Inhibition of stearoyl-CoA desaturase 1 in the mouse impairs pancreatic islet morphogenesis and promotes loss of ß-cell identity and α-cell expansion in the mature pancreas.

Abnormalities that characterize the pathophysiology of type 2 diabetes (T2D) include deficiencies of ß-cells and the expansion of α-cells in pancreatic islets, manifested by lower insulin release and glucagon oversecretion. The molecular mechanisms that determine intra-islet interactions between pancreatic α- and ß-cells are still not fully understood. The present study showed that stearoyl-coenzyme A (CoA) desaturase 1 (SCD1), an enzyme that is implicated in fatty acid metabolism, serves as a checkpoint in the control of endocrine cell equilibrium in pancreatic islets. Our data showed that SCD1 activity is essential for proper α-cell and ß-cell lineage determination during morphogenesis of the pancreas and the maintenance of mature ß-cell identity. The inhibition of SCD1 expression/activity led to both a decrease in the expression of ß-cell signature genes (e.g., Pdx1, Nkx6.1, MafA, and Neurod1, among others) and induction of the expression of the dedifferentiation marker Sox9 in mature pancreatic islets. The transcriptional repression of Pdx1 and MafA in SCD1-deficient ß-cells was related to the excessive methylation of promoter regions of these transcription factors. In contrast, SCD1 ablation favored the formation of α-cells over ß-cells throughout pancreas organogenesis and did not compromise α-cell identity in adult pancreatic islets. Such molecular changes that were caused by SCD1 downregulation resulted in the mislocalization of α-cells within the core of islets and increased the ratio of pancreatic α- to ß-cell mass. This was followed by islet dysfunction, including impairments in glucose-stimulated insulin release, simultaneously with elevations of basal glucagon secretion. Altogether, these findings provide additional mechanistic insights into the role of SCD1 in the pathogenesis of T2D.

gP2S, an Information Management System for CryoEM Experiments.

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not always achievable and cryoEM provides an alternative means to support structure-based ligand design. When dealing with a large number of distinct projects, and within each project a potentially large number of ligand-protein co-structures, accurate record keeping rapidly becomes challenging. Many experimental parameters are tuned for each target, including at the sample preparation, grid preparation, and microscopy stages. Therefore, accurate record keeping can be crucially important to enable long-term reproducibility, and to facilitate efficient teamwork, especially when steps of the cryoEM workflow are performed by different operators. To help deal with this challenge, we developed a web-based information management system for cryoEM, called gP2S. The application keeps track of each experiment, from sample to final atomic model, in the context of projects, a list of which is maintained in the application, or externally in a separate system. User-defined controlled vocabularies of consumables, equipment, protocols and software help describe each step of the cryoEM workflow in a structured manner. gP2S is widely configurable and, depending on the team's needs, may exist as a standalone product or be a part of a broader ecosystem of scientific applications, integrating via REST APIs with project management tools, applications tracking the production of proteins or of small molecules ligands, or applications automating data collection and storage. Users can register details of each grid and microscopy session including key experimental metadata and parameter values, and the lineage of each experimental artifact (sample, grid, microscopy session, map, etc.) is recorded. gP2S serves as a cryoEM experimental workflow organizer that enables accurate record keeping for teams, and is available under an open-source license.

Calcium Signaling in Glioma Cells: The Role of Nucleotide Receptors.

Calcium signaling is probably one of the evolutionary oldest and the most common way by which the signal can be transmitted from the cell environment to the cytoplasmic calcium binding effectors. Calcium signal is fast and due to diversity of calcium binding proteins it may have a very broad effect on cell behavior. Being a crucial player in neuronal transmission it is also very important for glia physiology. It is responsible for the cross-talk between neurons and astrocytes, for microglia activation and motility. Changes in calcium signaling are also crucial for the behavior of transformed glioma cells. The present chapter summarizes molecular mechanisms of calcium signal formation present in glial cells with a strong emphasis on extracellular nucleotide-evoked signaling pathways. Some aspects of glioma C6 signaling such as the cross-talk between P2Y1 and P2Y12 nucleotide receptors in calcium signal generation will be discussed in-depth, to show complexity of machinery engaged in formation of this signal. Moreover, possible mechanisms of modulation of the calcium signal in diverse environments there will be presented herein. Finally, the possible role of calcium signal in glioma motility is also discussed. This is a very important issue, since glioma cells, contrary to the vast majority of neoplastic cells, cannot spread in the body with the bloodstream and, at least in early stages of tumor development, may expand only by means of sheer motility.

Cross-Talk in Nucleotide Signaling in Glioma C6 Cells.

