Correction: Heterogeneous expression of CFTR in insulin-secreting ß-cells of the normal human islet.

[This corrects the article DOI: 10.1371/journal.pone.0242749.].

Heterogeneous expression of CFTR in insulin-secreting ß-cells of the normal human islet.

Cystic fibrosis (CF) is due to mutations in the CF-transmembrane conductance regulator (CFTR) and CF-related diabetes (CFRD) is its most common co-morbidity, affecting ~50% of all CF patients, significantly influencing pulmonary function and longevity. Yet, the complex pathogenesis of CFRD remains unclear. Two non-mutually exclusive underlying mechanisms have been proposed in CFRD: i) damage of the endocrine cells secondary to the severe exocrine pancreatic pathology and ii) intrinsic ß-cell impairment of the secretory response in combination with other factors. The later has proven difficult to determine due to low expression of CFTR in ß-cells, which results in the general perception that this Cl-channel does not participate in the modulation of insulin secretion or the development of CFRD. The objective of the present work is to demonstrate CFTR expression at the molecular and functional levels in insulin-secreting ß-cells in normal human islets, where it seems to play a role. Towards this end, we have used immunofluorescence confocal and immunofluorescence microscopy, immunohistochemistry, RT-qPCR, Western blotting, pharmacology, electrophysiology and insulin secretory studies in normal human, rat and mouse islets. Our results demonstrate heterogeneous CFTR expression in human, mouse and rat ß-cells and provide evidence that pharmacological inhibition of CFTR influences basal and stimulated insulin secretion in normal mouse islets but not in islets lacking this channel, despite being detected by electrophysiological means in ~30% of ß-cells. Therefore, our results demonstrate a potential role for CFTR in the pancreatic ß-cell secretory response suggesting that intrinsic ß-cell dysfunction may also participate in the pathogenesis of CFRD.

Impaired glucose tolerance, glucagon, and insulin responses in mice lacking the loop diuretic-sensitive Nkcc2a transporter.

The Na+K+2Cl- cotransporter-2 (Nkcc2, Slc12a1) is abundantly expressed in the kidney and its inhibition with the loop-diuretics bumetanide and furosemide has been linked to transient or permanent hyperglycemia in mice and humans. Notably, Slc12a1 is expressed at low levels in hypothalamic neurons and in insulin-secreting ß-cells of the endocrine pancreas. The present study was designed to determine if global elimination of one of the Slc12a1 products, i.e., Nkcc2 variant a (Nkcc2a), the main splice version of Nkcc2 found in insulin-secreting ß-cells, has an impact on the insulin and glucagon secretory responses and fuel homeostasis in vivo. We have used dynamic tests of glucose homeostasis in wild-type mice and mice lacking both alleles of Nkcc2a (Nkcc2aKO) and assessed their islet secretory responses in vitro. Under basal conditions, Nkcc2aKO mice have impaired glucose homeostasis characterized by increased blood glucose, intolerance to the sugar, delayed/blunted in vivo insulin and glucagon responses to glucose, and increased glycemic responses to the gluconeogenic substrate alanine. Further, we provide evidence of conserved quantitative secretory responses of Nkcc2aKO islets within a context of increased islet size related to hyperplastic/hypertrophic glucagon- and insulin-positive cells (α-cells and ß-cells, respectively), normal total islet Cl- content, and reduced ß-cell expression of the Cl- extruder Kcc2.

The neuronal K+Cl- co-transporter 2 (Slc12a5) modulates insulin secretion.

Intracellular chloride concentration ([Cl-]i) in pancreatic ß-cells is kept above electrochemical equilibrium due to the predominant functional presence of Cl- loaders such as the Na+K+2Cl- co-transporter 1 (Slc12a2) over Cl-extruders of unidentified nature. Using molecular cloning, RT-PCR, Western blotting, immunolocalization and in vitro functional assays, we establish that the "neuron-specific" K+Cl- co-transporter 2 (KCC2, Slc12a5) is expressed in several endocrine cells of the pancreatic islet, including glucagon secreting α-cells, but particularly in insulin-secreting ß-cells, where we provide evidence for its role in the insulin secretory response. Three KCC2 splice variants were identified: the formerly described KCC2a and KCC2b along with a novel one lacking exon 25 (KCC2a-S25). This new variant is undetectable in brain or spinal cord, the only and most abundant known sources of KCC2. Inhibition of KCC2 activity in clonal MIN6 ß-cells increases basal and glucose-stimulated insulin secretion and Ca2+ uptake in the presence of glibenclamide, an inhibitor of the ATP-dependent potassium (KATP)-channels, thus suggesting a possible mechanism underlying KCC2-dependent insulin release. We propose that the long-time considered "neuron-specific" KCC2 co-transporter is expressed in pancreatic islet ß-cells where it modulates Ca2+-dependent insulin secretion.

A highly efficient strategy to determine genotypes of genetically-engineered mice using genomic DNA purified from hair roots.

