504 Chloride currents underlie basal and alanine-induced alpha cell activity

Background and aims: Dysregulated glucagon secretion is central to diabetes with hyperglucagonaemia a key driver of hyperglycaemia. Despite this, the regulation of glucagon secretion remains enigmatic. Chloride (Cl^-) has been shown to play important roles in neighboring beta cells but has not been extensively studied in alpha cells. We therefore decided to assess the potential role of Cl^- in alpha cells, looking at the Cl^- permeable CFTR channel, which due to its dependence on ATP may represent a potential link between alpha cell metabolism and electrical activity, and the alanine/serine/cysteine transporters (ASCTs), that conduct Cl^- when transporting amino acids. Alanine was chosen to study these transporters due to its known potency in stimulating glucagon secretion.

Materials and methods: Experiments were performed on whole islets isolated from female c57b/6n mice. Calcium (Ca²⁺) activity was measured using live confocal microscopy with the dye Fluo4, alpha cells were identified by their responsiveness to adrenaline and activity at 1mM glucose. Sequential secretion was performed with glucagon and insulin secretion measured using MSD ELISA. All Ca⁺² imaging and secretion experiments were performed in Krebs’ ringer buffer supplemented with 0.36mM Non-esterified fatty acids (NEFA). Immunocytochemistry was performed to detect CFTR and glucagon in whole islets with images taken using a confocal microscope. Immunohistochemistry was used to assess glucagon and ASCT localization in pancreatic tissue sections using an epifluorescence microscope.

Results: The importance of Cl- conductance was underlined by experiments where extracellular Cl- was reduced. This induced Ca²⁺ activity in alpha cells with increases in peak frequency at 1mM glucose (p=0.0191). CFTR was found to co-localise with glucagon in pancreatic islets. Blocking of CFTR with CFTR-Inh172 at 5 μM or 10 μM caused cessation of Ca²⁺ dynamics with clear decreases in peak frequency at 1mM glucose (5μM: p<0.0001; 10μM: p<0.0001), indicating a central role for the channel in the maintenance of alpha cell Ca²⁺ dynamics. To assess the effects of alanine we used a physiological concentration of 500μM and 3mM alanine at 1mM and 5mM glucose. As expected, alanine increased Ca²⁺ peak frequency in alpha cells at 1mM glucose but significant effects were only observed with the higher concentration (500μM: p=0.0604; 3mM: p=0.0041). At 5mM glucose, both concentrations increased peak frequency (500μM: p=0.0004; 3mM: p=0.0005). In secretion assays, 3mM alanine was found to significantly increase glucagon secretion at 1mM glucose (p=0.0177). Immunostaining revealed that the isoform ASCT1 co-localised with glucagon in pancreatic tissue sections while ASCT2 was not found in alpha cells but was found in a small proportion of non-alpha cells. Inhibiting ASCTs with 4-chlorophenylglycine (100μM) reduced the effect of 3mM alanine on Ca²⁺ activity at 1mM glucose (p=0.002).

Conclusion: Cl^- plays a crucial role in α-cell regulation both in terms basal activity via CFTR but also the effect of alanine via ASCT1.