Cystic fibrosis-related diabetes (CFRD) worsens CF lung disease leading to early mortality. Loss of beta cell area, even without overt diabetes or pancreatitis is consistently observed. We investigated whether short-term CFTR inhibition was sufficient to impact islet morphology and function in otherwise healthy mice. CFTR was inhibited in C57BL/6 mice via 8-day intraperitoneal injection of CFTRinh172. Animals had a 7-day washout period before measures of hormone concentration or islet function were performed. Short-term CFTR inhibition increased blood glucose concentrations over the course of the study. However, glucose tolerance remained normal without insulin resistance. CFTR inhibition caused marked reductions in islet size and in beta cell and non-beta cell area within the islet, which resulted from loss of islet cell size rather than islet cell number. Significant reductions in plasma insulin concentrations and pancreatic insulin content were also observed in CFTR-inhibited animals. Temporary CFTR inhibition had little long-term impact on glucose-stimulated, or GLP-1 potentiated insulin secretion. CFTR inhibition has a rapid impact on islet area and insulin concentrations. However, islet cell number is maintained and insulin secretion is unaffected suggesting that early administration of therapies aimed at sustaining beta cell mass may be useful in slowing the onset of CFRD.
Replacement of pancreatic -cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional -cell mass has remained elusive. It has recently Accepted ArticleThis article is protected by copyright. All rights reserved.been proposed that dedifferentiation / plasticity towards other endocrine phenotypes may play an important role in stress-induced -cell dysfunction in type 2 diabetes. Here we report loss of enddifferentiated -cell phenotype in two intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin + / urocortin-3 -cells were seen in non-diabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate β-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β-cell sources, graft sites and ultimately fully-vascularised bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β-cell phenotype has been maintained.
β-Cell dysfunction in type 2 diabetes (T2D) is associated with loss of cellular identity and mis-expression of alternative islet hormones, including glucagon. The molecular basis for these cellular changes has been attributed to dysregulation of core β-cell transcription factors, which regulate β-cell identity through activating and repressive mechanisms. The TLE1 gene lies near a T2D susceptibility locus and, recently, the glucagon repressive actions of this transcriptional coregulator have been demonstrated in vitro. We investigated whether TLE1 expression is disrupted in human T2D, and whether this is associated with increased islet glucagon-expressing cells. Automated image analysis following immunofluorescence in donors with (n = 7) and without (n = 7) T2D revealed that T2D was associated with higher islet α/β cell ratio (Control: 0.7 ± 0.1 vs T2D: 1.6 ± 0.4; P < .05) and an increased frequency of bihormonal (insulin+/glucagon+) cells (Control: 0.8 ± 0.2% vs T2D: 2.0 ± 0.4%, P < .05). In nondiabetic donors, the majority of TLE1-positive cells were mono-hormonal β-cells (insulin+/glucagon–: 98.2 ± 0.5%; insulin+/glucagon+: 0.7 ± 0.2%; insulin–/glucagon+: 1.1 ± 0.4%; P < .001). TLE1 expression was reduced in T2D (Control: 36 ± 2.9% vs T2D: 24 ± 2.6%; P < .05). Reduced islet TLE1 expression was inversely correlated with α/β cell ratio (r = –0.55; P < .05). TLE1 knockdown in EndoC-βH1 cells was associated with a 2.5-fold increase in glucagon gene mRNA and mis-expression of glucagon in insulin-positive cells. These data support TLE1 as a putative regulator of human β-cell identity, with dysregulated expression in T2D associated with increased glucagon expression potentially reflecting β- to α-cell conversion.
Introduction: Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity in people with CF, occurring in 40-50% of adults. Whilst it is established that β-cell dysfunction in cystic fibrosis (CF) leads to diabetes, the mechanism by which the CF transmembrane conductance regulator (CFTR) channel influences insulin secretion remains debated. Currently, three major hypotheses have been proposed: 1. Intrinsic CFTR-dependent pathways of insulin secretion 2. Pancreas-extrinsic CFTR defects 3. Remodeling of islets following loss of exocrine tissue due to inflammation. Since the contribution of each to the pathogenesis of CFRD remains largely unknown, we sought to determine CFTR localisation within human pancreas using novel and highly sensitive approaches. Methods: Expression of chromogranin A (ChrA), CFTR and keratin 19 (K19) was assessed by immunofluorescence (IF) staining of tissue obtained from deceased donors without diabetes (n=10, age: 23-71 years). Two CFTR antibodies (ab576 and ab590) obtained from the CF Foundation were used to confirm specificity, with ductal and endocrine cells determined by K19 and ChrA respectively. CFTR RNA expression was determined by RNAscope® in situ hybridisation (ISH) and combined with either ChrA or insulin immunohistochemistry (IHC). Results: IF staining of pancreatic tissues indicated co-localisation of CFTR in K19+ ductal cells, but not in ChrA+ endocrine cells. These observations were confirmed by combined RNAscope® ISH and IHC (ChrA and insulin), which demonstrated the absence of CFTR RNA in human islets. Conclusion: Employment of these highly sensitive techniques has demonstrated absence of CFTR within normal β-cells or any other islet endocrine cell types. This is in line with recent observations in isolated human islets. We conclude that CFTR abnormalities do not directly impact beta-cell function and that CFRD is mediated by factors extrinsic to the pancreatic endocrine compartment. Disclosure R.R. Maheshwari: None. C.J. Jones: None. J.A.M. Shaw: Advisory Panel; Self; Novo Nordisk A/S. M.G. White: None.
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