Genotyping of genetically-engineered mice is necessary for the effective design of breeding strategies and identification of mutant mice. This process relies on the identification of DNA markers introduced into genomic sequences of mice, a task usually performed using the polymerase chain reaction (PCR). Clearly, the limiting step in genotyping is isolating pure genomic DNA. Isolation of mouse DNA for genotyping typically involves painful procedures such as tail snip, digit removal, or ear punch. Although the harvesting of hair has previously been proposed as a source of genomic DNA, there has been a perceived complication and reluctance to use this non-painful technique because of low DNA yields and fear of contamination. In this study we developed a simple, economic, and efficient strategy using Chelex® resins to purify genomic DNA from hair roots of mice which are suitable for genotyping. Upon comparison with standard DNA purification methods using a commercially available kit, we demonstrate that Chelex® efficiently and consistently purifies high-quality DNA from hair roots, minimizing pain, shortening time and reducing costs associated with the determination of accurate genotypes. Therefore, the use of hair roots combined with Chelex® is a reliable and more humane alternative for DNA genotyping.

Plasma Membrane Targeting of Endogenous NKCC2 in COS7 Cells Bypasses Functional Golgi Cisternae and Complex N-Glycosylation.

Na+K+2Cl- co-transporters (NKCCs) effect the electroneutral movement of Na+-K+ and 2Cl- ions across the plasma membrane of vertebrate cells. There are two known NKCC isoforms, NKCC1 (Slc12a2) and NKCC2 (Slc12a1). NKCC1 is a ubiquitously expressed transporter involved in cell volume regulation, Cl- homeostasis and epithelial salt secretion, whereas NKCC2 is abundantly expressed in kidney epithelial cells of the thick ascending loop of Henle, where it plays key roles in NaCl reabsorption and electrolyte homeostasis. Although NKCC1 and NKCC2 co-transport the same ions with identical stoichiometry, NKCC1 actively co-transports water whereas NKCC2 does not. There is growing evidence showing that NKCC2 is expressed outside the kidney, but its function in extra-renal tissues remains unknown. The present study shows molecular and functional evidence of endogenous NKCC2 expression in COS7 cells, a widely used mammalian cell model. Endogenous NKCC2 is primarily found in recycling endosomes, Golgi cisternae, Golgi-derived vesicles, and to a lesser extent in the endoplasmic reticulum. Unlike NKCC1, NKCC2 is minimally hybrid/complex N-glycosylated under basal conditions and yet it is trafficked to the plasma membrane region of hyper-osmotically challenged cells through mechanisms that require minimal complex N-glycosylation or functional Golgi cisternae. Control COS7 cells exposed to slightly hyperosmotic (~6.7%) solutions for 16 h were not shrunken, suggesting that either one or both NKCC1 and NKCC2 may participate in cell volume recovery. However, NKCC2 targeted to the plasma membrane region or transient over-expression of NKCC2 failed to rescue NKCC1 in COS7 cells where NKCC1 had been silenced. Further, COS7 cells in which NKCC1, but not NKCC2, was silenced exhibited reduced cell size compared to control cells. Altogether, these results suggest that NKCC2 does not participate in cell volume recovery and therefore, NKCC1 and NKCC2 are functionally different Na+K+2Cl- co-transporters.

Increased Slc12a1 expression in ß-cells and improved glucose disposal in Slc12a2 heterozygous mice.

The products of the Slc12a1 and Slc12a2 genes, commonly known as Na(+)-dependent K(+)2Cl(-) co-transporters NKCC2 and NKCC1, respectively, are the targets for the diuretic bumetanide. NKCCs are implicated in the regulation of intracellular chloride concentration ([Cl(-)]i) in pancreatic ß-cells, and as such, they may play a role in glucose-stimulated plasma membrane depolarization and insulin secretion. Unexpectedly, permanent elimination of NKCC1 does not preclude insulin secretion, an event potentially linked to the homeostatic regulation of additional Cl(-) transporters expressed in ß-cells. In this report we provide evidence for such a mechanism. Mice lacking a single allele of Slc12a2 exhibit lower fasting glycemia, increased acute insulin response (AIR) and lower blood glucose levels 15-30 min after a glucose load when compared to mice harboring both alleles of the gene. Furthermore, heterozygous expression or complete absence of Slc12a2 associates with increased NKCC2 protein expression in rodent pancreatic ß-cells. This has been confirmed by using chronic pharmacological down-regulation of NKCC1 with bumetanide in the mouse MIN6 ß-cell line or permanent molecular silencing of NKCC1 in COS7 cells, which results in increased NKCC2 expression. Furthermore, MIN6 cells chronically pretreated with bumetanide exhibit increased initial rates of Cl(-) uptake while preserving glucose-stimulated insulin secretion. Together, our results suggest that NKCCs are involved in insulin secretion and that a single Slc12a2 allele may protect ß-cells from failure due to increased homeostatic expression of Slc12a1.