Journal: The Journal of Biological Chemistry
Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice
doi: 10.1074/jbc.RA118.002290
Figure Lengend Snippet: SCP2 deficiency significantly reduces intestinal cholesterol absorption, and SCP2 knockdown in intestinal epithelial cells reduces cholesterol uptake. A, C57BL/6 (WT) or SCP2−/− mice were injected intravenously with the lipoprotein lipase inhibitor tyloxapol (500 mg/kg body weight) and gavaged with [3H]cholesterol in olive oil (2 μCi in 200 μl) after 5 min. Intestinal cholesterol absorption was assessed by monitoring the radiolabel associated with plasma collected at the time of euthanasia. Data are presented as plasma DPM per microliter of plasma for the indicated genotype and sex. B, the entire length of the intestine, from the base of the stomach to the tip of the cecum, was divided into four equal parts (P1 to P4), and total RNA was isolated. The mRNA levels of indicated genes were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. C, total protein extracts from ileum segments P1 to P4 of WT and SCP2−/− mice as well as the colon (C) were analyzed by Western blotting for expression of SCP2; β-actin was used as the loading control. Human intestinal epithelial cells (HT-29) were transfected with scrambled or SCP2-specific siRNA as described under “Experimental procedures.” Total protein or RNA was extracted and used to determine the levels of SCP2. D, top, a representative Western blot. Bottom, levels of SCP2 mRNA quantified by quantitative PCR and SCP2 protein by densitometric analyses of Western blots. Data are presented as percent scrambled siRNA-transfected controls for the indicated SCP2 siRNA concentrations used. E, HT-29 cells transfected with scrambled siRNA or 53.3 nm SCP2-specific siRNA were incubated with [3H]-cholesterol, and total cellular uptake was monitored as described under “Experimental procedures.” Data (mean ± S.D., n = 6) are presented as DPM normalized to total cellular protein. *, p < 0.05.
Article Snippet: Cells were transfected with a complex consisting of SCP2 siRNA (Santa Cruz Biotechnology, catalog no. sc-44636) and polyamidoamine (PAMAM) G5 (Sigma-Aldrich).
Techniques: Injection, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Incubation
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![SCP2 deficiency significantly reduces intestinal cholesterol absorption, and SCP2 knockdown in intestinal epithelial cells reduces cholesterol uptake. A, C57BL/6 (WT) or SCP2−/− mice were injected intravenously with the lipoprotein lipase inhibitor tyloxapol (500 mg/kg body weight) and gavaged with [3H]cholesterol in olive oil (2 μCi in 200 μl) after 5 min. Intestinal cholesterol absorption was assessed by monitoring the radiolabel associated with plasma collected at the time of euthanasia. Data are presented as plasma DPM per microliter of plasma for the indicated genotype and sex. B, the entire length of the intestine, from the base of the stomach to the tip of the cecum, was divided into four equal parts (P1 to P4), and total RNA was isolated. The mRNA levels of indicated genes were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. C, total protein extracts from ileum segments P1 to P4 of WT and SCP2−/− mice as well as the colon (C) were analyzed by Western blotting for expression of SCP2; β-actin was used as the loading control. Human intestinal epithelial cells (HT-29) were transfected with scrambled or SCP2-specific siRNA as described under “Experimental procedures.” Total protein or RNA was extracted and used to determine the levels of SCP2. D, top, a representative Western blot. Bottom, levels of SCP2 mRNA quantified by quantitative PCR and SCP2 protein by densitometric analyses of Western blots. Data are presented as percent scrambled siRNA-transfected controls for the indicated SCP2 siRNA concentrations used. E, HT-29 cells transfected with scrambled siRNA or 53.3 nm SCP2-specific siRNA were incubated with [3H]-cholesterol, and total cellular uptake was monitored as described under “Experimental procedures.” Data (mean ± S.D., n = 6) are presented as DPM normalized to total cellular protein. *, p < 0.05.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5425/pmc06005425/pmc06005425__zbc0251888600004.jpg)
![SCP2 deficiency significantly reduces lipid secretion from liver and isolated hepatocytes. A, C57BL/6 (WT) or SCP2−/− mice were injected with the lipoprotein lipase inhibitor tyloxapol (500 mg/kg of body weight), and blood samples were drawn at 0 and 3 h. Triglyceride secretion rates for indicated genotypes and sexes are presented. B and C, primary hepatocytes were prepared from C57BL/6 (WT) or SCP2−/− mice. Following incubation with [3H]oleic acid, radiolabel associated with secreted triglycerides (TG, B) or cholesteryl esters (C) was determined as described under “Experimental procedures” and normalized to cellular protein. Data are presented as DPM associated with the triglyceride or cholesteryl ester fraction in the total lipids extracted from the medium per milligram of total protein.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5425/pmc06005425/pmc06005425__zbc0251888600005.jpg)
![Effects of SCP2 deficiency on cholesterol accumulation in and cholesterol efflux from macrophages. Thioglycolate-elicited macrophages were isolated from C57BL/6 (WT) and SCP2−/− mice and incubated with AcLDL (25 μg/ml) for 48 h. A and B, following two washes with PBS, cells were either fixed and stained with Oil Red O (A) or used for total lipid extraction and cholesterol mass measurement (B). Total cholesterol mass was normalized to total cellular protein, and data (mean ± S.D., n = 6) are presented as nanomoles per milligram of protein. C, total protein extracts of macrophages were subjected to Western blot analyses to assess SR-A expression; β-actin was used as a loading control. D, for measurement of cholesterol efflux, cells were loaded with AcLDL and labeled with [3H]-cholesterol for 48 h. Following a 24-h equilibration, cholesterol efflux to 10% FBS in the growth medium was monitored over time. Data (mean ± S.D., n = 6) are presented as percent efflux. *, p < 0.05.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5425/pmc06005425/pmc06005425__zbc0251888600007.jpg)
