Enterocyte-specific ATGL overexpression affects intestinal and systemic cholesterol homeostasis

Figures

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  Fig. 1. Efficient intestine-specific overexpression of ATGL. (A) Atgl expression in tissues of WT mice: WAT, white adipose tissue; Duo, duodenum; Jej, jejunum; Ile, ileum; Col, colon; Kid, kidney. (B) Gene expression of Atgl in the small intestine and liver of WT and Atgl iTg mice after 8 weeks of HF/HCD feeding, normalized to cyclophilin A. (C) Western blot analysis of jejunal and hepatic tissue lysates to detect FLAG-tagged ATGL. The FLAG-tagged Ces2c construct served as a positive control (Ctl) and β-actin was determined as loading control. (D) Western blot analysis and (E) corresponding quantification of isolated jejunal enterocytes to detect total ATGL with exposure times of 1 s and 10 s. Tissue lysate of WT WAT served as a positive control (Ctl) and β-actin was determined as loading control. (F) Representative images of ATGL immunofluorescence staining (green) with DAPI (blue) on jejunal cryosections. Scale bar, 20 μm. (G) Jejunal neutral TG hydrolase activity and (H) fatty acid oxidation in jejunal enterocytes of chow diet-fed Atgl iTg mice and their corresponding WT littermates. Data represent mean values of 13–15 week-old female mice (n = 4–6) + SD. ** p ≤ 0.01; *** p ≤ 0.001.

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  Fig. 2. Increased CM-TC secretion in HF/HCD-fed Atgl iTg mice. Atgl iTg and control mice were fasted for 4 h prior to i.p. administration of tyloxapol. Secretion of CM-TG and CM-TC 2.5 and 5 h after an oral lipid bolus containing [3H]-triolein and [14C]-cholesterol in (A) chow diet- and (B) 5-week HF/HCD-fed WT and Atgl iTg mice. (C, D) VLDL-TG and VLDL-TC concentrations measured before, 2, 4, and 6 h after the administration of tyloxapol in (C) chow diet- and (D) 5-week HF/HCD-fed mice. (E) Plasma cholesterol levels in chow diet-fed mice under various feeding conditions. Data represent mean values of 16-21 week-old female mice (n=3-7) ± SD. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

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  Fig. 3. Atgl overexpression drastically reduces jejunal TG levels upon oral lipid bolus. Chow diet-fed WT and Atgl iTg mice were sacrificed 4 h after an oral oil bolus. (A) TG distribution among lipoprotein classes. (B) Duodenal lipid levels. (C) Representative oil red O staining and biochemical lipid quantification in jejuna. Scale bar, 50 μm. (D) Jejunal mRNA expression of LD-associated proteins normalized to cyclophilin A. (E) Jejuna of mice sacrificed 4 h post-gavage of BODIPY®-C12 (red) were stained with anti-PLIN2 (green) to detect LDs. Nuclei were visualized by DAPI staining (blue). Scale bar, 20 μm. (F) Jejunal mRNA expression of genes involved in cholesterol metabolism normalized to cyclophilin A. (G) TC distribution among lipoprotein classes. Data represent mean values of 33 week-old female mice (n = 3-4) + SD. * p < 0.05; *** p ≤ 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 4. Accelerated cholesterol uptake into enterocytes of Atgl iTg mice. (A, B) Chow diet-fed mice were fasted for 4 h prior to intraperitoneal injection of [14C]-acetate in 200 μl PBS and sacrificed 1 h after injection. (A) Distribution of total cholesterol (solid line) and radioactivity (dashed line) in plasma lipoprotein fractions, (B) intestine, and liver. (C) Secretion of orally administered [14C]-cholesterol into the circulation and (D) distribution of radioactivity in intestinal and hepatic tissues. Data represent mean values of 21 week-old female mice (n = 4) ± SD. (E) Cryosections of jejunal swiss rolls (middle to outer layer: proximal to distal part) 2 h after oral administration of fluorescently-labeled cholesterol (red) with DAPI staining for nuclei (blue) in 14 week-old female mice. Triangle, proximal end; star, distal end. Scale bar, 1000 μm; magnification of square: scale bar, 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 5. Accelerated dietary cholesterol absorption in HF/HCD-fed Atgl iTg mice. (A) Secretion of orally administered [14C]-cholesterol into the circulation and (B) distribution of the radioactivity in intestinal and hepatic tissues in 5-week HF/HCD-fed Atgl iTg mice. (C) Gene expression analysis of LD-associated proteins, PPARα target genes, and Mttp in jejuna of WT and Atgl iTg mice after 5 weeks of HF/HCD feeding, normalized to cyclophilin A. Data represent mean values of 12-13 week-old female mice (n = 4-5) ± SD. * p < 0.05; ** p ≤ 0.001; *** p ≤ 0.001.

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  Fig. 6. Increased energy expenditure and fatty acid utilization in HF/HCD-fed Atgl iTg mice. Jejunal (A) TG hydrolase activity and (B) lipid levels. (C) mRNA expression analysis of Plin2, Plin3, and genes involved in basolateral lipid uptake of HF/HCD-fed mice after 4 h of fasting, normalized to cyclophilin A. (D—F) After 10 weeks of HF/HCD feeding, mice were housed in metabolic cages, and fatty acid oxidation (FOX) and energy expenditure (EE) were analyzed. Data represent mean values of 16—18 week-old female mice (n = 3—5) ± SD. * p < 0.05; ** p ≤ 0.001; *** p ≤ 0.001.

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  Fig. 7. Refeeding for 2 h decreases HDL in HF/HCD-fed Atgl iTg mice. WT and Atgl iTg mice fed HF/HCD for 8 weeks were sacrificed 2 h after refeeding. (A) Plasma lipid parameters and lipoprotein profile. (B) Representative oil red O-stained sections and biochemical lipid quantification of jejuna from WT and Atgl iTg mice. Scale bar, 50 μm. Jejunal mRNA expression of (C) genes involved in intestinal cholesterol metabolism, including LXR target genes (underlined), (D) nuclear receptors, and (E) PPARβ/δ target genes, normalized to cyclophilin A as housekeeping gene. Data represent mean values of 18 week-old female mice (n = 3–5) + SD. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

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  Fig. 8. LXR agonist treatment increases plasma HDL in Atgl iTg mice. (A) Chow diet-fed mice were orally treated with the LXR agonist GW3965 (GW) in corn oil containing 1% cholesterol for 9 subsequent days and body weight was monitored daily. Blood was collected from 4-h fasted mice and (B) plasma lipid parameters before and after GW3965 treatment and (C) lipoprotein profiles after 9 days of GW3965 treatment were determined. (D) Jejunal mRNA expression of GW3965-treated WT and Atgl iTg mice normalized to β-actin as housekeeping gene. Data represent mean values of 27 week-old female mice (n = 4–6) ± SD. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001. §§ p ≤ 0.01; §§§ p ≤ 0.001 between pre- and post-treatment within the genotypes.