Unperturbed islet α‐cell function examined in mouse pancreas tissue slices

Abstract

Non-technical summary Critical investigation into pancreatic islet α-cell biology in health and diabetes has been sparse and inconsistent because of technical difficulties in islet isolation and dispersion into single cells. We have circumvented these difficulties by employing the pancreas slice preparation. We functionally characterized (electrophysiologically) the α-cells in their in situ native state, then loaded the tested cells with biocytin dye to subsequently confirm the cell identities by immunocytochemistry. We characterized a very large number of α-cells, which showed a wide-range distribution in the electrophysiological parameters of several ion channels (ATP-sensitive K + , Na + and Ca 2+ currents) and capacitance changes as measure of exocytosis of glucagon granules. This could explain the apparent inconsistency of previous reports on α-cells that inadvertently showed skewed data due to insufficient sampling of α-cells. Our innovative approaches will enable future studies into elucidating α-cell dysregulation in diabetes.Abstract Critical investigation into α-cell biology in health and diabetes has been sparse and at times inconsistent because of the technical difficulties with employing conventional strategies of isolated islets and dispersed single cells. An acute pancreas slice preparation was developed to overcome the enzymatic and mechanical perturbations inherent in conventional islet cell isolation procedures. This preparation preserves intra-islet cellular communication and islet architecture in their in situ native state. α-Cells within tissue slices were directly assessed by patch pipette and electrophysiologically characterized. The identity of the patched cells was confirmed by biocytin dye labelling and immunocytochemistry. α-Cells in mouse pancreas slices exhibited well-described features of I Na (excitable at physiological membrane potential), I KATP , small cell size, low resting membrane conductance, and inducible low and high voltage-activated I Ca , the latter correlating with exocytosis determined by capacitance measurements. In contrast to previous reports, our large unbiased sampling of α-cells revealed a wide range distribution of all of these parameters, including the amount of K ATP conductance, Na + and Ca 2+ current amplitudes, and capacitance changes induced by a train of depolarization pulses. The proposed pancreas slice preparation in combination with standard patch-clamping technique allowed large sampling and rapid assessment of α-cells, which revealed a wide distribution in α-cell ion channel properties. This specific feature explains the apparent inconsistency of previous reports on these α-cell ion channel properties. Our innovative approach will enable future studies into elucidating islet α-cell dysregulation occurring during diabetes.

Diethyldithiocarbamate-mediated zinc ion chelation reveals role of Cav2.3 channels in glucagon secretion

“…One could argue that this is due to compensatory up-regulation of other (less Zn 2+ sensitive) α-cell VGCCs. However, recent large scale patch clamp analysis of identified mouse α-cells in a pancreatic slice preparation indicates that there is actually a wide range of electrophysiological properties due to heterogeneous α-cell ion channel arrangement [36]. Thus, insufficient or biased α-cell sampling could also explain the apparently unchanged whole-cell Ca 2+ current as well as other inconsistencies in the reported ion channel arrangement of mouse α-cells.…”

“…Due to their intermediate voltage-range for activation Ca v 2.3 channels have been proposed to provide sufficient depolarization after K ATP channel closure for subsequent Na + -and L-type Ca 2+ channel-dependent AP firing [33]. This might be especially important in mouse α-cells, which have very low K ATP channel densities [35,36]. The particular importance of Ca v 2.3 channels for suppressing glucagon release during hyperglycemia could be explained by the fact that high glucose concentrations can directly affect α-cell oxidative metabolism and thereby close K ATP channels [35,37], which has led to the proposal that K ATP channel dependent α-cell depolarization may actually prevent secretion by inactivating Na + and Ca 2+ channels [34,35,37].…”

Physiological and Pathophysiological Control of Glucagon Secretion by Pancreatic α-Cells

“…This kind of comparison should however be taken with care because the results were obtained by different groups with different experimental conditions and a limited number of cells. Indeed, a more recent study performed on a larger number of α-cells in mouse pancreatic slices has reported that the density of the K ATP channels was extremely variable between α-cells and in average slightly lower than that of β-cells (Huang et al 2011b). In the mouse, the sensitivity of the K ATP channels to ATP is higher in α-than β-cells (Huang et al 2011b;Leung et al 2006a), whereas in the rat, it is similar in both cell types (Gromada et al 2007).…”

ATP-regulated potassium channels and voltage-gated calcium channels in pancreatic alpha and beta cells: similar functions but reciprocal effects on secretion

“…Nevertheless, hormone release measurements using the P/Q-type Ca 2+ channel inhibitor ω-agatoxin 1 indicate that the P/Q-type Ca 2+ channels are particularly important for glucagon secretion evoked by low glucose concentrations [26]. In addition, mouse alpha cells express low-threshold T-type Ca 2+ channels that may be involved in the initiation ('pace-making') of action potential firing [47,48].…”