The chapter is focused on the mechanism of action of metabotropic P2Y nucleotide receptors: P2Y1, P2Y2, P2Y12, P2Y14 and the ionotropic P2X7 receptor in glioma C6 cells. P2Y1 and P2Y12 both respond to ADP, but while P2Y1 links to PLC and elevates cytosolic Ca2+ concentration, P2Y12 negatively couples to adenylate cyclase, maintaining cAMP at low level. In glioma C6, these two P2Y receptors modulate activities of ERK1/2 and PI3K/Akt signaling and the effects depend on physiological conditions of the cells. During prolonged serum deprivation, cell growth is arrested, the expression of the P2Y1 receptor strongly decreases and P2Y12 becomes a major player responsible for ADP-evoked signal transduction. The P2Y12 receptor activates ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulates Akt activity, contributing to glioma invasiveness. In contrast, P2Y1 has an inhibitory effect on Akt pathway signaling. Furthermore, the P2X7 receptor, often responsible for apoptotic fate, is not involved in Ca2+elevation in C6 cells. The shift in nucleotide receptor expression from P2Y1 to P2Y12 during serum withdrawal, the cross talk between both receptors and the lack of P2X7 activity shows the precise self-regulating mechanism, enhancing survival and preserving the neoplastic features of C6 cells.

Microglia is a key player in the reduction of stroke damage promoted by the new antithrombotic agent ticagrelor.

The ADP-responsive P2Y12 receptor is expressed on both platelets and microglia. Clinical data show that ticagrelor, a direct-acting, reversibly binding P2Y12-receptor antagonist, reduces total cardiovascular events, including stroke. In our present study, we investigated the expression of P2Y12 receptors and the effects of ticagrelor on brain injury in Sprague-Dawley rats subjected to a permanent middle cerebral artery occlusion (MCAo). Rats were treated per os with ticagrelor 3 mg/kg or vehicle at 10 minutes, 22, and 36 hours after MCAo and killed after 48 hours. Immunofluorescence analysis showed an ischemia-related modulation of the P2Y12 receptor, which is constitutively expressed in Iba1(+) resting microglia. After MCAo, activated microglia was mainly concentrated around the lesion, with fewer cells present inside the ischemic core. Ticagrelor significantly attenuated the evolution of ischemic damage-evaluated by magnetic resonance imaging (MRI) at 2, 24, and 48 hours after MCAo-, the number of infiltrating cells expressing the microglia/monocyte marker ED-1, the cerebral expression of proinflammatory mediators (interleukin 1 (IL-1), monocyte chemoattractant protein 1 (MCP-1), nitric oxide synthase (iNOS)) and the associated neurologic impairment. In transgenic fluorescent reporter CX3CR1-green fluorescent protein (GFP) mice, 72 hours after MCAo, ticagrelor markedly reduced GFP(+) microglia and both early and late infiltrating blood-borne cells. Finally, in primary cultured microglia, ticagrelor fully inhibited ADP-induced chemotaxis (P<0.01). Our results show that ticagrelor is protective against ischemia-induced cerebral injury and this effect is mediated, at least partly, by inhibition of P2Y12-mediated microglia activation and chemotaxis.

[Recently identified GPR17 receptors--characteristics and role in pathology of the nervous system]. / Nowopoznane receptory GPR17--charakterystyka i udzial w patologii ukladu nerwowego.

Newly identified metabotropic GPR17 receptors are structurally related to purinergic P2Y receptors and cysteinyl leukotriene receptors. Re- cent studies showed that they play an important role in maturation of oligodendrocyte precursor cells. This review describes structure and pharmacological characterization of GPR17 receptors. It also summarizes knowledge about role of GPR17 receptors in physiology and patho- logy of nervous system, with special attention to remyelination processes.

Cross-talk in nucleotide signaling in glioma C6 cells.

The chapter is focused on the mechanism of action of metabotropic P2Y nucleotide receptors: P2Y(1), P2Y(2), P2Y(12), P2Y(14) and the ionotropic P2X(7) receptor in glioma C6 cells. P2Y(1) and P2Y(12) both respond to ADP, but while P2Y(1) links to PLC and elevates cytosolic Ca(2+) concentration, P2Y(12) negatively couples to adenylate cyclase, maintaining cAMP at low level. In glioma C6, these two P2Y receptors modulate activities of ERK1/2 and PI3K/Akt signaling and the effects depend on physiological conditions of the cells. During prolonged serum deprivation, cell growth is arrested, the expression of the P2Y(1) receptor strongly decreases and P2Y(12) becomes a major player responsible for ADP-evoked signal transduction. The P2Y(12) receptor activates ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulates Akt activity, contributing to glioma invasiveness. In contrast, P2Y(1) has an inhibitory effect on Akt pathway signaling. Furthermore, the P2X(7) receptor, often responsible for apoptotic fate, is not involved in Ca(2+)elevation in C6 cells. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) during serum withdrawal, the cross talk between both receptors and the lack of P2X(7) activity shows the precise self-regulating mechanism, enhancing survival and preserving the neoplastic features of C6 cells.

