Cystic fibrosis-related diabetes is caused by islet loss and Cystic fibrosis-related diabetes is caused by islet loss and inflammation inflammation

Cystic fibrosis–related (CF-related) diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting approximately 35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of b cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine b cells did not affect b cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of b cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by b cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation. β cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine β cells did not affect β cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/ CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of β cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by β cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation. islets is limited (11, 15, 35), we examined CFTR mRNA and protein expression in human islet endocrine cells, whether CFTR regulates human islet insulin secretion, and the hormone secretory profile and transcriptome of human CF islets. Our studies indicate that the CFTR does not intrinsically regulate α or β cell function and that the etiology of CFRD is largely dependent on islet loss and intraislet inflammation in the setting of a complex and progressive multiorgan disease. Stimulated insulin and glucagon secretion and key islet regulatory genes were maintained in CF islets . To determine the secretory profile CF islets, we simultaneously measured in vitro CF islet insulin and glucagon secretion by perifusion. Following normalization of islet secretion to islet equivalents (IEQs), total hormone content, and conditions of maximal stimulation, 5 of 5 CF donors displayed phasic GSIS, pGSIS, hypoglycemic epinephrine inhibition of insulin secretion, and KCl-mediated insulin secretion (Supple-mental Figure 8, A–E). CF islet insulin secretory profiles (normalized to IEQ and insulin content) displayed insulin secretory responses above, within, or below the range of normal islets (Figure 5, A and C, and Supplemental Figure 9). There was a statistical difference between CF and normal islet insulin secretory values (normalized to IEQ or percentage content) when all values ( n = 5 donors/group × 50 values/perifusion = 250 insulin secretory values) were compared (IEQ: P = 0.0021; percentage content: P < 0.0001). To further investigate possible differences in CF and normal islets, we performed additional statistical analyses and found that insulin secretion was not equal between CF and normal islets at all time points ( P < 0.0001); there was only one significantly different time point in the IEQ normalization ( t = 126 minutes, during perifusion with 5.6 G + KCl) and there were no significantly different time points in the percentage content normalization, using Sidak’s multiple comparison test. However, when aggregating insulin secretory values across all time points, there were no significant differences between CF and normal insulin secretory responses (IEQ: P = 0.8231; percentage content: P > 0.999). Important-ly, these analyses indicate that the mean CF insulin secretory response to a number of conditions was similar to that of normal islets, Because there is limited and contradictory information regarding CFTR expression and function in islets, we used two murine models of β cell CFTR deletion as well as healthy and CF pancreata and islets to study expression of CFTR in human islets, whether CFTR controls islet hormone secretion, and CF pancreas and islet pathology. Specific deletion of CFTR from β cells in two murine models did not alter glucose tolerance or in vitro β cell function, indicating CFTR is not involved in murine β cell stimulus-secretion coupling. Studies of normal human pancreata and islets found that expression of CFTR mRNA was minimal and that CFTR protein and function were not detectable in islet endocrine cells. We also report for the first time to our knowledge that in vitro insulin and glucagon secretion by human CF/CFRD islets was similar to normal human islets and the CF/CFRD islet-regulatory transcriptome was essentially intact. In addi-tion, we observed profound pancreatic pathology in human CF donors (observed by others, refs. 17, 18, 44, 53, 54) with loss of β cell area, islet dysmorphia, and now reported details of islet-infiltrating immune cells. These studies demonstrate that CFTR does not directly regulate insulin or glucagon secretion and that CFRD is primarily caused by pancreatic damage, intraislet inflammation, in vivo islet dysfunction, and islet loss in the setting of a progressive multiorgan disease (Figure 7). In CF loss of β cell function with animal glucose tolerance and β cell with age-related β cell and worsening both been CF ferret recapitulates the pancreatic exocrine pathology of glucose and mixed intolerance and islet been islets also maintained expression of many key islet-regulatory transcripts, indicating that islet cell identity was preserved. Taken together, these results not only indicate that CFTR does not intrinsically regulate human islet hormone secretion, but that the remnant CF islets are functional in vitro, which may be clinically significant. We present for the first time to our knowledge a detailed characterization of an increased inflammatory potential within the human CF islet that is evidenced by increased expression of several cytokines/ chemokines (including IL6 , IL1B , CXCL10 , TNFA and IFNG ) and high levels of TNF- α and IFN- γ production by stimulated T cells isolated from CF islets. We postulate that islet isolation from the CF pancreas removes the islet from the limiting the effect of cytokines/chemokines, allow-ing for relatively normal in vitro islet secretion of insulin and glucagon. Thus, we propose that islet-proximal immune cells are stimulated in by tissue and and secrete chemokines/cytokines, which to impair islet secretory function analysis to determine the layout and spread of the samples. Differential expression between conditions was calculated on the basis of fold change (cutoff ≥ 2.0), and the P value was estimated by Z -score calculations (cutoff 0.05), as determined by the Benjamini-Hochberg false discovery rate method (85). Differential-ly expressed genes underwent gene set enrichment analysis, gene ontology analysis, and pathway analysis using DAVID and Ingenuity pathway analysis. Sequencing data are available on the Gene Expression Omnibus repository (accession GSE110935). Statistics . One-way ANOVA followed by Tukey multiple-comparisons test and unpaired 2-tailed Student’s t test were used to compare outcomes in mice of different genotypes. Unpaired 2-tailed Student’s t tests were used to compare human samples with and without CF. An ordinary 2-way ANOVA with a Sidak’s multiple comparison test was used to compare CF and normal islet hormone secretion during islet perifusion. A P value of less than 0.05 was considered significant.