Deletion of CGI-58 or adipose triglyceride lipase differently affects macrophage function and atherosclerosis

Abstract

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58(-/-) mice). CGI-58(-/-) macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL(-/-) macrophages. In contrast to ATGL(-/-) macrophages, however, CGI-58(-/-) macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58(-/-) macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE(-/-) mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor(-/-) mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency.

Keywords: comparative gene identification-58; inflammation; lipid droplets; storage diseases.

Fig. 1.

Unchanged plasma lipid parameters, body weight, glucose concentrations, and glucose tolerance in macCGI-58^−/− mice. Plasma parameters (A) and body weight (B) of overnight fasted 12–14-week-old female Wt and macCGI-58^−/− mice fed a standard chow diet. C: Glucose concentrations in female mice aged 11–46 weeks of age. D: Glucose tolerance test of 34–35-week-old female Wt and macCGI-58^−/− mice fed WTD. Data are shown as mean values ± SEM (n = 4–5).

Fig. 2.

Decreased TG hydrolase activity and TG-rich lipid droplet accumulation in CGI-58^−/− macrophages. A: mRNA expression of CGI-58 in Wt and CGI-58^−/− macrophages determined by real time PCR, including normalization to HPRT, expressed as mean values + SEM, performed in duplicate (n = 3–4). B: Western blot analysis of CGI-58 protein expression in Wt white adipose tissue (WAT) as well as Wt and CGI-58^−/− macrophages (50 μg protein per lane). C: TG hydrolase activities in cell lysates of Wt and CGI-58^−/− macrophages are presented as mean values + SEM, performed in duplicate (n = 4). D: Representative fluorescent microscopy images after Nile Red staining and electron micrographs of Wt and CGI-58^−/− macrophages. Lipid droplets in the CGI-58^−/− macrophages are indicated by arrows. E: TG, TC, and unesterified FC concentrations in lipid extracts of macrophages presented as mean values + SEM (n = 4–5). F: FA composition in TG of macrophages after separation by thin layer chromatography determined by GC-flame ionization detection. Data are presented as mean values + SEM (n = 4). Inset: TG-FA distribution in percentage. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 3.

Apoptosis, mitochondrial fragmentation, and ER stress are not induced in CGI-58^−/− macrophages. A: mRNA expression of the anti-apoptotic genes Bcl-XL and Mcl-1 in Wt and CGI-58^−/− macrophages determined by real time PCR, including normalization to HPRT. Expression in Wt macrophages was arbitrarily set to one. Data are expressed as mean values + SEM, performed in duplicate (n = 5). B: Western blot analysis of Bax protein expression in macrophages (40 μg protein per lane). The cytosolic fraction was blotted for cytochrome C expression. C: Representative electron micrographs of mitochondria (indicated by arrows) in Wt, ATGL^−/−, and CGI-58^−/− macrophages. D: Quantifications of the total amount of annexin V-positive (early apoptotic), annexin V/PI-positive (late apoptotic), and PI-positive (necrotic) cells shown as mean values + SEM, performed in duplicate (n = 5). Data represent the percentage of cells stained with annexin V and/or PI. E: mRNA expression of the ER-resident chaperones Pdi and Erdj4. Data are expressed as mean values + SEM, performed in duplicate (n = 5). IRE1α (F) and CHOP (G) protein expression determined by Western blotting analyses. As positive control for CHOP expression Wt macrophages were treated with the ER stress inducer, tunicamycin. LD, lipid droplet; n.s., not significant.

Fig. 4.

Reduced phagocytosis capacity and mitochondrial respiration of CGI-58^−/− macrophages. A: Macrophages from Wt and macCGI-58^−/− mice were cultivated in DMEM/10% LPDS containing 0, 6, and 25 mM glucose for 1 h. Phagocytosis of fluorescein-labeled E. coli particles is presented as mean values + SEM of two independent experiments performed in triplicate (n = 8–9). Phagocytosis of Wt cells was arbitrarily set to 100%. *P < 0.05; **P ≤ 0.01. B: mRNA expression of the PPARα target genes Ctp1α, Aox, Vlcad, and Mcad, including normalization to HPRT, was determined by real time PCR. Data are expressed as means + SEM, performed in duplicate (n = 5). C: The OCR of Wt (continuous lines) and CGI-58^−/− macrophages (dotted lines) in the presence (black) or absence (gray) of 25 mM glucose and 2 mM L-glutamine normalized to protein content. As indicated, cells were treated with 10 μM oligomycin, 0.3 μM FCCP, and 2.5 μM antimycin A. Data are presented as mean values ± SEM of triplicate repeats (n = 3–4). D, E: OCR calculated as percentage of maximal mitochondrial respiration of Wt and CGI-58^−/− macrophages in the presence (D) or absence (E) of 25 mM glucose and 2 mM L-glutamine. Data are presented as mean values ± SEM of triplicate repeats (n = 3–4). F: Fluorescein-labeled E. coli particles (200 μl) were injected into Wt and macCGI-58^−/− mice. After 2 h, macrophages were isolated and assayed for internalized fluorescence after quenching of extracellular fluorescence by trypan blue. Phagocytosis of Wt macrophages was arbitrarily set to 100%. Relative phagocytosis is presented as mean values + SEM (n = 5).

