Extended Data Fig. 2: Strategy for Gpr132 deficiency in pancreatic islet macrophages.

(a) Representative blots (left) and quantification (right) showing the expression of 12-LOX in islet from NCD mice or HFD mice. Consistent with the increase in the level of total 9(S)-HODE, the level of 12-LOX, an enzyme that converts linoleic acid (LA) to 9(S)-HODE, was also significantly increased in pancreatic islet cells under HFD conditions compared with NCD conditions. 600 islets from 8–12 NCD-fed mice or 4–6 HFD-fed mice were grouped (200 islets/group). Data from three independent experiments (n = 3). (P = 0.0010). (b) Generation of conditional knockout mice of GPR132 and specifically ablated GPR132 in macrophage using Cre-LoxP recombination system. The single exon of GPR132 is deleted using Lyz2-Cre-mediated recombination. (c) Schematic description of the PCR strategy for the genotyping of the conditional knockout mice targeting to Lyz2-cre and Gpr132^fl/fl, respectively. (d) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from GPR132^fl/fl mice or Lyz2-cre^+/−Gpr132^fl/fl mice in response to stimulations with the SN or lysate from HFD mice islets. The supernatants were collected from HFD mice islets after stimulation with 20 mM glucose for 1h in mKRBB solutions. Approximately 6*10⁴ CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from 18–24 NCD-fed mice (1*10⁴ CD11c⁺Ly6C⁻F4/80^low cells/group), Data are from three independent experiments (n = 3). (from left to right, P = 0.0025, P = 0.0047, P = 0.0002, P = 0.0003, P = 0.0105, P = 0.0252, P = 0.0002, P = 0.0013, P = 0.0003, P = 0.0028, P = 0.0002, P = 0.0022, P = 0.0018, P = 0.0040, P < 0.0001, P < 0.0001). (e, f) Plasma glucose (E) and insulin (F) levels in Gpr132^fl/fl or Lyz2-cre^+/− Gpr132^fl/fl mice fed with NCD or HFD for 12 weeks fasted for 16 hours. (n = 6). (from left to right, E, P = 0.9091, P < 0.0001; F, P = 0.5582, P < 0.0001). (g, h) Plasma glucose (G) and insulin levels (H) of the Gpr132^fl/fl or Lyz2-cre^+/− Gpr132^fl/fl mice fed with NCD or HFD for 12 weeks (4h after refeeding) (n = 6). (from left to right, G, P = 0.6290, P = 0.0003; H, P = 0.8824, P = 0.0054). (i) The expression of Gpr132 and Cx3cr1 in CD11c⁺-labeled islet cells was examined by single-cell RT‒PCR. The results indicated that all GPR132⁺ and CD11c⁺ cells expressed Cx3cr1. Approximately 1*10⁴ CD11c⁺-labeled islet cells isolated from 4–6 NCD-fed mice were grouped (1*10⁴ CD11c⁺-labeled islet cells /group). Twelve individual cells were randomly selected from each group and then subjected to single-cell RT‒PCR. Agarose electrophoresis gel of the RT‒PCR product showing the correct molecular weights of Gpr132 (217 bp), Cd11c (185 bp) and Cx3cr1 (125 bp). (j) We constructed a Cx3cr1-cre adeno-associated virus (AAV-Cx3cr1-cre) to specifically knock down the expression of GPR132 in Cx3cr1+ macrophages by infecting the Gpr132^fl/fl mice with AAV-Cx3cr1-cre. Representative immunostaining of GFP (green) cells and CX3CR1 (red) cells in pancreatic sections from Gpr132^fl/fl mice infected with pAAV-Cx3cr1 promoter-Cre-P2A-eGFP (AAV-Cx3cr1^Cre). Scale bar: 50 μm. n = 6 mice; each mouse was used for slicing and counted as an independent experiment; 4–7 random areas were selected from each section, and 10 sections were randomly selected from each mouse. (k, l) Representative western blots (K) and Quantification (L) showing the protein levels of GPR132 in pancreatic islets isolated from Gpr132^fl/fl mice infected with pAAV-Cx3cr1 promoter-Cre-P2A-eGFP (AAV-Cx3cr1^Cre) or pAAV-Cx3cr1 promoter-eGFP (AAV-Cx3cr1^Con). Infection of pancreatic islets derived from Gpr132^fl/fl mice by AAV-Cx3cr1-cre showed a more than 80% reduction in Gpr132 expression in islets. Approximately 600 islets isolated from 8–12 Gpr132^fl/fl mice infected with AAV-Cx3cr1^Con or AAV-Cx3cr1^Cre were grouped (200 islets/group). Data are from three independent experiments (n = 3). (L, P = 0.0018). (m) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in Cx3cr1⁺-labeled islet cells isolated from Gpr132^fl/fl mice infected with AAV-Cx3cr1^Con or AAV-Cx3cr1^Cre treated with 1 μM 9(S)-HODE or Vehicle for 24 h. The increased mRNA levels of Il-1, Tnf-α, Ccl-2 and Cxcl1 induced by 9(S)-HODE were significantly reduced in GPR132 downregulated Cx3cr1⁺ islet macrophages in AAV-Cx3cr1-cre Gpr132^fl/fl mice. 2*10⁴ Cx3cr1⁺-labeled islet cells isolated from 4–6 Gpr132^fl/fl mice infected with AAV-Cx3cr1^Con or AAV-Cx3cr1^Cre were grouped (1* Cx3cr1⁺-labeled islet cells / group) stimulated with 1 μM 9(S)-HODE or vehicle, and subjected for RNA extraction for an independent experiment. The data are from three independent experiments (n = 3). (from left to right, P = 0.8032, P < 0.0001, P = 0.0004, P = 0.4189, P = 0.0006, P = 0.0007, P = 0.7717, P = 0.0002, P = 0.0003). (n, o) The epididymal fat mass (N) and the liver mass (O) of Gpr132^fl/fl or Lyz2-cre^+/−Gpr132^fl/fl mice fed with NCD or HFD for 12 weeks (n = 6). (from left to right, N, P = 0.8564, P < 0.0001; O, P = 0.9214, P = 0.0001). (p) Hepatic triglyceride content of Gpr132^fl/fl or Lyz2-cre^+/− Gpr132^fl/fl mice fed with NCD or HFD for 12 weeks. The data are from six independent experiments (n = 6). (from left to right, P = 0.6916, P < 0.0003). (q) Food intake of wild-type mice, Lyz2-cre^+/− mice, GPR132^fl/fl mice and Lyz2-cre^+/−GPR132^fl/fl mice fed a NCD or HFD (n = 6). (from left to right, P = 0.9201, P = 0.5767, P = 0.6661, P = 0.7913, P = 0.7328, P = 0.5660). (B) **P < 0.01, protein level of 12-LOX of islets isolated from HFD mice compared with those isolated from NCD mice; (D) *P < 0.05; **P < 0.01; ***P < 0.001, ns, no significant difference; CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from Gpr132^fl/fl mice treated with islet lysate or SN from HFD mice islets compared with vehicle; ^#P < 0.05; ^##P < 0.01; ^###P < 0.001, ns, no significant difference; CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from Lyz2-cre^+/−Gpr132^fl/fl mice compared with those from Gpr132^fl/fl mice. (E-H) ns, no significant difference; **P < 0.01; ***P < 0.001. Lyz2-cre^+/− Gpr132^fl/fl mice were compared with Gpr132^fl/fl mice fed on the same diet. (L) **P < 0.01; the protein levels of GPR132 in pancreatic islets from Gpr132^fl/fl mice infected with AAV-Cx3cr1^Con compared with AAV-Cx3cr1^Cre. (M) *** P < 0.001; Gpr132^fl/fl mice injected with AAV-Cx3cr1^Con treated with 1 μM 9(S)-HODE compared with vehicle. Ns, no significant; ^###P < 0.001; Gpr132^fl/fl mice infected with AAV-Cx3cr1^Con compared with Gpr132^fl/fl mice infected with AAV-Cx3cr1^Cre. (N-P) ns, no significant difference; ***P < 0.001. Gpr132^fl/fl mice and were compared with Lyz2-cre^+/− Gpr132^fl/fl mice fed on the same diet. (Q) Ns, no significant difference, for Lyz2-cre^+/− mice, GPR132^fl/fl mice and Lyz2-cre^+/−GPR132^fl/fl mice compared with wild-type mice. The bars indicate the mean ± SEM values. Data were statistically analyzed using one-way ANOVA with Dunnett’s post hoc test (A, L) or two-way ANOVA with Dunnett’s post hoc test (H and M-Q).