Effects of HFD or isoprenaline on Pld2 mRNA levels in ingWAT, BAT, and brown adipocytes and on metabolic parameters in adipocyte-specific Pld2 KO or PLD2-inhibited mice. (A) Pld2 mRNA expression in BAT and ingWAT from HFD-fed mice for 0, 4, and 12 wk (n = 6/group). (B) Quantitative RT-PCR for Pld2 mRNA expression in vehicle- or isoprenaline (1 µM)- or bortezomib (20 nM, 1 h pretreat)-treated primary brown adipocytes for 24 h (n = 4/group). (C) Generation of Pld2 Ad-KO mice. Details of the KO generation strategy are described in the Materials and methods section. (D) Western blot analysis of PLD2 and β-Actin in various adipose tissue depots and liver from 8-wk-old control and Pld2 Ad-KO mice. (E) Fat and lean mass, and body weight (BW) of 8-wk-old control and Pld2 Ad-KO mice (n = 6/group). (F) Representative Western blot images of Cytochrome C (Cyto C), DIO2, PGC1-α, and GAPDH in BAT and ingWAT of 8-wk-old mice injected with vehicle or CAY10594 for 7 consecutive days at 22°C. (G–I) The level of EE (G), RER (H), and locomotor activity (I) of 8-wk-old mice injected with vehicle or CAY10594 for 7 consecutive days at 22°C (n = 5/group). (J–L) The level of EE (J), RER (K), and locomotor activity (L) of 8-wk-old mice injected with vehicle or CAY10594 for 7 consecutive days at 30°C (n = 5/group). (M) Oxygen uptake (VO₂) levels of CL-injected 8-wk-old control or Pld2 Ad-KO mice at thermoneutral condition (n = 6/group). (N) EE of HFD-fed (6 wk) control and Pld2 Ad-KO mice (n = 5/group). The data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student’s t test (A, B, E, G–L, and N) and two-way ANOVA (M). Data are representative of two (B and F) or three independent experiments (A and D). Veh, vehicle; VCO₂, carbon dioxide production.