Gene Expression Analysis of Environmental Temperature and High-Fat Diet-Induced Changes in Mouse Supraclavicular Brown Adipose Tissue

Figure 1. The effect of cold exposure on the adipocyte morphology and gene expression in scBAT, iBAT, iWAT, and eWAT. (a) The body temperatures of male C57BL/6 mice undergoing cold exposure (4 °C). n = 4. h: hour. (b) Representative images of the H&E stained sections of scBAT, iBAT, iWAT, and eWAT isolated from mice housed for 6 h at ambient temperature (22 °C) or exposed to cold (4 °C). n = 3. Scale bar = 50 µm. (c–f) RT-qPCR analysis of adipocyte differentiation marker genes, thermogenic genes, glucose uptake genes and lipid processing genes in scBAT, iBAT, iWAT, and eWAT depots from mice housed at ambient temperature and under acute cold exposure (4 °C for 6 h). (c) Adipocyte differentiation markers, Pparg, Prdm16, Fabp4, and Adipoq. (d) Thermogenic genes, Ucp1, Pgc-1α, and Dio2. (e) Glucose transporters, Glut4 and Glut1. (f) Lipid uptake and processing genes, Lpl and Hsl. The expression of these genes is normalized to the housekeeping gene β-actin. Data are presented as mean ± SEM. n = 4–8 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = nonsignificant.

Figure 2. The effect of HFD on the adipocyte morphology and gene expression in scBAT, iBAT, iWAT, and eWAT. (a) Schematic diagram of the HFD study design. P0: postnatal day 0, wks: weeks. Mice were subjected to 10 to 16 weeks of RD or HFD. (b) Representative images of H&E-stained sections of scBAT, iBAT, iWAT, and eWAT from mice treated with RD or HFD for 16 weeks. n = 3–4. Scale bar = 50 µm. (c) Body weight (BW), adipose tissue mass, and ratio of adipose tissue to body weight of mice described in (b) (RD, n = 4 and HFD, n = 4). (d–h) RT-qPCR analysis of adipocyte differentiation marker genes, thermogenic genes, glucose uptake genes, and lipid processing genes in scBAT, iBAT, iWAT, and eWAT depots from mice fed 10 weeks with RD or 10 weeks with HFD. (d) Adipocyte differentiation markers, Pparg, C/ebpβ, Prdm16, Pparα, Fabp4, and Adipoq. (e) Thermogenic genes, Ucp1, Pgc-1α, and Dio2. (f) Glucose transporters, Glut4 and Glut1. (g) Lipid uptake and processing genes, Lpl and Hsl. (h) Lipogenesis genes, Fas and Acc1. Data are presented as mean ± SEM. The expression of these genes is normalized to the housekeeping gene β-actin. n = 5–6 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, ****…

Figure 3. The effect of thermoneutrality on the adipocyte morphology and gene expression in scBAT, iBAT, iWAT, and eWAT. (a) Schematic diagram of the thermoneutral housing study design. Mice were housed at the thermoneutral temperature for 8 weeks. (b) Representative images of H&E-stained sections of scBAT, iBAT, iWAT, and eWAT from mice housed under ambient and thermoneutral temperatures. n = 3. Scale bar = 50 µm. (c–g) RT-qPCR analysis of adipocyte differentiation marker genes, thermogenic genes, glucose uptake genes, and lipid-processing genes in scBAT, iBAT, iWAT, and eWAT depots from mice housed under ambient and thermoneutral temperatures. (c) Adipocyte differentiation markers, Pparg, C/ebpβ, Ebf2, Prdm16, Pparα, Fabp4, and Adipoq. (d) Thermogenic genes, Ucp1, Pgc-1α, and Dio2. (e) Glucose transporters, Gult4 and Glut1. (f) Lipid uptake and processing genes, Lpl and Hsl. (g) Lipogenesis genes, Fas and Acc1. The expression of these genes is normalized to the housekeeping gene β-actin. Data are presented as mean ± SEM. n = 4–6. for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = nonsignificant.

Figure 4. The effect of HFD on the adipocyte morphology and gene expression in scBAT, iBAT, iWAT, and eWAT under thermoneutrality. (a) Schematic diagram of the thermoneutral housing study design under HFD. 3-week-old mice were housed at 28 °C for 8 weeks. Mice were fed with RD for 1week for adaptation and then fed with HFD for an additional 7 weeks. (b) Representative images of H&E-stained sections of scBAT, iBAT, iWAT, and eWAT from mice fed RD or HFD under thermoneutral temperatures. n = 5. Scale bar = 50 µm. (c) Body weight, adipose tissue mass, and ratio of adipose tissue to body weight of mice described in B (RD, n = 5 and HFD, n = 5). (d–h) RT-qPCR analysis of adipocyte differentiation marker genes, thermogenic genes, glucose uptake genes, and lipid processing genes, and lipogenesis genes in scBAT, iBAT, iWAT, and eWAT depots from mice housed under ambient and thermoneutral temperatures. (d) Adipocyte differentiation markers, Pparg, C/ebpβ, Prdm16, Pparα, Fabp4, and Adipoq. (e) Thermogenic genes, Ucp1, Pgc-1α, and Dio2. (f) Glucose transporters, Glut4 and Glut1. (g) Lipid uptake and processing genes, Lpl and Hsl. (h) Lipogenesis genes, Fas, Acc1. The expression of these genes is…

Figure 5. The effect of thermoneutrality and cold exposure on the adipocyte morphology and gene expression of dnBAT in C57BL/6 mice. (a) Representative image of the anatomical location of dnBAT in 2-month-old mice. tr: trachea, t: thyroid, m: sternocleidomastoid muscle, jv: external jugular vein. dnBAT is outlined by the black dotted line. (b) Representative images of H&E-stained sections of dnBAT from mice housed at ambient, thermoneutral, and cold temperatures. n = 3. Scale bar = 50 µm. (c–f). RT-qPCR analysis of adipocyte differentiation marker genes, thermogenic genes, glucose uptake genes, and lipid-processing genes in scBAT, iBAT, iWAT, and eWAT depots from mice housed at ambient and thermoneutral temperatures and under acute cold exposure. (c) Adipocyte differentiation markers, Pparg, Prdm16, Fabp4, and Adipoq. (d) Thermogenic genes, Ucp1, Pgc-1α, and Dio2. (e) Glucose transporters, Glut4 and Glut1. (f) Lipid uptake and processing genes, Lpl and Hsl. The expression of these genes is normalized to the housekeeping gene β-actin. Data are presented as mean ± SEM. n = 4–5 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = nonsignificant.