CFTR Influences Beta Cell Function and Insulin Secretion Through Non-Cell Autonomous Exocrine-Derived Factors.
ABSTRACT: Although ?-cell dysfunction in cystic fibrosis (CF) leads to diabetes, the mechanism by which the cystic fibrosis transmembrane conductance regulator (CFTR) channel influences islet insulin secretion remains debated. We investigated the CFTR-dependent islet-autonomous mechanisms affecting insulin secretion by using islets isolated from CFTR knockout ferrets. Total insulin content was lower in CF as compared with wild-type (WT) islets. Furthermore, glucose-stimulated insulin secretion (GSIS) was impaired in perifused neonatal CF islets, with reduced first, second, and amplifying phase secretion. Interestingly, CF islets compensated for reduced insulin content under static low-glucose conditions by secreting a larger fraction of islet insulin than WT islets, probably because of elevated SLC2A1 transcripts, increased basal inhibition of adenosine triphosphate-sensitive potassium channels (K-ATP), and elevated basal intracellular Ca2+. Interleukin (IL)-6 secretion by CF islets was higher relative to WT, and IL-6 treatment of WT ferret islets produced a CF-like phenotype with reduced islet insulin content and elevated percentage insulin secretion in low glucose. CF islets exhibited altered expression of INS, CELA3B, and several ?-cell maturation and proliferation genes. Pharmacologic inhibition of CFTR reduced GSIS by WT ferret and human islets but similarly reduced insulin secretion and intracellular Ca2+ in CFTR knockout ferret islets, indicating that the mechanism of action is not through CFTR. Single-molecule fluorescent in situ hybridization, on isolated ferret and human islets and ferret pancreas, demonstrated that CFTR RNA colocalized within KRT7+ ductal cells but not endocrine cells. These results suggest that CFTR affects ?-cell function via a paracrine mechanism involving proinflammatory factors secreted from islet-associated exocrine-derived cell types.
Project description: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 ? cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine ? cells did not affect ? 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 ? 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 ? cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.
Project description:Cystic fibrosis (CF) is a genetic disorder caused by defective CF Transmembrane Conductance Regulator (CFTR) function. Insulin producing pancreatic islets are located in close proximity to the pancreatic duct and there is a possibility of impaired cell-cell signaling between pancreatic ductal epithelial cells (PDECs) and islet cells as causative in CF. To study this possibility, we present an in vitro co-culturing system, pancreas-on-a-chip. Furthermore, we present an efficient method to micro dissect patient-derived human pancreatic ducts from pancreatic remnant cell pellets, followed by the isolation of PDECs. Here we show that defective CFTR function in PDECs directly reduced insulin secretion in islet cells significantly. This uniquely developed pancreatic function monitoring tool will help to study CF-related disorders in vitro, as a system to monitor cell-cell functional interaction of PDECs and pancreatic islets, characterize appropriate therapeutic measures and further our understanding of pancreatic function.
Project description:Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (<i>CFTR</i>). Cystic fibrosis-related diabetes (CFRD) is the most common comorbidity, affecting more than 50% of adult CF patients. Despite this high prevalence, the etiology of CFRD remains incompletely understood. Studies in young CF children show pancreatic islet disorganization, abnormal glucose tolerance, and delayed first-phase insulin secretion suggesting that islet dysfunction is an early feature of CF. Since insulin-producing pancreatic β-cells express very low levels of CFTR, CFRD likely results from β-cell extrinsic factors. In the vicinity of β-cells, CFTR is expressed in both the exocrine pancreas and the immune system. In the exocrine pancreas, CFTR mutations lead to the obstruction of the pancreatic ductal canal, inflammation, and immune cell infiltration, ultimately causing the destruction of the exocrine pancreas and remodeling of islets. Both inflammation and ductal cells have a direct effect on insulin secretion and could participate in CFRD development. CFTR mutations are also associated with inflammatory responses and excessive cytokine production by various immune cells, which infiltrate the pancreas and exert a negative impact on insulin secretion, causing dysregulation of glucose homeostasis in CF adults. In addition, the function of macrophages in shaping pancreatic islet development may be impaired by CFTR mutations, further contributing to the pancreatic islet structural defects as well as impaired first-phase insulin secretion observed in very young children. This review discusses the different factors that may contribute to CFRD.
