Figure 5. UCP1 is essential for the beneficial effects of pharmacological inhibition of α₄ integrin on beige adipogenesis.

a–b) After establishment of DIO, C57BL/6 mice were treated with α₄-inhibitor (α₄-inh.) or vehicle (Con) for 6 more weeks. a) Expression of mRNA of thermogenesis-related genes in SAT of control- or α₄-inhibitor-treated mice (n=6 mice per group). 18S expression was used for normalization and the respective gene expression of control-treated mice was set as 1. b) Representative cropped blot images showing immunoblotting (left) of UCP1 (and actin) in SAT of 3 control- and 3 α₄-inhibitor-treated mice. Densitometric analysis (right) of UCP1 immunoblotting from a total of 6 control- and 6 α₄-inhibitor-treated mice is shown. The protein amounts of UCP1 were normalized against actin; the UCP1 amounts (normalized over actin) in SAT from control-treated mice were set as 1. c–f) After 4 weeks on HFD, wild-type C57BL/6 mice were implanted with an Alzet osmotic pump, delivering α₄-inhibitor (α₄-inh.) or vehicle (Con). After pump implantation, each group of mice (α₄-inh. or Con) was divided into two subgroups and the feeding was continued for another 4 weeks at 22°C or 30°C (thermoneutrality). c–d) Insulin tolerance tests (ITT) from mice 4 weeks after pump implantation are shown (n=6 mice/group and n=8 mice/group for 22°C and 30°C, respectively). e) The mRNA expression of thermogenic genes in SAT of α₄-inh.-treated or control-treated mice at 22°C or 30°C was analyzed at the end of the feeding period. 18S expression was used for normalization and the respective gene expression of control-treated mice maintained at 22°C was set as 1 (22°C - Con, n=6 mice; 22°C - α₄-inh., n=6 mice; 30°C - Con, n=8 mice; 30°C - α₄-inh., n=8 mice). f) The SVF from SAT and VAT of control- or α₄-inhibitor-treated obese wild-type mice maintained under thermoneutrality conditions, was isolated at the end of the experiment and M1-like macrophages (M1MΦ, F4/80⁺CD11b⁺CD206⁻iNOS⁺) were analyzed by flow cytometry. Data are presented as relative to control; the numbers of M1MΦ/gram tissue from control-treated mice were set as the 100% control in each case (Con, n=6 mice; α₄-inh., n=7 mice in SAT; Con, n=5 mice; α₄-inh., n=7 mice in VAT).

Data are presented as mean ± SEM. Data in (a)–(b) are representative of 2 experiments; data in (c)–(f) are from one experiment. *P < 0.05. Mann-Whitney U-test in (a)–(f). Abbreviations: Ucp1, uncoupling protein 1; Cidea, cell death-inducing DNA fragmentation factor-like effector A; Cox8b, cytochrome c oxidase subunit 8b; Cox7a1, Cytochrome c oxidase subunit 7A1; Acsl1, acyl-CoA synthetase long-chain family member 1; Prdm16, PR (PRD1-BF1-RIZ1 homologous)-domain containing 16