Extended Data Fig. 5: Adipsin/C3a increases insulin secretion and protects from beta cell death by inhibiting DUSP26.
(a) Ins1 beta cells were subjected to a glucose-stimulated insulin secretion assay at 0 or 17 mM glucose with or without C3a (n = 8 per group). Results are representative of three independent experiments. Data were analyzed by two-tailed unpaired t-test. (Glc 0 mM vs glc 17 mM p = 0.0019, glc 17 mM vs glc 17 mM + C3a p = 0.009). (b) Representative gating strategy for islet cells. (c) Heatmap of genes significantly changed by palmitate treatment and counter-regulated by C3a in WT islets (n = 3 per group) Colors show raw z-scores of mean normalized counts. (d) GO biological process analysis of genes whose expression were downregulated by palmitate and counter-regulated (increased) by C3a. Data were analyzed by Fisher/binominal test with Bonferroni-adjusted P value (n = 33 genes) (e) GO biological process analysis of genes whose expression were upregulated by palmitate and counter-regulated (decreased) by C3a. Data were analyzed by Fisher/binominal test with Bonferroni-adjusted P value (n = 43 genes) (f) Pathway analysis from the DEPOD database depicting significant phosphatase substrates in islets that are regulated at the gene expression level by C3a from Fig. 3d. (n = 76 genes) Data were analyzed by Fisher/binominal test with Bonferroni-adjusted P value (g) Ins2 and Gcg were determined by qPCR in isolated pancreatic islets from WT mice transduced with Dusp26 or control lentivirus (n = 4 GFP, n = 3 Dusp26). Data are representative of 3 independent experiments. Data were analyzed by two-tailed unpaired t-test. (Ins2 p = 0.0284, Gcg p = 0.033). (h) Cell viability was determined in INS-1 cells transduced with Dusp26 and controls (n = 6 per group). Data are representative of 3 independent experiments. Data were analyzed by two-tailed unpaired t-test. (GFP in veh vs GFP in Pa p = 0.022, Dusp26 in veh vs Dusp26 in Pa p = 0.0000047, GFP in Pa vs Dusp26 in Pa p = 0.0004). (i) Representative western blot analysis of DUSP26 in INS-1 cells treated with shRNA against Dusp26 versus control shRNA. Data are representative of 3 independent experiments. (j) Quantification of DUSP26 expression for Supplementary Figure 8d from 3 independent experiments. Data were analyzed by two-tailed unpaired t-test. (p = 0.028). (k) Body weight in db/db mice treated for 2 weeks with NSC-87877 and controls at the indicated time points (n = 15 NSC-87877 group, n = 16 saline group). (l) Representative western blot analysis for FLAG after immunoprecipitation of the DUSP26 protein complex using FLAG-M2 antibodies in lysates of INS-1 cells overexpressing control GFP, intact DUSP26 (D26) and catalytically inactive DUSP26 mutant (C152S). Data are representative of at least 3 independent experiments. (m) KEGG pathway analysis of proteins enriched in DUSP26 immunoprecipitation experiment. Proteins with log2 ≥ 1.5 between control GFP and DUSP26 mutant (C152S) were included in the analysis. Top and selected pathways are shown. Data were analyzed by Fisher/binominal test with Bonferroni-adjusted P value (n = 253 genes) (n) Heatmap of selected proteins belonging to relevant pathways and significantly enriched in the DUSP26 mutant (C152S) pulldown. (n = 2 per group). Colors show log2 fractional intensity. Data are expressed as mean ± s.e.m. *P < 0.05, ** P < 0.01, *** P < 0.001.