Project description:Diabetes is a common comorbidity in cystic fibrosis (CF) that worsens prognosis. The lack of an animal model for CF-related diabetes (CFRD) has made it difficult to dissect how the onset of pancreatic pathology influences the emergence of CFRD. We evaluated the structure and function of the neonatal CF endocrine pancreas using a new CFTR-knockout ferret model. Although CF kits are born with only mild exocrine pancreas disease, progressive exocrine and endocrine pancreatic loss during the first months of life was associated with pancreatic inflammation, spontaneous hyperglycemia, and glucose intolerance. Interestingly, prior to major exocrine pancreas disease, CF kits demonstrated significant abnormalities in blood glucose and insulin regulation, including diminished first-phase and accentuated peak insulin secretion in response to glucose, elevated peak glucose levels following glucose challenge, and variably elevated insulin and C-peptide levels in the nonfasted state. Although there was no difference in lobular insulin and glucagon expression between genotypes at birth, significant alterations in the frequencies of small and large islets were observed. Newborn cultured CF islets demonstrated dysregulated glucose-dependent insulin secretion in comparison to controls, suggesting intrinsic abnormalities in CF islets. These findings demonstrate that early abnormalities exist in the regulation of insulin secretion by the CF endocrine pancreas.
Project description:Cystic fibrosis (CF)-related diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting ~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/electrical activity was not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion with few changes in key islet-regulatory transcripts. Furthermore, ~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 not caused by intrinsic islet dysfunction from CFTR mutation, but rather, by b cell loss and intra-islet inflammation in the setting of a complex pleiotropic disease
Project description:Fibroblast growth factor (FGF) 21 is an endocrine factor that normalizes glucose homeostasis and reduces insulin resistance in diabetes. Although the pancreas is an FGF21 target organ, its role in pancreatic islets remains obscure. This study aimed to elucidate the physiological role of FGF21 in pancreatic islets using FGF21-knockout (FGF21-KO) mice. Twenty-four-week-old male global FGF21-KO mice were used in this study. Glucose and insulin tolerance were assessed. Expression of genes and proteins related to islet function and underlying mechanisms were also examined. Islet morphology and insulin-secreting capacity were further evaluated. FGF21-KO mice exhibited insulin resistance while being normoglycemic, associated with increases in beta-cell proliferation and insulin synthesis, acting as compensatory responses. This phenotype probably results from enhanced growth hormone (GH) sensitivity in FGF21-KO mouse islets. In addition, ex vivo FGF21 treatment in normal C57BL/6J mouse islets reduced GH signaling, probably via upregulation of peroxisome proliferator-activated receptor gamma (PPAR?) and cytokine-inducible SH-2 containing (CIS) protein, whereas KO mouse islets displayed reduced PPAR? and CIS expression. FGF21 treatment also reversed GH-induced insulin expression, beta-cell proliferation and GH-impaired glucose-stimulated insulin secretion (GSIS) in islets. Furthermore, distorted islet morphology and impaired GSIS were observed in KO mice, suggestive of islet dysfunction, whereas the enhanced insulin expression and impaired GSIS in FGF21-KO mouse islets could be reversed by blockade of GH signaling. Our data indicate that FGF21 is important in the regulation of beta-cell proliferation and insulin synthesis, probably via modulation of GH signaling. These findings provide evidence that FGF21 is an obligatory metabolic regulator in pancreatic islets and shed new light onto the role of endogenous FGF21 in the pathogenesis of insulin resistance and islet dysfunction.
