Intestine-specific DGAT1 deficiency improves atherosclerosis in apolipoprotein E knockout mice by reducing systemic cholesterol burden

Figures

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  Fig. 1. Altered intestinal and plasma lipid concentrations in iDgat1−/−ApoE−/− mice. Male iDgat1 −/− ApoE −/− mice and controls were fed a WTD for 9 weeks. (A) Body weight gain (n = 4–6). (B) Biochemical estimation of intestinal lipids (n = 8–9), and (C) representative images of oil red O-stained intestinal sections 4 h after gavaging with 100 μl corn oil containing 0.25% cholesterol. Lipoprotein profiles of (D) TC and (E) TG in pooled plasma samples (n = 8) of 12 h-fasted mice. Data represent means ± SD; *p < 0.05.

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  Fig. 2. Reduced acute cholesterol absorption and CM size in iDgat1−/−ApoE−/− mice. Mice were fed a WTD for 9–12 weeks. Acute cholesterol absorption determined as radioactivity distribution in (A) gastrointestinal tissues, (B) plasma, and (C) liver after corn oil gavage containing [3H]cholesterol (n = 7–12). (D and E) Four hour-fasted mice were i.p. injected with P407 and gavaged with 100 μl corn oil. After 90 min, blood was taken for (D) CM size measurement by light-scattering (n = 3) and (E) plasma TG and TC estimation (n = 6–8). (F) Intestinal mRNA expression analyzed in duplicate by real-time PCR and normalized to cyclophilin A as reference gene. Expression profiles and associated statistical parameters were determined by the 2—ΔΔCt method (n = 3–4). Data represent means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 3. Increased fecal cholesterol excretion and decreased fractional cholesterol absorption in iDgat1−/−ApoE−/− mice. Male mice were fed a WTD for 9–12 weeks. Radioactivity in (A) plasma, (B) tissues and feces, and (C) bile 24 h after i.v. injection of [3H]cholesterol-enriched LDL (n = 5–6). (D and F) Hepatic mRNA expression analyzed in duplicate by realtime PCR and normalized to cyclophilin A expression as reference gene. Expression profiles and associated statistical parameters were determined by the 2—ΔΔCt method (n = 4). (E) TLC separation of lipid classes after hepatic incorporation of i.p. injected [14C]acetate into lipids (n = 5). (G) Fractional cholesterol absorption determined in chow diet-fed mice by the fecal-dual isotope method (n = 7–8). (H) Fecal caloric output measured by bomb calorimetry (n = 8–10). Data represent means ± SD; *p < 0.05, **p < 0.01.

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  Fig. 4. Reduced atherosclerotic plaque formation in aortas of iDgat1−/−ApoE−/− mice. Female mice were fed a WTD for 9 weeks. Oil red O staining of en face aortas and quantitation of lesion sizes in (A) aortic arch and (B) total thoracic aortas. Data represent means (n = 8–9) ± SD. ***p < 0.001.

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  Fig. 5. Delayed atherosclerosis in aortic valves of iDgat1−/−ApoE−/− mice with reduced lesion size and improved plaque stability. Female mice were fed WTD for 9 weeks. (A) Oil red O-stained lipids and total lesion size, (B) MoMa-2 immunohistochemically-stained macrophages (red), (C) Masson’s Trichrome-stained collagen (blue), and (D) acellular compartments of necrotic core. (E) Quantitation of collagen per necrotic area (n = 8–9). (A–E) Data represent mean values of 3 aortic valve sections in the area of maximal plaque size for each mouse. (F) Morphometric scoring of plaque stage development according to Whitman et al. [22]. Data represent means (n = 8–9) ± SD. *p < 0.05, **p < 0.01.