The ability of the pancreatic β-cell to secrete insulin in response to changes in glucose concentration is the cornerstone of glucose homeostasis. The β-cell couples glucose metabolism to electrical activity via the modulation of ion channel activity. Defects in β-cell ion channels can result in the dysregulation of insulin secretion and, consequently, in glucose homeostasis. Congenital hyperinsulinism of infancy (CHI) is an inherited disorder of inappropriate insulin secretion often caused by gene defects in the subunits of KATP channels (ABCC8, KCNJ11). In 50% of CHI patients, no genetic basis has yet been discovered. I aimed to characterise the expression, and function, of two novel ion channel families in the healthy human pancreas and the hyperinsulinaemic pancreas.Hyperpolarisation-activated cyclic nucleotide-gated channels (HCN channels) are selective for K+ /Na+ under physiological conditions and are responsible for the rhythmic electrical behaviour (pacemaker behaviour) in the heart and brain. Two-pore potassium channels (K2P channels) are potassium-selective channels whose activity contributes to, and maintains, the resting membrane potential of living cells. HCN and K2P gene and protein expression was investigated by RT-PCR and immunofluorescence. HCN1, 2, 3, and 4 mRNA and mRNA from six K2P channels was detected in isolated mouse islets. Immunofluorescence revealed the presence of HCN1, HCN2 and HCN4 in mouse β-cells. The role of HCN channels in glucose-stimulated insulin secretion (GSIS) was assessed in isolated mouse islets. The HCN channel agonist lamotrigine had no effect on GSIS (n=3), but GSIS was significantly inhibited by the HCN channel blocker zatebradine (n=3) thereby confirming that HCN channels may play a role in glucose homeostasis. Tissue was isolated from adult control human pancreas and from six patients following pancreatectomy for CHI. HCN and K2P channel expression was also assessed in pancreatic tissue from ABCC8-knockout mice. HCN1-4 mRNA and the mRNA of four K2P channels were detected in islets isolated from adult control and CHI patient pancreatic tissue. Co-expression of HCN channel isoforms with insulin confirmed that HCN1, -2 and -4 were β-cell-specific in control human islets. By contrast, in all patient tissues expression of HCN channels was altered, and in 5 out of 6 cases, HCN channels were not expressed in β-cells. The K2P channel TWIK-1 was found to be expressed predominantly in the healthy human exocrine pancreas. In contrast, its expression was consistently detected in the β-cells of CHI patients. We found similar data for both HCN channels and TWIK-1 in islets of ABCC8-knockout mice, but not their litter-mate controls (n=3).These studies provide the first data on the expression of HCN and K2P channels in the human pancreas and reveal that CHI is associated with an altered expression profile of both ion channel families. Significantly, we found that HCN and K2P expression in β-cells is altered in CHI patients and we speculate that this is as a consequence of physiological remodelling.
|Date of Award||1 Aug 2011|
- The University of Manchester
|Supervisor||Mark Dunne (Supervisor)|