Calcium signaling in glioma cells--the role of nucleotide receptors.

Calcium signaling is probably one of the evolutionary oldest and the most common way by which the signal can be transmitted from the cell environment to the cytoplasmic calcium binding effectors. Calcium signal is fast and due to diversity of calcium binding proteins it may have a very broad effect on cell behavior. Being a crucial player in neuronal transmission it is also very important for glia physiology. It is responsible for the cross-talk between neurons and astrocytes, for microglia activation and motility. Changes in calcium signaling are also crucial for the behavior of transformed glioma cells. The present Chapter summarizes molecular mechanisms of calcium signal formation present in glial cells with a strong emphasis on extracellular nucleotide-evoked signaling pathways. Some aspects of glioma C6 signaling such as the cross-talk between P2Y(1) and P2Y(12) nucleotide receptors in calcium signal generation will be discussed in-depth, to show complexity of machinery engaged in formation of this signal. Moreover, possible mechanisms of modulation of the calcium signal in diverse environments there will be presented herein. Finally, the possible role of calcium signal in glioma motility is also discussed. This is a very important issue, since glioma cells, contrary to the vast majority of neoplastic cells, cannot spread in the body with the bloodstream and, at least in early stages of tumor development, may expand only by means of sheer motility.

P2Y1 nucleotide receptor silencing and its effect on glioma C6 calcium signaling.

In our earlier studies of the signaling cross-talk between nucleotide receptors in an in vitro glioma model (C6 cell line) under prolonged serum deprivation conditions, a growth arrest of the cells and expression shift from P2Y(1) to P2Y(12) receptors was found. The aim of the present work was to test if siRNA silencing of P2Y(1) receptor changes P2Y(12) expression similarly as following the serum deprivation and which physiological downstream pathways it affects. Here we demonstrate for the first time the efficiency of siRNA technology in silencing P2Y nucleotide receptors in glioma C6 cell line. Moreover, P2Y(12) proved to be insensitive to the P2Y(1) receptor silencing. The effect of the P2Y(1) silencing on calcium signaling was less pronounced then the extent of the protein change itself, exactly as was the case for the serum starvation experiments. Phosphorylation of ERK and Akt kinases were studied as the downstream effect of P2Y(1)-evoked signaling and similar effects as in the case of serum deprivation were found for ERK, and even stronger ones for Akt phosphorylation.

Is MLC phosphorylation essential for the recovery from ROCK inhibition in glioma C6 cells?

Inhibition of Rho-associated protein kinase (ROCK) activity in glioma C6 cells induces changes in actin cytoskeleton organization and cell morphology similar to those observed in other types of cells with inhibited RhoA/ROCK signaling pathway. We show that phosphorylation of myosin light chains (MLC) induced by P2Y2 receptor stimulation in cells with blocked ROCK correlates in time with actin cytoskeleton reorganization, F-actin redistribution and stress fibers assembly followed by recovery of normal cell morphology. Presented results indicate that myosin light-chain kinase (MLCK) is responsible for the observed phosphorylation of MLC. We also found that the changes induced by P2Y2 stimulation in actin cytoskeleton dynamics and morphology of cells with inhibited ROCK, but not in the level of phosphorylated MLC, depend on the presence of calcium in the cell environment.

2', 3'-O-(4-benzoylbenzoyl)-ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y(2) nucleotide receptor.

In this study, we examined the response of glioma C6 cells to 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X(7) receptor activity. We show here that in the absence of extracellular Ca(2+), BzATP-generated increase in [Ca(2+)](i)via Ca(2+) release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca(2+) response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X(7) receptor, did not reduce any of the two components of the Ca(2+) response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca(2+) elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y(2) but not the P2X(7) receptors.

Regulation of subcellular localization of muscle FBPase in cardiomyocytes. The decisive role of calcium ions.

Glyconeogenesis, the synthesis of glycogen from carbohydrate precursors like lactate, seems to be an important pathway participating in replenishing glycogen in cardiomyocytes. Fructose-1,6-bisphosphatase (FBPase), an indispensible enzyme of glyconeogenesis, has been found in cardiomyocytes on the Z-line, in the nuclei and in the intercalated discs. Glyconeogenesis may proceed only when FBPase accumulates on the Z-line. Searching for the mechanism of a FBPase regulation we investigated the effects of the calcium ionophore A23187, a muscle relaxant dantrolene, glucagon, insulin and medium without glucose on the subcellular localization of this enzyme in primary culture of neonatal rat cardiomyocytes. Immunofluorescence was used for protein localization and the intracellular calcium concentration was measured with Fura. We found that the concentration of calcium ions was the decisive factor determining the localization of muscle FBPase on the Z-line. Calcium ions had no effect on the localization of the enzyme in the intercalated discs or in the nuclei, but accumulation of FBPase in the nuclei was induced by insulin.