Acetylcholine-synthesizing macrophages in subcutaneous fat are regulated by β2-adrenergic signaling (2021)
Figure 4

Figure 4. ChAMs function selectively via activation of the β2-AR.

A-C - ChAT-eGFP mice were treated with (A) veh (n = 4) or β1-AR agonist (1 mg/kg dobutamine, Dob) (n = 3), (B) veh (n = 7) or β2-AR agonist (1 mg/kg formoterol, Form) (n = 7) or (C) veh (n = 4) or β3-AR agonist (1 mg/kg CL 316,243, CL) (n = 4 for MΦ; n = 3 for T cells and B cells) for 4 h and the percentages of total IWAT SVF cells and of total T cells, B cells and MΦ that were ChAT-eGFP+ were measured by flow cytometry.

D-E - Percentage of (D) total IWAT SVF cells and (E) IWAT MΦ expressing ChAT-eGFP in reporter mice with genetic deletion combinations of β-ARs 1, 2 and 3 following treatment with veh (white bar: n = 7) or 1 mg/kg NE (black bars: from left to right, n = 7, 5, 4, 5, 4, 7) for 2 h.

F - LC/MS-MS quantification of acetylcholine levels secreted by SVF cells derived from IWAT of β2WT and β2KO mice treated with veh or 1 mg/kg Form for 2 h (n = 3).

G-I - BMDMs were isolated and grown from WT and β‑less mice then treated for 2 h with (G) veh (n = 6 WT, n = 6 β-less) or β1-AR agonist (2.5 μM Dob) (n = 6 WT, n = 6 β-less), (H) veh (n = 6 WT, n = 4 β‑less) or β2-AR agonist (2.5 μM Form) (n = 6 WT, n = 4 β-less) or (I) veh (n = 6 WT, n = 6 β‑less) or β3-AR agonist (2.5 μM CL) (n = 5 WT, n = 6 β-less). Chat mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.

J - Total number of ChAT-eGFP+ cells in BMDMs derived from ChAT-eGFP mice. BMDMs were treated for 2 h with veh or β1-AR agonist (2.5 μM Dob), β2-AR agonist (2.5 μM Form) or β3-AR agonist (2.5 μM CL) then an equal number of events (50,000) were analyzed by flow cytometry (n = 4).

K - Total number of ChAT-eGFP+ cells in BMDMs derived from ChAT-eGFP and β-less ChAT-eGFP mice. BMDMs were treated for 2 h with veh or β2-AR agonist (2.5 μM Form) then an equal number of events (50,000) were analyzed by flow cytometry (n = 4). n.s: not significant.

L - Primary MΦ were isolated from IWAT by FACS then seeded into cell culture plates and treated for 2 h with veh (n = 6) or β2-AR agonist (2.5 μM Form) (n = 4). Chat mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.

M - Left: BMDMs were treated for 2 h with veh or pan β-AR agonist (100 μM NE), β2-AR antagonist (5 μM butoxamine, Buto) or a combination of NE and β2 antagonist (Buto) (n = 6). Chat mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: Total number of ChAT-eGFP+ BMDMs. ChAT-eGFP BMDMs were treated for 2 h with veh or pan β-AR agonist (100 μM NE), β2-AR antagonist (5 μM butoxamine, Buto) or a combination of NE and β2-AR antagonist (Buto) (n = 4). An equal number of events (50,000) were analyzed by flow cytometry.

N - Left: Bicompartmental co-culture system with media alone (Ctrl) or WT SVF cells isolated from IWAT in the upper compartment (transwell insert) and freshly isolated IWAT explants from β-less mice in the lower compartment. Cells were co-cultured for 4 h in the presence or absence of β2-AR agonist (2.5 μM Form). 150 μM rivastigmine was added to the media to prevent degradation of Ach. Right: qPCR analyses of Chat and Ucp1 mRNA levels in β‑less explants following co-culture with media (n = 4), vehicle (n = 10) or β2-AR agonist (n = 10) treated SVF cells. mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.

Data information: In (A-C, F-I, K-L and N), data are presented as mean ± SEM where *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed Student's t-test). In (D-E), data are presented as mean ± SEM where ***P < 0.001 compared to vehicle-treated (two-tailed Student's t-test). In (J and M), data are presented as mean ± SEM and the letters "a", "b" and "c" indicate P < 0.05 between groups (one-way ANOVA).

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