MitoNEET in Perivascular Adipose Tissue Blunts Atherosclerosis under Mild Cold Condition in Mice

Figures

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  Figure 1. mitoNEET is reduced in PVAT of Pgc1 knockout mice. Real-time PCR shows Pgc1α mRNA levels in BAT and PVAT of Pgc1α knockout mice (Pgc1α KO) (A), and Pgc1β mRNA levels in BAT and PVAT of Pgc1β knockout mice (Pgc1β KO) (B). The relative mRNA levels were normalized by 18S, respectively. Data shown are mean ± SD. n = 5 mice/group. (C) Intravascular (thoracic aorta) temperature in anesthetized wild type (WT), Pgc1α KO and Pgc1β KO mice in response to 4°C stimuli. The lag time reflects the time of dipping of the hind feet and tail in 4°C cold water. The zero represents the start time point of immersion in the cold water. Data shown are mean ± SD. n = 6 mice/group.*p < 0.05 Pgc1α KO vs. WT, **p < 0.01 Pgc1α KO vs. WT; #p < 0.05 Pgc1β KO vs. WT, ##p < 0.01 Pgc1β KO vs. WT. (D) RNA deep sequencing identified eight common downregulated genes in PVAT of both Pgc1α KO and Pgc1β KO mice, as indicated in the Venn diagrams. (E) mitoNEET mRNA levels in PVAT of Pgc1α KO and Pgc1β KO mice. The relative mitoNEET mRNA level was normalized by 18S, respectively. Data shown are mean ± SD. n = 5 mice/group. (F) Western blots show Pgc1α, Pgc1β and mitoNEET protein levels in PVAT of WT, Pgc1α KO and Pgc1β…

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  Figure 2. mitoNEET is up-regulated in PVAT upon cold stimuli. (A) mitoNEET mRNA levels in BAT, PVAT, gonadal WAT (gWAT), and subcutaneous WAT (sWAT) in 10-week old C57BL/6J mice which were housed at 22°C. The relative mitoNEET mRNA level was normalized by 18S, and the expression level of mitoNEET mRNA in gWAT was set as 1. Data shown are mean ± SD. n = 5 mice/group. (B) Western blots show mitoNEET protein levels in PVAT, BAT, sWAT, and gWAT at 22°C and after 24-h 4°C cold stimuli. n = 3 mice per temperature condition. (C) Quantitative data of blots in (B) expressed as the ratio of densitometry of mitoNEET/β-Tubulin. Data shown as mean ± SD of 3 blots either at 22°C or 4°C.

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  Figure 3. Cold tolerance in mitoNEET-Tg mice. (A) Schema of the construct used for generating transgenic mice with brown adipocyte-specific overexpression of the human mitoNEET driven by the mouse Ucp-1 promoter (top). Identification of four human mitoNEET positive founders in the C57BL/6J background (#8, #17, #27, and #28) were identified (bottom). The transgenic mice used in this study are from #8 line. (B) Western blot shows that mitoNEET is overexpressed in PVAT and BAT of the transgenic mice. (C) Representative H.E. staining showing the morphology of thoracic aortic PVAT and interscapular BAT in 10-week old wild type and mitoNEET-Tg mice. Magnification bar = 20 μm. (D) Body temperature of conscious wild type and mitoNEET-Tg mice in response to 4°C stimuli. The body temperatures were collected at 9 a.m., 12 p.m., and 4 p.m. when the mice were housed either in a 22°C or a 4°C chamber. Data shown as mean ± SD, n = 5 mice per group,*p < 0.05 vs. WT mice. (E) Intravascular (thoracic aorta) temperature in the anesthetized mice with interscapular BAT removal was recorded for 90 s as described in Materials and Methods Section. Data shown as mean ± SD. n = 6 mice/group.**p < 0.01 vs. WT.

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  Figure 4. Increase in thermogenesis-related genes in PVAT of mitoNEET-Tg mice. RT-PCR was used to determine the mRNA levels (relative to 18S) of thermogenesis-related genes in PVAT of wild type and mitoNEET-Tg mice housed at 16°C for 1-week. Data shown as mean ± SD. n = 6 mice/group.

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  Figure 5. Energy expenditure in mitoNEET-Tg mice. Wild type and mitoNEET-Tg mice with interscapular BAT removed were single-housed in metabolic cages. The food intake (A), body weight and body composition (B), total locomotor activity (C), energy expenditure (D), oxygen consumption (E) and carbon dioxide production (F) were recorded when the chamber temperatures were adjusted to 22°C or 4°C. Data shown as mean ± S.E.M (n = 6) and histogram figures are the 24-h average.

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  Figure 6. Atherosclerosis in mitoNEET-Tg mice. Twelve-week-old ApoE KO and ApoE/mitoNEET-Tg mice were housed at and fed a high cholesterol diet (HCD) at 16°C for 3 months. (A) mRNA levels of thermogenesis genes in PVAT of mice at the end-point of the atherosclerosis study. Data shown are mean ± S.E.M. n = 5–6 in each group. (B) Representative Oil Red O staining showing atherosclerotic lesions in whole aortic trees. (C) Quantitative analysis of the ratio of atherosclerotic lesion area to total aortic tree area. Data shown are mean ± SD. n = 15 in each group. (D) Body weights of mice during mild cold challenge. Data shown are mean ± S.E.M. n = 15 in each group. (E) Plasma total cholesterol and triglyceride levels in ApoE knockout and ApoE/mitoNEET-Tg mice at the end-point of the HCD challenge at 16°C for 3 months. Data shown are mean ± SD. n = 15 in each group. (F) mRNA levels of inflammatory genes in PVAT of mice at the end-point of HCD challenge at 16°C for 3 months. Data shown are mean ± S.E.M. n = 5–6 in each group. (G) Macrophage infiltration detected by F4/80 staining (brown) in PVAT of mice at the end-point of HCD challenge at 16°C for 3 months. (H) Quantitative data of F4/80 positive…