Loss of tetraspanin-7 expression reduces pancreatic ß-cell exocytosis Ca2+ sensitivity but has limited effect on systemic metabolism.

BACKGROUND: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate ß-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic ß-cell function is not yet fully understood. METHODS: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. ß-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. RESULTS: Tspan7 is expressed in insulin-containing granules of pancreatic ß-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- ß-cells, but ß-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent ß-cells, exocytotic Ca2+ sensitivity was reduced in a human ß-cell line (EndoC-ßH1) following Tspan7 KD. CONCLUSION: Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in ß-cells. Its function is more significant in human ß-cells than their rodent counterparts.

The endoplasmic reticulum plays a key role in α-cell intracellular Ca2+ dynamics and glucose-regulated glucagon secretion in mouse islets.

Glucagon is secreted by pancreatic α-cells to counteract hypoglycaemia. How glucose regulates glucagon secretion remains unclear. Here, using mouse islets, we studied the role of transmembrane and endoplasmic reticulum (ER) Ca2+ on intrinsic α-cell glucagon secretion. Blocking isradipine-sensitive L-type voltage-gated Ca2+ (Cav) channels abolished α-cell electrical activity but had little impact on its cytosolic Ca2+ oscillations or low-glucose-stimulated glucagon secretion. In contrast, depleting ER Ca2+ with cyclopiazonic acid or blocking ER Ca2+-releasing ryanodine receptors abolished α-cell glucose sensitivity and low-glucose-stimulated glucagon secretion. ER Ca2+ mobilization in α-cells is regulated by intracellular ATP and likely to be coupled to Ca2+ influx through P/Q-type Cav channels. ω-Agatoxin IVA blocked α-cell ER Ca2+ release and cell exocytosis, but had no additive effect on glucagon secretion when combined with ryanodine. We conclude that glucose regulates glucagon secretion through the control of ER Ca2+ mobilization, a mechanism that can be independent of α-cell electrical activity.

Acetyl-CoA-carboxylase 1 (ACC1) plays a critical role in glucagon secretion.

Dysregulated glucagon secretion from pancreatic alpha-cells is a key feature of type-1 and type-2 diabetes (T1D and T2D), yet our mechanistic understanding of alpha-cell function is underdeveloped relative to insulin-secreting beta-cells. Here we show that the enzyme acetyl-CoA-carboxylase 1 (ACC1), which couples glucose metabolism to lipogenesis, plays a key role in the regulation of glucagon secretion. Pharmacological inhibition of ACC1 in mouse islets or αTC9 cells impaired glucagon secretion at low glucose (1 mmol/l). Likewise, deletion of ACC1 in alpha-cells in mice reduced glucagon secretion at low glucose in isolated islets, and in response to fasting or insulin-induced hypoglycaemia in vivo. Electrophysiological recordings identified impaired KATP channel activity and P/Q- and L-type calcium currents in alpha-cells lacking ACC1, explaining the loss of glucose-sensing. ACC-dependent alterations in S-acylation of the KATP channel subunit, Kir6.2, were identified by acyl-biotin exchange assays. Histological analysis identified that loss of ACC1 caused a reduction in alpha-cell area of the pancreas, glucagon content and individual alpha-cell size, further impairing secretory capacity. Loss of ACC1 also reduced the release of glucagon-like peptide 1 (GLP-1) in primary gastrointestinal crypts. Together, these data reveal a role for the ACC1-coupled pathway in proglucagon-expressing nutrient-responsive endocrine cell function and systemic glucose homeostasis.

Somatostatin secretion by Na+-dependent Ca2+-induced Ca2+ release in pancreatic delta-cells.

Pancreatic islets are complex micro-organs consisting of at least three different cell types: glucagon-secreting α-, insulin-producing ß- and somatostatin-releasing δ-cells1. Somatostatin is a powerful paracrine inhibitor of insulin and glucagon secretion2. In diabetes, increased somatostatinergic signalling leads to defective counter-regulatory glucagon secretion3. This increases the risk of severe hypoglycaemia, a dangerous complication of insulin therapy4. The regulation of somatostatin secretion involves both intrinsic and paracrine mechanisms5 but their relative contributions and whether they interact remains unclear. Here we show that dapagliflozin-sensitive glucose- and insulin-dependent sodium uptake stimulates somatostatin secretion by elevating the cytoplasmic Na+ concentration ([Na+]i) and promoting intracellular Ca2+-induced Ca2+ release (CICR). This mechanism also becomes activated when [Na+]i is elevated following the inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin in vivo 6. Islets from some donors with type-2 diabetes hypersecrete somatostatin, leading to suppression of glucagon secretion that can be alleviated by a somatostatin receptor antagonist. Our data highlight the role of Na+ as an intracellular second messenger, illustrate the significance of the intraislet paracrine network and provide a mechanistic framework for pharmacological correction of the hormone secretion defects associated with diabetes that selectively target the δ-cells.

Glucose stimulates somatostatin secretion in pancreatic δ-cells by cAMP-dependent intracellular Ca2+ release.

Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+ , increasing glucose from 1 mM to 20 mM produced an ∼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.

Modulation of large dense core vesicle insulin content mediates rhythmic hormone release from pancreatic beta cells over the 24h cycle.

The rhythmic nature of insulin secretion over the 24h cycle in pancreatic islets has been mostly investigated using transcriptomics studies showing that modulation of insulin secretion over this cycle is achieved via distal stages of insulin secretion. We set out to measure ß-cell exocytosis using in depth cell physiology techniques at several time points. In agreement with the activity and feeding pattern of nocturnal rodents, we find that C57/Bl6J islets in culture for 24h exhibit higher insulin secretion during the corresponding dark phase than in the light phase (Zeitgeber Time ZT20 and ZT8, respectively, in vivo). Glucose-induced insulin secretion is increased by 21% despite normal intracellular Ca2+ transients and depolarization-evoked exocytosis, as measured by whole-cell capacitance measurements. This paradox is explained by a 1.37-fold increase in beta cell insulin content. Ultramorphological analyses show that vesicle size and density are unaltered, demonstrating that intravesicular insulin content per granule is modulated over the 24h cycle. Proinsulin levels did not change between ZT8 and ZT20. Islet glucagon content was inversely proportional to insulin content indicating that this unique feature is likely to support a physiological role. Microarray data identified the differential expression of 301 transcripts, of which 26 are miRNAs and 54 are known genes (including C2cd4b, a gene previously involved in insulin processing, and clock genes such as Bmal1 and Rev-erbα). Mouse ß-cell secretion over the full course of the 24h cycle may rely on several distinct cellular functions but late night increase in insulin secretion depends solely on granule insulin content.