Global inactivation of carboxylesterase 1 (Ces1/Ces1g) protects against atherosclerosis in Ldlr -/- mice

Abstract

Atherosclerotic cardiovascular disease is a leading cause of death in the western world. Increased plasma triglyceride and cholesterol levels are major risk factors for this disease. Carboxylesterase 1 (Ces1/Ces1g) has been shown to play a role in metabolic control. So far, the role of mouse Ces1/Ces1g deficiency in atherosclerosis is not elucidated. We generated Ces1/Ces1g ^-/- mice. Compared to wild-type mice, Ces1/Ces1g ^-/- mice had reduced plasma cholesterol levels. We then generated Ces1g ^-/- Ldlr ^-/- double knockout (DKO) mice, which were fed a Western diet for 16 weeks. Compared to Ldlr ^-/- mice, DKO mice displayed decreased plasma cholesterol and TG levels and reduced atherosclerotic lesions. Interestingly, knockdown of hepatic Ces1/Ces1g in Apoe ^-/- mice resulted in hyperlipidemia and exacerbated Western diet-induced atherogenesis. Mechanistically, global inactivation of Ces1/Ces1g inhibited intestinal cholesterol and fat absorption and Niemann-Pick C1 like 1 expression, and increased macrophage cholesterol efflux by inducing ATP-binding cassette subfamily A member 1 (ABCA1) and ABCG1. Ces1/Ces1g ablation also promoted M2 macrophage polarization and induced hepatic cholesterol 7α-hydroxylase and sterol 12α-hydroxylase expression. In conclusion, global loss of Ces1/Ces1g protects against the development of atherosclerosis by inhibiting intestinal cholesterol and triglyceride absorption and promoting macrophage cholesterol efflux.

Figure 1

Generation of global Ces1/Ces1g ^−/− mice and Ces1/Ces1g tissue distribution. (A) A diagram showing the Ces1/Ces1g wild-type (WT) allele and the targeting allele. Exons 1 and 2 are replaced by a nucleus LacZ (nLacZ) cassette and neomycin cassette. K, KpnI. B, BamHI. (B) Genomic DNA from WT or heterozygous ES cells was used for Southern blotting using a 5′ probe (left panel) or 3′ probe (right panel). (C) Mouse genomic DNA was genotyped by PCR. (D) Hepatic mRNA levels of different Ces1 isoforms were determined (n = 4). (E) LacZ staining of various tissues isolated from Ces1/Ces1g ^+/− mice. (F) Hepatic proteins were isolated from 8-weeks-old male control littermates (Ces1/Ces1g ^+/+ mice) or Ces1/Ces1g ^−/− mice. Protein levels were determined by Western blotting. The top band is Ces1/Ces1g. (G) LacZ staining of peritoneal macrophages isolated from Ces1/Ces1g ^+/− mice. (H) Protein levels in the liver (serve as control) or peritoneal macrophages of Ces1g ^+/+ or Ces1g ^−/− mice were determined by Western blotting. BAT, brown adipose tissue. WAT, white adipose tissue. GB, gallbladder. The Western blots are cropped from larger gels.

Figure 2

Ces1/Ces1g ^−/− mice have reduced plasma cholesterol levels. (A and B) Male Ces1/Ces1g ^−/− mice and their control littermates (Ces1/Ces1g ^+/+) were fed a chow diet for 6 months (n = 8). Plasma TG (A) and cholesterol (B) were determined. (C-G) 8-weeks old male Ces1/Ces1g ^−/− mice and their control littermates (Ces1/Ces1g ^+/+) were fed a Western diet for 16 weeks (n = 8). Plasma TG (C) and cholesterol (D) levels were determined. FPLC was performed to determine cholesterol distribution in lipoproteins (E). Representative liver images of oil red O staining are presented (F). Hepatic mRNA levels of inflammatory marker were analyzed (G). *P < 0.05, **P < 0.01.

