Figure 5. miR-182-5p promotes PKA activation via the nAChR signaling pathway.

PKA substrate phosphorylation (A) in sWAT of mice injected with miR-182-5p agomir or control agomir, (B) in sWAT of miR-182-5p^+/– and WT control mice, and (C) in miR-182-5p–overexpressed primary adipocytes cocultured with or without PMs (n = 3/group). Choline acetyltransferase (Chat) mRNA levels were determined by qRT-PCR in (D) SVFs and (E) PM treated with or without FGF21 (n = 3–4/group). (F) Chat mRNA levels in sWAT of mice injected with miR-182-5p agomir- or control agomir. ELISA analysis of acetylcholine levels secreted from the (G) sWAT and (H) inguinal SVF of mice injected with miR-182-5p agomir or control agomir (n = 4/group). (I) Chat mRNA levels in PMs cocultured with primary white adipocytes overexpressing 182-mimic or nc-mimic were determined by qRT-PCR (n = 3/group). (J) Chat mRNA levels in βKlotho-siRNA or nc-siRNA–treated PMs cocultured with primary white adipocytes overexpressing miR-182-5p mimic were determined by qRT-PCR (n = 3/group). (K) UCP1 protein levels in sWAT of βKlotho^mKO mice and Loxp control mice injected with miR-182-5p agomir (-182) or control agomir (-nc) (n = 4/group). The mRNA (L) and protein (M) levels of UCP1 or PKA substrate phosphorylation (M) in primary adipocytes incubated with CM from control (nc-siRNA) or βKlotho-suppressed PMs treated with or without FGF21 were determined by qRT-PCR or Western blot (n = 3 biological replicates). The mRNA (N) and protein (O) levels of UCP1 or PKA substrate phosphorylation (O) in Chrna2-siRNA– or nc-siRNA–treated primary adipocytes incubated with CM from FGF21-treated or nontreated PMs were determined by qRT-PCR or Western blot (n = 3 biological replicates). Data represent mean ± SEM. Significance determined by unpaired 2-tailed Student’s t test (D–J) and by 1-way ANOVA (L and N). *P < 0.05; **P < 0.01.