Figure 5: SENP1 deletion alters pancreatic adipocyte phenotype and augments NF-κB-dependent inflammation.

(a,b) Pancreatic adipose was collected from 7 and 14 weeks old Ctrl and SENP1-aP2KO male (male, n=6). Morphology was visualized by haematoxylin and eosin stain staining. Scale bar, 20 μm (a). Cell sizes were quantified in (b). Three sections from each adipose tissue. Data are representative for three independent experiments. (c) Transcript levels of adipocyte differentiation markers (fatty acid synthase, adipose triglyceride lipase and lipoprotein lipase) in PATs were quantified by quantitative PCR with reverse transcription with GAPDH for normalization. n=6, male for each group. (d) Increased IKK-NF-κB activities in SENP1-aP2KO PATs. A representative western blot was from Ctrl and SENP1-aP2KO mice at the age of 7 weeks. Data are representative from three independent experiments is shown (n=6, male). Relative protein levels were quantified from three blots by taking Ctrl as 1.0 (n=6, male). (e–h) NF-κB activation is specifically detected in PATs of SENP1-aP2KO mice. (e) Phosphor-p65 staining (red) in PATs but not in pancreas. (f) Co-immunostaining of phosphor-p65 (green) and adipocyte marker FABP4 (red). (g) High-power images show co-staining of phosphor-p65 (red) in the nucleus of FABP4⁺ adipocytes (green). (h) Co-staining of phosphor-p65 (green) with APC-conjugated adipocyte marker FABP4 (green; yellow arrowheads), but not with macrophage marker F4/80 (red; white arrowheads). Representative images are from one of three sections from SENP1-aP2KO PATs and n=3 male mice. Scale bar, 20 μm. (i,j) ChIP assay. ChIP assays with p65/RelA antibody were performed in the adipocyte isolated from Ctrl (n=6, male) and SENP1-aP2KO mice (n=6, male) with an IgG isotype as a control. Bindings of p65/RelA to the IL-6, IL-1β and TNF-α gene promoters were quantified with the ratio of IP/input for each promoter (j). (k,l) Effects of SENP1 knockdown on NF-κB and cytokine expression in adipocytes. 3T3-L1 adipocytes were transfected with control or SENP1-specific siRNA for 24 h. IKK-NF-κB p65/RelA signalling molecules were detected by western blotting. A representative blot from three experiments is shown and protein levels are quantified by taking Ctrl as 1.0 (k). Protein levels of IL-6, TNF-α, IFN-γ and IL-1β levels present in adipocyte cultures were detected with ELISA after 24-h culture (l). All data are means±s.e.m. of three independent experiments. The two-tailed Student’s paired t-test was used for the statistical analysis. *P<0.05; **P<0.01, ***; P<0.001; DAPI, 4′,6-diamidino-2-phenylindole; IP, immunoprecipitation; NS, non-significance.