Figure 4: SENP1 deletion augments PAT inflammation and islet immunogenicity before onset of diabetes.

(a,b) Cytokine expression, concentrations of IL-6, TNF-α, IFN-γ and IL-1β proteins in serum (a) and PATs (b) were measured by ELISArray kits in six pairs of age-matched Ctrl and SENP1-aP2KO mice at the age of 7 weeks, n=6, male. (c) Cytokine expression in adipose tissues. Transcript levels of IL-6, TNF-α, IFNγ and IL-1β in pancreatic, gonadal, peri-renal and subcutaneous inguinal adipose tissue (PAT, GAT, KAT and SAT, respectively) were quantified by quantitative PCR with reverse transcription (qRT–PCR). GAPDH was used for normalization. n=6 male mice at the age of 7 weeks. (d) Total T cells (CD3⁺) and macrophages (CD11b⁺F4/80⁺) in PAT of Ctrl (n=6, male) and SENP1-aP2KO (n=6, male) mice were detected by FACS at the age of 7 weeks. Quantifications of total T cells and macrophages are presented. (e) Protein levels of IL-6, TNF-α, IFN-γ and IL-1β levels present in culture supernatant of isolated PAT adipocytes, T cells and macrophages were detected with ELISA after 24 h culture. Data are means±s.e.m. n=6, male. All data are means±s.e.m. from n=6 male mice per group. The two-tailed Student’s paired t-test was used for the statistical analysis. *P<0.05; **P<0.01; NS, non-significance. (f–h) CCL5 expression. mRNAs of chemokines in islets which isolated from Ctrl (n=10, male) and SENP1-aP2KO mice (n=10, male ) were quantified by TaqMan PCR with normalization by HPRT (f). Sections of pancreases from Ctrl and SENP1-aP2KO mice were co-immunostaining with CCL5 (green) and β-cell marker insulin (red). Representative images of the pancreases from Ctrl and SENP1-aP2KO mice at the age of 7 weeks are shown (g). Scale bar, 20 μm. Three sections from each mice, n=6 male mice per group at each age. (h) Mice pancreatic islets (100 islets per well) were incubated with culture supernatant of adipocyte collected from SENP1-aP2KO mice at the age of 7 weeks with or without IL-6, TNF-α or IL-1β neutralization antibody as indicated. After 24 h, mRNA level of CCL5 were quantified by qRT–PCR. n=6 male mice per group at each age. (i,j) Direct effects of culture supernatant of peri-pancreatic adipocytes on islets. Mice pancreatic islets were isolated from Ctrl mice. Islets (100 islets per well) were cultured with culture supernatant of peri-pancreatic adipocytes from Ctrl, SENP1-aP2KO mice at the age of 5 weeks. After 72-h incubation, islet structure and apoptosis were monitored by Live/Dead viability/Cytotoxicity kit and detected by fluorescence microscopy. Representative images from each group are shown. Scale bar, 200 μm. Data are representative for three independent experiments. Three images from each mice, n=6 male mice per group. (j) Quantifications of intact islets (green), partial damaged islets (yellow) and completed damaged islets (red). Total 300 islets from each group were counted. The two-tailed Student’s paired t-test was used for the statistical analysis. All data are means±s.e.m., *P<0.05; **P<0.01; ***P<0.001. Abs, antibiodies; Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole; HPRT, hypoxanthine-guanine phosphoribosyltransferase; NS, non-significance; .