Figure 6. Genetic ablation of Atg12 or Atg5 maintains beige adipocyte characteristics after removal of β3-AR agonist.

(A) Schematic illustration of experiments. Control (Atg12^flox/flox or Atg5^flox/flox), Atg12^Ucp1 KO (Ucp1^Cre/+;Atg12^flox/flox), and Atg5^Ucp1 KO (Ucp1^Cre/+;Atg5^flox/flox) mice were treated with the β3-AR agonist CL316,243 for seven consecutive days. Interscapular BAT and inguinal WAT depots were harvested for molecular analyses at day 0 and 15 following β3-AR agonist withdrawal.

(B) Immunoblotting for UCP1 and mitochondrial complexes (as indicated) in the inguinal WAT depots of control (Atg12^flox/flox) and Atg12^Ucp1 KO mice at day 0 and day 15 following β3-AR agonist withdrawal. Inguinal WAT depots from control and Atg12^Ucp1 KO mice treated with saline were included as a reference of basal expression of UCP1 and mitochondrial complexes. β-actin was used as a loading control. Molecular weight (MW) is shown on the right.

(C) Immunoblotting for UCP1 and mitochondrial complexes (as indicated) in the inguinal WAT depots of control (Atg5^flox/flox) and Atg5^Ucp1 KO mice. Samples were harvested as illustrated in (B)

(D) Left; Mitochondrial DNA (mtDNA) transcripts (as indicated) were quantified in the inguinal WAT depots of control and Atg12^Ucp1 KO mice at day 15 following β3-AR agonist withdrawal. Right; mRNA levels of nuclear-coded beige-enriched markers (as indicated) are shown. * P <0.05 by two-tailed Student’s t-test. n = 5. Data are expressed as means ± s.e.m.

(E) Top panel: Wild-type mice were housed at 6°C for 7 days and subsequently kept under thermoneutrality (30°C) for 15 days. During the re-warming period, the mice were treated with chloroquine at a dose of 60 mg kg⁻¹ or saline. Inguinal WAT depots were harvested for molecular analysis. Bottom panel; Immunoblotting for UCP1 and mitochondrial complexes (as indicated) in the Inguinal WAT of mice. Molecular weight (MW) is shown on the right.

(F) Oxygen consumption rate (OCR) in the inguinal WAT depots of control and Atg12^Ucp1 KO mice at day 15 following β3-AR agonist withdrawal. The isolated tissues were treated with isoproterenol or vehicle (basal). OCR data were shown per 1 mg of tissue. * P < 0.05, ** P < 0.01 by two-tailed Student’s t-test. n = 4. Data are expressed as means ± s.e.m.

(G) Quantification of whole-body oxygen consumption rate (VO₂) of control and Atg12^Ucp1 KO mice during day 17–18 following β3-AR agonist withdrawal. VO₂ was measured by CLAMS during day and night time. ** P <0.01 by two-tailed Student’s t-test. n = 5 per genotype. Data are expressed as means ± s.e.m.