Macrophage fatty-acid synthase deficiency decreases diet-induced atherosclerosis

Abstract

Fatty acid metabolism is perturbed in atherosclerotic lesions, but whether it affects lesion formation is unknown. To determine whether fatty acid synthesis affects atherosclerosis, we inactivated fatty-acid synthase (FAS) in macrophages of apoE-deficient mice. Serum lipids, body weight, and glucose metabolism were the same in FAS knock-out in macrophages (FASKOM) and control mice, but blood pressure was lower in FASKOM animals. Atherosclerotic extent was decreased 20-40% in different aortic regions of FASKOM as compared with control mice on Western diets. Foam cell formation was diminished in FASKOM as compared with wild type macrophages due to increased apoAI-specific cholesterol efflux and decreased uptake of oxidized low density lipoprotein. Expression of the anti-atherogenic nuclear receptor liver X receptor alpha (LXRalpha; Nr1h3) and its downstream targets, including Abca1, were increased in FASKOM macrophages, whereas expression of the potentially pro-atherogenic type B scavenger receptor CD36 was decreased. Peroxisome proliferator-activated receptor alpha (PPARalpha) target gene expression was decreased in FASKOM macrophages. PPARalpha agonist treatment of FASKOM and wild type macrophages normalized PPARalpha target gene expression as well as Nr1h3 (LXRalpha). Atherosclerotic lesions were more extensive when apoE null mice were transplanted with LXRalpha-deficient/FAS-deficient bone marrow as compared with LXRalpha-replete/FAS-deficient marrow, consistent with anti-atherogenic effects of LXRalpha in the context of FAS deficiency. These results show that macrophage FAS deficiency decreases atherosclerosis through induction of LXRalpha and suggest that FAS, which is induced by LXRalpha, may generate regulatory lipids that cause feedback inhibition of LXRalpha in macrophages.

FIGURE 1.

Targeting FAS in macrophages. A, top shows the targeted allele (FAS locus with loxP-flanked exons 4–8 (with the loxP sites indicated as triangles)) and the post-Cre allele. P1 and P2 represent primers used to detect a 317-bp fragment indicating Cre excision at the FAS locus. The bottom shows a simplified breeding scheme (omitting intermediate progeny) to produce FASKOM mice. B, PCR analyses of DNA from FASKOM and control mice using P1 and P2. DNA sources for both FASKOM and WT were as follows: lane 1, liver; lane 2, spleen; lane 3, lung; lane 4, kidney; lane 5, heart; lane 6, aorta; lane 7, adipose tissue; lane 8, peritoneal macrophages; and lane 9, negative control. C, FAS mRNA expression in peritoneal macrophages by quantitative RT-PCR. Data are expressed relative to L32 mRNA. *, p < 0.05. D, FAS enzyme activity in peritoneal macrophages. *, p < 0.001. The inset shows FAS enzyme activity in liver. Each bar in C and D represents the mean ± S.E. of eight mice for each genotype.

FIGURE 2.

Blood pressure, lipoproteins, and atherosclerosis in FASKOM and littermate control mice. A, base-line blood pressures on chow (top) and after 8 weeks of high fat diet (HFD, bottom). Systolic (SBP) and diastolic (DBP) blood pressures are shown for FASKOM (black bars) and littermate WT mice (open bars). *, p < 0.05; **, p < 0.01 versus WT. B, lipoprotein profiles for WT (open symbols) and FASKOM littermates (solid symbols) on chow (triangles) and after 8 weeks of high fat diet (squares). Each tracing is presented as the average of serum from n ≥5 mice. C, atherosclerosis as assayed by the en face technique for the same mice in A after 8 weeks of high fat diet.

FIGURE 3.

