Figure 2. ATF4 activation in brown adipocytes is necessary for thermogenic phenotypes in the Lrpprc^BKO mice.

(A) List of enriched Transcript factors (TF) in commonly up-regulated DEGs in the BAT of CON and Lrpprc^BKO mice at RT and 30°C. Name, motif sequence and number of targets of each TF shown. (B) ATF4 signaling network. GO terms of ATF4 targets shown. (C) Scatter plots showing the correlation of log2 fold changes (Log2 FC)) of mRNA and protein levels of mitochondria-localized proteins from BAT of Lrpprc^BKO mice housed at RT and 30°C. mtDNA-encoded ETC, nuclear-encoded ETC, and ATF4 targets circled. (D) Left: Immunoblots showing amounts of Lrpprc, p-eIF2α, total eIF2α, Atf4 and Hsp90 in the BAT of ~8–12-week-old male CON and Lrpprc^BKO mice at normal chow at both RT and 30°C. Right: Relative protein abundance (to Hsp90) or phosphorylation (to total protein) shown. Significance between genotypes indicated. (E) Clustering analysis of log2 fold changes of known ATF4 target genes in the BAT of mouse models with defective mitochondrial function in BAs (Lrpprc^BKO, Tfam^BKO, Gnas^BKO and betaless mice, ~8–12-week-old male) at normal chow at both RT and 30°C. Sample size: n=5 for each genotype/condition. (F) Representative H&E staining of BAT from ~8–10-week-old male CON, Lrpprc^BKO, Atf4^BKO, and Lrpprc;Atf4^BKO mice housed at RT. Scale bar: 50 μm. (G) Violin plot of log2 fold change (FC) of 8 mtDNA-encoded ETC (mt ETC, listed in Fig.1A), 15 nuclear-encoded ETC (nuclear ETC, listed in Fig.1A) and 22 ATF4 targets (listed in Fig.2E) in the BAT of ~8–12-week old male Lrpprc^BKO and Lrpprc;Atf4^BKO mice to their relative controls (Lrpprc^f/f and Lrpprc;Atf4^f/f) housed at RT. Red and black bars: median and quartiles. Hourly CL-induced VO2 (H) and EE (I) in ~10-week old male CON, Lrpprc^BKO and Lrpprc;Atf4^BKO mice at RT and 30°C. Sample size: CON/RT(n=8), Lrpprc^BKO/RT (n=3), Lrpprc;Atf4^BKO/RT(n=6), CON/30°C(n=13), Lrpprc^BKO/30°C (n=6) and Lrpprc;Atf4^BKO/30°C (n=7). (J) The Temp (Neck-Back) of ~12-week-old male and female CON, Lrpprc^BKO, Atf4^BKO, and Lrpprc;Atf4^BKO mice at thermoneutrality. Sample size: CON (n=15), Lrpprc^BKO (n=9), Atf4^BKO (n=3) and Lrpprc;Atf4^BKO (n=10). (K) CTT of ~8–12-week old male and female CON, Lrpprc^BKO, Atf4^BKO, and Lrpprc;Atf4^BKO mice housed at RT. Sample size: CON (n=6), Lrpprc^BKO (n=9), Atf4^BKO (n=4), and Lrpprc;Atf4^BKO (n=8). Data were presented as average ± SEM. Unpaired t-test. n.s.: non-significant; *: p<0.05 and **: p<0.01.