Figure 3

Loss of Ces1/Ces1g in Ldlr ^−/− mice causes hypolipidemia and protects against atherosclerosis. (A–H) 8-weeks-old male Ces1 ^−/− Ldlr ^−/− (DKO) mice and control littermates Ces1 ^+/+ Ldlr ^−/− (Ldlr ^−/−) mice were fed a Western diet for 16 weeks (n = 8). Hepatic Ces1/Ces1g and LDLR protein levels were determined (A). Plasma TG (B) and cholesterol (C) levels were determined. Plasma TG (D) and cholesterol (E) lipoprotein profiles were analyzed by FPLC. En face aortas were stained by oil red O and representative images are shown (F). En face aorta lesion size was quantified (G). Aortic roots were also stained with oil red O and representative images are shown (H). Aortic root lesion size was quantified (I). **P < 0.01.

Figure 4

Knockdown of Ces1/Ces1g in Apoe ^−/− mice results in hyperlipidemia and increases atherosclerosis. (A–K) 12-weeks-old male Apoe ^−/− mice were fed a Western diet for one week, followed by i.v. injection of Ad-shLacZ and Ad-shCes1 (n = 7). The mice continued to be fed a Western diet for another three weeks. mRNA (left panel) and protein (right panel) levels of Ces1/Ces1g were determined three weeks post adenovirus injection (A). Plasma levels of TG (B) and cholesterol (C) as well as hepatic levels of TG (D) and cholesterol (E) were determined. Plasma TG (F) and cholesterol (G) lipoprotein profiles were analyzed by FPLC. Representative en face aorta images are shown (H) and aortic lesion size was quantified (I). Representative aortic root images are also shown (J) and lesion size was quantified (K). *P < 0.05, **P < 0.01.

Figure 5

Loss of Ces1/Ces1g inhibits intestinal cholesterol absorption. (A–C) 8-weeks-old male WT and Ces1/Ces1g ^−/− mice (A) or 8-weeks-old male Ldlr ^−/− and Ces1 ^−/− Ldlr ^−/− (DKO) mice (B) were fed a Western diet for 8 weeks. Cholesterol absorption was then performed (n = 8). Fat absorption was determined in male Ldlr ^−/− vs DKO mice (n = 8) (C). (D) mRNA levels in the intestine of 6-months-old male WT mice and Ces1 ^−/− mice were quantified (n = 8). *P < 0.05, **P < 0.01.

Figure 6

Ces1/Ces1g ^−/− mice have increased Cyp7a1 and Cyp8b1 expression. (A–F) 6-months-old male chow-fed mice were sacrificed (n = 6). Hepatic and intestinal mRNA levels were quantified by qRT-PCR (A). Bile acids (BA) in gallbladder (B), intestine (C) or liver (D) were quantified. Total BA levels were calculated by addition of BA levels in gallbladder, intestine and liver (E). Cholesterol concentration in bile was analyzed (F). (G–I) 8-weeks-old male Ces1 ^−/− Ldlr ^−/− (DKO) mice and Ldlr ^−/− control littermates were fed a Western diet for 16 weeks (n = 8). Hepatic and intestinal mRNA levels were quantified by qRT-PCR (G). Bile acids in liver, gallbladder, intestine as well as total bile acid levels were determined (H). Biliary cholesterol levels were also determined (I). *P < 0.05, **P < 0.01.

Figure 7

Loss of Ces1/Ces1g increases cholesterol efflux from macrophages. (A and B) Cholesterol efflux to ApoA-I (A) or HDL (B) was performed in the presence or absence of 25 μg/L Ac-LDL (n = 6). (C) mRNA levels were quantified in peritoneal macrophages in the presence of PBS for 24 h (n = 6). (D and E) Peritoneal macrophages were isolated and treated with 10 μM T-0901317 for 24 h (n = 6). mRNA levels of Abca1 (D) and Abcg1 (E) were quantified. (F,G) Peritoneal macrophages were treated in the presence or absence of 25 μg/L Ac-LDL for 24 h (n = 5). Intracellular free cholesterol (F) and total cholesterol (G) were quantified. *P < 0.05, **P < 0.01.

Figure 8

Loss of Ces1/Ces1g promotes M2 macrophage polarization. (A–E) Peritoneal macrophages were isolated from wild-type or Ces1/Ces1g ^−/− mice and subjected to induction to M1 or M2 macrophages (n = 4). mRNA levels were determined by qRT-PCR. (F) Mechanism for Ces1/Ces1g inactivation to repress atherosclerosis. ^{a,b,c, or d} P < 0.05.