The objective of this study is to investigate the role of intracellular chloride concentration ([Cl¿¿¿¿¿¿]i) in insulin secretion in vivo and in vitro.
The maintenance of high [Cl¿¿¿¿¿¿]i in insulin-secreting pancreatic β-cells plays an important role in glucose-induced depolarization and insulin secretion. Insulinotropic glucose concentrations i.e., >6 mM induces electrical activity in β-cells which results in insulin secretion. This electrical activity is mainly due to electrogenic efflux of Cl¿¿¿¿¿¿ from β-cells through volume-regulated anion channels (VRAC). The solute carrier family 12 group A member 1 (Slc12a1) and 2 (Slc12a2) encode several splice variants of the kidney-specific and the ubiquitous isoforms, respectively, of bumetanide (BTD)-sensitive Na+-dependent K+2Cl¿¿¿¿¿¿co-transporters. The Slc12a2 co-transporter, also known as NKCC1, is involved in the maintenance of a high [Cl¿¿¿¿¿¿]i in β-cells and its pharmacological inhibition impairs insulin secretion in response to glucose in vivo and in vitro. We have used mice lacking both functional alleles of the Slc12a2 gene (NKCC1 KO) to demonstrate that absence of NKCC1 correlates with better fasting glycaemia and improved glucose tolerance when compared to wild-type mice (NKCC1 WT). These observations correlated with increased insulin secretion in response to glucose in vivo and in vitro in primary culture of islets. Further, morphometric analysis of pancreas obtained from NKCC1 KO mice had increased β-cell mass. Moreover, administration of BTD to NKCC1 KO mice worsened their glucose tolerance. This effect of BTD in vivo correlated with a direct inhibition of insulin secretion by BTD from islets lacking functional Slc12a2 genes, therefore suggesting the presence of a BTD-sensitive mechanism involved in insulin secretion, which is different from Slc12a2. We used reverse transcription coupled to the polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence analysis to determine the molecular expression pattern of Slc12a1 and Slc12a2 genes in β-cells. We demonstrate that these cells express a specific splice variant of NKCC1 and the "kidney-specific" BTD-sensitive Slc12a1 gene. Altogether, our results provide for the first time evidence for a functional role of these co-transporters on insulin secretion.
Conclusions:
1) Functional alleles of the Slc12a2 gene are not necessary for insulin secretion
2) Bumetanide impairs insulin secretion and glucose homeostasis in vivo
3) Bumetanide impairs insulin secretion by a direct effect on insulin secreting β-cells
4) Pancreatic β-cells express specific splice variants of Slc12a2 and Slc12a1