Project description:<h4>Background</h4>Notch signaling pathway plays an important role in regulating pancreatic endocrine and exocrine cell fate during pancreas development. Notch signaling is also expressed in adult pancreas. There are few studies on the effect of Notch on adult pancreas. Here, we investigated the role of Notch in islet mass and glucose homeostasis in adult pancreas using Notch1 antisense transgenic (NAS).<h4>Methods</h4>Western blot analysis was performed for the liver of 8-week-old male NAS mice. We also conducted an intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test in 8-week-old male NAS mice and male C57BL/6 mice (control). Morphologic observation of pancreatic islet and ?-cell was conducted in two groups. Insulin secretion capacity in islets was measured by glucose-stimulated insulin secretion (GSIS) and perifusion.<h4>Results</h4>NAS mice showed higher glucose levels and lower insulin secretion in IPGTT than the control mice. There was no significant difference in insulin resistance. Total islet and ?-cell masses were decreased in NAS mice. The number of large islets (?250 µm) decreased while that of small islets (<250 µm) increased. Reduced insulin secretion was observed in GSIS and perifusion. Neurogenin3, neurogenic differentiation, and MAF bZIP transcription factor A levels increased in NAS mice.<h4>Conclusion</h4>Our study provides that Notch1 inhibition decreased insulin secretion and decreased islet and ?-cell masses. It is thought that Notch1 inhibition suppresses islet proliferation and induces differentiation of small islets. In conclusion, Notch signaling pathway may play an important role in ?-cell mass determination and diabetes.
Project description:Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous beta-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL(-/-) mice indicated the presence of other TG lipase(s) in the beta-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K(ATP)-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL(-/-) mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL(-/-) mice. Accordingly, isolated islets from ATGL(-/-) mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL(-/-) islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.
Project description:Isolated human islets do not always meet the quality standards required for transplant survival and reliable functional in vitro studies. The formation of pseudoislets, i.e. the reaggregation of a defined number of islet cells after dissociation, improves insulin secretion. We present a simple method of pseudoislet formation from human islet cells and assess the transcriptome and function of isolated human islets and pseudoislets from the same organ donors. Following pseudoislet formation, insulin content/DNA and mRNA/RPS13 resembled that of islets. In pseudoislets, glucose-stimulated insulin secretion (GSIS) was significantly higher (8-13-fold) than in islets (2-4-fold). GSIS of pseudoislets was partly inhibited by the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin-9. The stimulatory effects of palmitate and forskolin at 12?mM glucose were also significantly higher in pseudoislets than in islets. Further analysis of pseudoislets revealed that regulation of secretion and insulin and glucagon content was maintained over a longer culture period (6-14 d). While adrenaline inhibited GSIS, adrenaline together with palmitate stimulated glucagon secretion 2-fold at low glucose, an effect suppressed by high glucose. Transcriptome analysis revealed that, unlike islets, pseudoislets were deprived of exocrine and endothelial cells. In conclusion, pseudoislet formation restores functional integrity of human islet cells and allows long-term in vitro testing.
Project description:Excessive caloric intake leading to obesity is associated with insulin resistance and dysfunction of islet ? cells. High-fat feeding decreases desnutrin (also called ATGL/PNPLA2) levels in islets. Here we show that desnutrin ablation via RIP-Cre (?KO) or RIP-CreER results in hyperglycemia with impaired glucose-stimulated insulin secretion (GSIS). Due to decreased lipolysis, islets have higher TAG content but lower free FA levels. ?KO islets exhibit impaired mitochondrial respiration and lower production of ATP required for GSIS, along with decreased expression of PPAR? target genes involved in mitochondrial oxidation. Furthermore, synthetic PPAR?, but not PPAR?, agonist restores GSIS and expression of mitochondrial oxidative genes in ?KO mice, revealing that desnutrin-catalyzed lipolysis generates PPAR? ligands. Finally, adenoviral expression of desnutrin in ?KO islets restores all defects of ?KO islet phenotype and function, including GSIS and mitochondrial defects, demonstrating the critical role of the desnutrin-PPAR?-mitochondrial oxidation axis in regulating islet ? cell GSIS.