Cholesterol metabolism in peritoneal macrophages. A, Oil Red O staining of peritoneal macrophages. Magnification ×40 with bar indicating 50 μm. B, lipid content of peritoneal macrophages. *, p < 0.05. Similar results were seen in three independent experiments. C, representative electrospray ionization-mass spectrometry analysis of fatty acids extracted from WT and FASKOM peritoneal macrophages. The vertical axis represents relative abundance and the horizontal axis m/z values. The peak with m/z 311.3 is an internal standard. D, apoAI-specific cholesterol efflux using macrophages from n ≥3 mice of each genotype. Cells were loaded with acetylated LDL in the presence of [³H]cholesterol and then treated with apoAI for 48 h. *, p < 0.05. E, Western blot of proteins extracted from WT and FASKOM macrophages and detected with an anti-ABCA1 antibody (top panel) and an anti-actin antibody (bottom panel). F, cholesterol uptake at 4 and 16 h after exposure to fluorescently labeled oxidized LDL using cells from n ≥3 mice of each genotype. *, p < 0.05.

FIGURE 4.

Lipid content in atherosclerotic lesions. WT and FASKOM mice were fed a high fat diet for 8 weeks; animals were visually selected for differences in atherosclerosis and then the aortic arch was isolated without fixation, cleaned, and subjected to lipid extraction. For these animals, serum cholesterol at the time of sacrifice was 1747 ± 217 mg/dl for WT and 1842 ± 170 mg/dl for FASKOM (mean ± S.E.). A, cholesterol content in the aortic arch for each genotype. Data are expressed as mean ± S.E. for 4 WT and 6 FASKOM mice. *, p < 0.05. B, total lipid phosphorus in aortic arches. C, total triglyceride content in aortic arches. D, representative electrospray ionization-mass spectrometry analysis of triacylglycerol species extracted from the aortic arches of WT and FASKOM mice. The horizontal axis represents m/z values. The vertical axis (not labeled) represents arbitrary units of relative abundance. The internal standard is not shown to simplify data presentation.

FIGURE 5.

Cell type distribution in atherosclerotic lesions. WT and FASKOM mice were fed a high fat diet for 8 weeks, and then the aortic root was sectioned using a crytostat. Slides were subjected to Oil Red O staining for lesion quantification by computerized image processing, and to fluorescent immunocytochemical quantification using an anti-macrophage antibody (MOMA) and an anti-smooth muscle antibody (SMA). A, representative immunocytochemistry images from WT (top panels) and FASKOM (bottom panels) sections. B, atherosclerotic lesion area for four WT and five FASKOM mice. A.U., arbitrary units. *, p < 0.05. C, macrophage content normalized to lesion area in the same lesions quantified in B.

FIGURE 6.

Gene expression in peritoneal macrophages. Message expression was assayed by quantitative RT-PCR using cells from WT (open symbols) and FASKOM (solid symbols) mice for LXRα (Nr1h3) (A), Abca1 (B), SREBP1 (Srebf1) (C), Ppara (D), Cpt1a (E), Acox1 (F), Pparg (G), aP2 (Fabp4) (H), and Cd36 (I). Data are expressed relative to L32 (Rpl32) mRNA as mean ± S.E. using RNA from cells derived from three to five mice per condition. *, p < 0.05. Positive results were replicated in one or more independent experiments.

FIGURE 7.

Effect of PPARα activation in peritoneal macrophages. Cells from FASKOM (solid bars) and WT controls (open bars) were treated with 100 μm Wy14,643 in DMSO (Wy, right side of panels) or DMSO alone (Control, left side of panels) for 24 h. A, Acox1 message assayed by RT-PCR. B, LXRα (Nr1h3) message assayed by RT-PCR. n ≥3 for each condition; *, p < 0.05 versus WT cells treated with DMSO only (Control).

FIGURE 8.

Effects of bone marrow LXRα deficiency on atherosclerosis in FASKOM mice. FASKOM littermates were transplanted with marrow from littermates that were LXRα^+/+ (solid squares) or LXRα^−/− (open diamonds), allowed to recover, fed a Western diet for 8 weeks, and then atherosclerosis by the en face technique was assayed.

FIGURE 9.

Model of the effects of macrophage FAS inactivation on atherosclerosis. WT macrophages with normal FAS expression are shown on the left, and FASKOM macrophages with FAS deficiency are shown on the right.