Although β-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 β-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 β-cell function via a paracrine mechanism involving proinflammatory factors secreted from islet-associated exocrine-derived cell types.

These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.

Cystic fibrosis (CF)-related diabetes in humans is intimately related to exocrine pancreatic insufficiency, yet little is known about how these 2 disease processes simultaneously evolve in CF. In this context, we examined CF ferrets during the evolution of exocrine pancreatic disease. At 1 month of age, CF ferrets experienced a glycemic crisis with spontaneous diabetic-level hyperglycemia. This occurred during a spike in pancreatic inflammation that was preceded by pancreatic fibrosis and loss of β-cell mass. Surprisingly, there was spontaneous normalization of glucose levels at 2-3 months, with intermediate hyperglycemia thereafter. Mixed meal tolerance was impaired at all ages, but glucose intolerance was not detected until 4 months. Insulin secretion in response to hyperglycemic clamp and to arginine was impaired. Insulin sensitivity, measured by euglycemic hyperinsulinemic clamp, was normal. Pancreatic inflammation rapidly diminished after 2 months of age during a period where β-cell mass rose and gene expression of islet hormones, peroxisome proliferator-activated receptor-γ, and adiponectin increased. We conclude that active CF exocrine pancreatic inflammation adversely affects β-cells but is followed by islet resurgence. We predict that very young humans with CF may experience a transient glycemic crisis and postulate that pancreatic inflammatory to adipogenic remodeling may facilitate islet adaptation in CF.

Previous studies indicate that stress damages oocytes with increased secretion of glucorticoids. However, although injection of female mice with cortisol decreased oocyte competence, exposure of mouse oocytes directly to physiological or stress-induced concentrations of glucorticoids did not affect oocyte maturation and embryo development. This study has explored the mechanisms by which glucocorticoids impair oocyte competence. Female mice were injected with cortisol and the effects of cortisol-injection on oocyte competence, ovarian cell apoptosis and Fas/FasL activation were observed. The results showed that cortisol-injection decreased (a) oocyte developmental potential, (b) the E2/P4 ratio in serum and ovaries, and (c) expression of insulin-like growth factor 1, brain-derived neurotrophic factor and glucocorticoid receptor in mural granulosa cells (MGCs), while increasing levels of (a) cortisol in serum and ovaries, (b) apoptosis in MGCs and cumulus cells (CCs), (c) FasL secretion in ovaries and during oocyte maturation in vitro, and (d) Fas in MGCs, CCs and oocytes. The detrimental effects of cortisol-injection on oocyte competence and apoptosis of MGCs and CCs were significantly relieved when the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations were observed. Together, the results suggested that glucocorticoids impair oocyte competence by triggering apoptosis of ovarian cells via activating the Fas system.

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The domestic ferret (Mustela putorius furo) has proven to be a useful species for modeling human genetic and infectious diseases of the lung and brain. However, biomedical research in ferrets has been hindered by the lack of rapid and cost-effective methods for genome engineering. Here, we utilized CRISPR/Cas9-mediated, homology-independent insertion at the ROSA26 “safe harbor” locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase attP sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the ROSA26 intron-1 sgRNA, there were 23 births of which 5 had targeted integration events (22% efficiency). The encoded tdTomato transgene was highly expressed in all tissues evaluated. Targeted integration was verified by PCR analyses, Southern blot, and germ-line transmission. Function of the ROSA26-CAG-LoxPtdTomatoStopLoxPEGFP (ROSA-TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase attP sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the ROSA26 locus. These methods and the model generated will greatly enhance biomedical research involving lineage tracing, the evaluation of stem cell therapy, and transgenesis in ferret models of human disease.

While effects of gestational, neonatal or adolescent stress on psychological alterations in progeny have been extensively studied, much less is known regarding the effects of adult pre-gestational life events on offspring behavior. Although full siblings often display behavioral differences, whether the different parental life events prior to different pregnancies contribute to these behavioral differences among siblings is worth studying. In this study, male and female adult mice were restrained for 60 days before mating with unstressed or stressed partners. F1 offspring were examined for anxiety or mated to generate F2. Both F1 females and males from restrained mothers and/or fathers showed significantly reduced anxiety and serum cortisol and increased mRNA levels of glucocorticoid receptor and brain-derived neurotrophic factor compared to control offspring from unstressed parents. Similar behavioral and molecular changes were also observed in F2 females and males. Although restraint of adolescent mice reduced anxiety in F1 of both sexes, social instability of them increased anxiety predominantly in F1 females. Thus, adult pre-gestational restraint reduced offspring’s anxiety across generations; different stressors on parents may cause different phenotypes in offspring; individual behaviors can depend on adult life experiences of parents.