sex difference was observed in the incorporation of [³H]cholesterol oleate–ß-VLDL. That into female C57-H macrophages was 1.3-fold greater than that into female C57-B at 36 hours of incubation. The incorporation of [³H]cholesterol oleate–ß-VLDL into female C3H-B macrophages was two thirds of that into female C57-B macrophages. The incorporation into female C3H-H macrophages was almost the same as that into female C3H-B macrophages. The above results indicated that there were no remarkable sex differences in terms of lipid metabolism in macrophages.

	Discussion

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It has been reported that total cholesterol levels did not differ greatly between C57 and C3H strains either with basal chow (4% fat) or atherogenic diet (15% fat, 1.25% cholesterol, and 0.5% cholic acid) and that HDL-C levels were decreased only in the C57 strain with atherogenic diet.^{3 4} In this study we observed a difference in HDL-C levels between C57 and C3H strains either with basal chow or high-cholesterol diet. Despite such a difference, the ratios of HDL-C to LDL-C+VLDL-C were very low in all groups of mice fed the high-cholesterol diet compared with those fed the basal diet. Accordingly, we observed differences in the characteristics of macrophages between the two strains of mice. The results suggested that there are genetic differences in the cholesterol metabolism at a cellular level between the two strains with high-cholesterol feeding.

Five weeks after the start of the high-cholesterol diet, male and female C57-H macrophages showed the characteristic of accumulating more cholesterol ester by increased ß-VLDL incorporation and decreased release of free cholesterol from the cells compared with other macrophages (Figs 2 and 3). Enzyme changes were in accordance with this metabolism. That is, ACAT activity was induced only in macrophages of C57 mice by feeding of a high-cholesterol diet. However, neutral cholesterol esterase activity was not induced by such a diet in C57 macrophages. These activities are quite similar to those reported in rat thioglycollate-elicited peritoneal macrophages, rabbit atherosclerotic lesion cells,⁶ and blood monocyte–derived macrophages from rabbits fed a high-cholesterol diet.²² All these macrophages easily accumulate cholesterol ester in the cells. Therefore, it could be concluded that C57 mouse macrophages possess the feature of accumulating more cholesterol ester and forming foam cells, and that this may contribute to the formation of atheroma when loaded with lipoproteins.

However, macrophage cholesterol metabolism in C3H-H macrophages was almost the same as that in C3H-B macrophages, and cholesterol ester content in C3H-H macrophages incubated with ß-VLDL was less than that in C57-H macrophages. These results suggested that macrophages from C3H mice did not change their cholesterol metabolism in response to a high-cholesterol diet. The C3H macrophages had a high activity of cholesterol metabolism, like rat and rabbit alveolar macrophages⁶ and THP-1–derived macrophages treated with macrophage colony–stimulating factor⁷ ; ie, C3H-H macrophages preserved high ACAT activity and high neutral cholesterol esterase activity. These macrophages (C3H-B and C3H-H) must have a high capacity to catabolize exogenous cholesterol and to release cholesterol from the cells, and these cells accumulate less cholesterol ester. These properties may explain why the C3H mouse is resistant to atherosclerosis induced by an atherogenic diet.

Of the various differences, the most remarkable one between C57 and C3H mice was the activity of neutral cholesterol esterase in normal diet– and high-cholesterol diet–fed mice (Fig 6). Probably this enzyme, in combination with ACAT, is critical for the release of cholesterol from macrophages. The lack of induction of this enzyme coupled with induction of ACAT in C57 mice by cholesterol feeding leads to a low level of release and accumulation of cholesterol ester. In the case of C57-B mice, the release of cholesterol was high despite low neutral cholesterol esterase activity. Under conditions of low ACAT activity, release of cholesterol depending on both acid and neutral cholesterol esterase could be stimulated.²³

Female C57 mice become atherosclerotic more easily than male mice. When testosterone was administered to female C57 mice, atherosclerotic lesions decreased.⁴ In this study, total cholesterol and HDL-C levels in female C57-B mice were significantly lower than those in male C57-B mice (Table 2). However, the ratio of HDL-C to LDL-C+VLDL-C was higher in female C57-B mice than in the males. After high-cholesterol diet feeding, both total cholesterol and LDL-C+VLDL-C levels increased in both sexes. Absolute HDL-C level and the ratio of HDL-C to LDL-C+VLDL-C were not significantly different between both sexes of C57-H mice. These results suggested that plasma lipids do not explain sex differences in terms of atherogenicity. In addition, there was no difference in ß-VLDL–C metabolism between male and female mouse macrophages, indicating that our results cannot explain sex differences at the cellular level either. Other factor(s) related to sex may contribute to the atherogenicity of female C57 mice together with abnormalities of plasma lipid and macrophage lipid metabolism.

In summary, our findings lead to the conclusion that enzyme activities in cellular lipid metabolism and their responses to a high-cholesterol diet are genetically regulated in C57 and C3H mice.

	Selected Abbreviations and Acronyms

 

ACAT = acyl CoA:cholesterol acyltransferase ß-VLDL–C = ß-VLDL cholesterol DMEM = Dulbecco's modified Eagle medium FBS = fetal bovine serum HDL-C = HDL cholesterol LDL-C = LDL cholesterol

Received November 3, 1994; accepted May 22, 1995.

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