Cytoplasmic ATP and Ca2+ are implicated in current models of glucose's control of glucagon and insulin secretion from pancreatic α-and β-cells, respectively, but little is known about ATP and its rel...

Aims/hypothesis Glucagon is critical for normal glucose homeostasis and aberrant secretion of the hormone aggravates dysregulated glucose control in diabetes. However, the mechanisms by which glucose controls glucagon secretion from pancreatic alpha cells remain elusive. The aim of this study was to investigate the role of the intracellular messenger cAMP in alpha-cell-intrinsic glucose regulation of glucagon release. Methods Subplasmalemmal cAMP and Ca²⁺ concentrations were recorded in isolated and islet-located alpha cells using fluorescent reporters and total internal reflection microscopy. Glucagon secretion from mouse islets was measured using ELISA. Results Glucose induced Ca²⁺-independent alterations of the subplasmalemmal cAMP concentration in alpha cells that correlated with changes in glucagon release. Glucose-lowering-induced stimulation of glucagon secretion thus corresponded to an elevation in cAMP that was independent of paracrine signalling from insulin or somatostatin. Imposed cAMP elevations stimulated glucagon secretion and abolished inhibition by glucose elevation, while protein kinase A inhibition mimicked glucose suppression of glucagon release. Conclusions/interpretation Glucose concentrations in the hypoglycaemic range control glucagon secretion by directly modulating the cAMP concentration in alpha cells independently of paracrine influences. These findings define a novel mechanism for glucose regulation of glucagon release that underlies recovery from hypoglycaemia and may be disturbed in diabetes.

The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca²⁺ and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca²⁺ signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca²⁺ oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca²⁺- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations ("spiking") at the plasma membrane because of autocrine activation of P2Y₁ purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K⁺ depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca²⁺ concentration ([Ca²⁺]_pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca²⁺]_pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca²⁺]_pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function.

Zinc finger BED domain containing protein 6 ( Zbed6 ) has evolved from a domesticated DNA transposon and encodes a transcription factor unique to placental mammals. The aim of the present study was to investigate further the role of ZBED6 in insulin‐producing cells, using mouse MIN6 cells, and to evaluate the effects of Zbed6 knockdown on basal β‐cell functions, such as morphology, transcriptional regulation, insulin content, and release. Zbed6‐ silenced cells and controls were characterized with a range of methods, including RNA sequencing, chromatin immunoprecipitation sequencing, insulin content and release, subplasma membrane Ca ²⁺ measurements, cAMP determination, and morphologic studies. More than 700 genes showed differential expression in response to Zbed6 knockdown, which was paralleled by increased capacity to generate cAMP, as well as by augmented subplasmalemmal calcium concentration and insulin secretion in response to glucose stimulation. We identified >4000 putative ZBED6‐binding sites in the MIN6 genome, with an enrichment of ZBED6 sites at upregulated genes, such as the β‐cell transcription factors v‐maf musculoaponeurotic fibrosarcoma oncogene homolog A and Nk6 homeobox 1. We also observed altered morphology/growth patterns, as indicated by increased cell clustering, and in the appearance of axon‐like Neurofilament, medium polypeptide and tubulin β 3, class III‐positive protrusions. We conclude that ZBED6 acts as a transcriptional regulator in MIN6 cells and that its activity suppresses insulin production, cell aggregation, and neuronal‐like differentiation.—Wang, X., Jiang, L., Wallerman, O., Younis, S., Yu, Q., Klaesson, A., Tengholm, A., Welsh, N., Andersson, L. ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells. FASEB J. 33, 88–100 (2019). www.fasebj.org