Fig. 5.

CGI-58^−/− macrophages polarize toward a pro-inflammatory M1-like phenotype. A: mRNA expression of Gro-1, Mcp1, Mcp2, Ccl5, Mrc-1, and Arg-1 in Wt and CGI-58^−/− macrophages determined by real time PCR, including normalization to HPRT. Data are presented as mean values + SEM, performed in duplicate (n = 5). *P < 0.05; ***P ≤ 0.001. B: Macrophages were treated with saline (control) or LPS (100 ng/ml) for 16 h. IL-6 secretion in the supernatant was determined by ELISA. Data represent mean values + SEM (n = 3–5). **P ≤ 0.01.

Fig. 6.

Unchanged atherosclerosis susceptibility by CGI-58 deficiency. ApoE^−/− and macCGI-58/ApoE-DKO mice were challenged with a HF/HCD for 10 weeks. A: Representative images of aortic valve sections stained with Oil Red O, Moma-2, and Masson’s trichrome for the detection of lipids, macrophages, and collagen, respectively. Magnification, ×40. Data represent mean values of 10 aortic valve sections per mouse. Bars represent the mean values of 9–11 mice per group. B: Representative images and quantification of Oil Red O-stained en face aorta. Data represent mean values ± SEM (n = 10–11). *P < 0.05. C: Cholesterol efflux to the extracellular acceptors ApoA-I and HDL₃ expressed as the percentage of [³H]cholesterol transferred from macrophages to the medium. Data show the mean values ± SEM (n = 7).

Fig. 6.

Unchanged atherosclerosis susceptibility by CGI-58 deficiency. ApoE^−/− and macCGI-58/ApoE-DKO mice were challenged with a HF/HCD for 10 weeks. A: Representative images of aortic valve sections stained with Oil Red O, Moma-2, and Masson’s trichrome for the detection of lipids, macrophages, and collagen, respectively. Magnification, ×40. Data represent mean values of 10 aortic valve sections per mouse. Bars represent the mean values of 9–11 mice per group. B: Representative images and quantification of Oil Red O-stained en face aorta. Data represent mean values ± SEM (n = 10–11). *P < 0.05. C: Cholesterol efflux to the extracellular acceptors ApoA-I and HDL₃ expressed as the percentage of [³H]cholesterol transferred from macrophages to the medium. Data show the mean values ± SEM (n = 7).

Fig. 7.

ASO-mediated knockdown of CGI-58 does not alter atherosclerosis susceptibility. A: ASO treatment effectively reduces macrophage expression of CGI-58. C57BL/6 mice maintained on a chow diet in conjunction with weekly injections (50 mg/kg) of either a nontargeting control ASO or CGI-58 for 6 weeks. Thereafter, thioglycolate-elicited peritoneal macrophages were isolated from control and CGI-58 ASO-treated mice (n = 5 per group), plated for 2 h, and Western blotting was conducted to examine CGI-58 protein expression in isolated macrophages. B–H: For atherosclerosis studies, 6-week-old male LDLR^−/− mice were fed a diet enriched in 0.2% (w/w) cholesterol and 20% of energy as lard for 16 weeks in conjunction with biweekly injections (25 mg/kg) of either a nontargeting control ASO or CGI-58 ASO. Total plasma cholesterol (TC) (B) and TG (C) concentrations during atherosclerosis progression. VLDL (D), LDL (E), and HDL (F) cholesterol levels following 16 weeks of diet induction. G: Representative images of Oil Red O-stained aortic valve sections (magnification, ×40) and quantification of cross-sectional aortic valve lesion area. H: En face morphometric analysis of total aortic lesion area. Data represent the mean ± SEM from four to ten mice per group.