Opioid precursor protein isoform is targeted to the cell nuclei in the human brain

Background: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the kappa-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. Methods: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence activated nuclei sorting (FANS) from postmortem human striatal tissue. lmmunofluorescence staining and con focal microscopy was performed for human caudate nucleus. Results: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ASP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). Delta SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. Conclusions and general significance: High levels of alternatively spliced Delta SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum. (C) 2016 Elsevier B.V. All rights reserved

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes. Pancreatic islets derived from stem cells are benchmarked against primary cells.Peer reviewe

Ca2+-induced Ca2+ Release via Inositol 1,4,5-trisphosphate Receptors Is Amplified by Protein Kinase A and Triggers Exocytosis in Pancreatic β-Cells

Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca2+-induced Ca2+ release (CICR) in pancreatic β-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca2+ spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca2+ concentration ([Ca2+]i). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca2+ spiking at low or even in the absence of depolarization-dependent elevation of [Ca2+]i. The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca2+, failed to mobilize intracellular Ca2+. On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP3Rs). Therefore, the cell-permeable IP3R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic β-cells were followed by [Ca2+]i spikes in neighboring human erythroleukemia cells, used to report secretory events in the β-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP3Rs is part of the mechanism by which cAMP amplifies insulin release

Ca2+-induced Ca2+ Release via Inositol 1,4,5-trisphosphate Receptors Is Amplified by Protein Kinase A and Triggers Exocytosis in Pancreatic β-Cells

Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca2+-induced Ca2+ release (CICR) in pancreatic β-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca2+ spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca2+ concentration ([Ca2+]i). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca2+ spiking at low or even in the absence of depolarization-dependent elevation of [Ca2+]i. The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca2+, failed to mobilize intracellular Ca2+. On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP3Rs). Therefore, the cell-permeable IP3R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic β-cells were followed by [Ca2+]i spikes in neighboring human erythroleukemia cells, used to report secretory events in the β-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP3Rs is part of the mechanism by which cAMP amplifies insulin release

Store-operated influx of Ca2+ in the pancreatic β-cells exhibits graded dependence on the filling of the endoplasmic reticulum

The store-operated pathway for Ca2+ entry was studied in individual mouse pancreatic β-cells by measuring the cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mn2+ ([Mn2+]i) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca2+ sequestration. To avoid interference with voltage-dependent Ca2+ entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca2+ depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca2+]i or from the rate of increase in [Mn2+]i due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn2+ depended on store filling in a graded manner. The store-operated influx of Ca2+/Mn2+ was inhibited by Gd3+ and 2-aminoethoxydiphenyl borate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the β-cell has direct implications for the control of membrane potential and insulin secretion

Store-operated influx of Ca2+ in the pancreatic β-cells exhibits graded dependence on the filling of the endoplasmic reticulum

The store-operated pathway for Ca2+ entry was studied in individual mouse pancreatic β-cells by measuring the cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mn2+ ([Mn2+]i) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca2+ sequestration. To avoid interference with voltage-dependent Ca2+ entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca2+ depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca2+]i or from the rate of increase in [Mn2+]i due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn2+ depended on store filling in a graded manner. The store-operated influx of Ca2+/Mn2+ was inhibited by Gd3+ and 2-aminoethoxydiphenyl borate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the β-cell has direct implications for the control of membrane potential and insulin secretion

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic β-cells

The effect of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) inhibition on the cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in primary insulin-releasing pancreatic β-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca2+-deficient medium the individual primary β-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca2+]i followed by an explosive transient elevation. The [Ca2+]i transients were preferentially observed at low intracellular concentrations of the Ca2+ indicator fura-2 and were unaffected by pre-treatment with 100 μM ryanodine. Whereas 20 mM caffeine had no effect on basal [Ca2+]i or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca2+]i transients as well as inositol 1,4,5-trisphosphate-mediated [Ca2+]i transients in response to carbachol. In striking contrast to the primary β-cells, caffeine readily mobilized intracellular Ca2+ in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca2+ from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca2+-induced Ca2+ release (CICR). In primary pancreatic β-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal β-cells

Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells

Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we have investigated the influence of Ca2+ on receptor-triggered PLC activity in individual insulin-secreting β-cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP2/IP3-binding pleckstrin homology domain from PLCδ1, and the cytoplasmic Ca2+ concentration ([Ca2+]i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 β-cells with the muscarinic-receptor agonist carbachol induced rapid and sustained PLC activation. By contrast, only transient activation was observed after stimulation in the absence of extracellular Ca2+ or in the presence of the non-selective Ca2+ channel inhibitor La3+. The Ca2+-dependent sustained phase of PLC activity did not require voltage-gated Ca2+ influx, as hyperpolarization with diazoxide or direct Ca2+ channel blockade with nifedipine had no effect. Instead, the sustained PLC activity was markedly suppressed by the store-operated channel inhibitors 2-APB and SKF96365. Depletion of intracellular Ca2+ stores with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca2+ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca2+-deficient medium. Analogous suppressions were observed after loading cells with the Ca2+ chelator BAPTA. Stimulation of primary mouse pancreatic β-cells with glucagon elicited pronounced [Ca2+]i spikes, reflecting protein kinase A-mediated activation of Ca2+-induced Ca2+ release via IP3 receptors. These [Ca2+]i spikes were found to evoke rapid and transient activation of PLC. Our data indicate that receptor-triggered PLC activity is enhanced by positive feedback from Ca2+ entering the cytoplasm from intracellular stores and via store-operated channels in the plasma membrane. Such amplification of receptor signalling should be important in the regulation of insulin secretion by hormones and neurotransmitters

Functional differences between aggregated and dispersed insulin-producing cells

Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets. MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, alpha-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca2+ concentration ([Ca2+](c); microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured. Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca2+](c) response. We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca2+](c)

Indicator-dependent differences in detection of local intracellular Ca2+release events evoked by voltage-gated Ca2+entry in pancreatic & beta;-cells

Genetically encoded Ca2+ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca2+ concentration ([Ca2+]i) changes in insulin-secreting & beta;-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K+ depolarizationtriggered submembrane [Ca2+]i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca2+]i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca2+]i spikes from an elevated level. [Ca2+]i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y1-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca2+ dyes. The indicator-dependent response patterns were unrelated to Ca2+ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca2+ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca2+ events originating outside this volume. The study highlights that voltage-dependent Ca2+ entry in & beta;-cells is tightly linked to local intracellular Ca2+ release mediated via an autocrine route that may be more important than previously reported direct Ca2+ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release