scholarly journals Inhibition of autophagic proteolysis by inhibitors of phosphoinositide 3-kinase can interfere with the regulation of glycogen synthesis in isolated hepatocytes

2002 ◽  
Vol 368 (3) ◽  
pp. 827-833 ◽  
Author(s):  
Peter F. DUBBELHUIS ◽  
Daphne A. VAN SLUIJTERS ◽  
Edward F.C. BLOMMAART ◽  
Lori A. GUSTAFSON ◽  
George M. VAN WOERKOM ◽  
...  
Keyword(s):  
Amino Acids ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Regulatory Volume Decrease ◽  
Isolated Hepatocytes ◽  
Cell Swelling ◽  
Phosphoinositide 3 Kinase

Amino acid-induced cell swelling stimulates conversion of glucose into glycogen in isolated hepatocytes. Activation of glycogen synthase (GS) phosphatase, caused by the fall in intracellular chloride accompanying regulatory volume decrease, and activation of phosphoinositide 3-kinase (PI 3-kinase), induced by cell swelling, have been proposed as underlying mechanisms. Because PI 3-kinase controls autophagic proteolysis, we examined the possibility that PI 3-kinase inhibitors interfere with glycogen production due to their anti-proteolytic action. The PI 3-kinase inhibitor wortmannin inhibited endogenous proteolysis, the production of glycogen from glucose and the activity of active (dephosphorylated) GS (GSa) in the absence of added amino acids. The stimulation by amino acids of glycogen production and of GSa was only slightly affected by wortmannin. These effects of wortmannin could be mimicked by proteinase inhibitors. A combination of leucine, phenylalanine and tyrosine, which we showed previously to stimulate PI 3-kinase-dependent phosphorylation of ribosomal protein S6, did not stimulate glycogen production from glucose. In contrast with wortmannin, LY294002, another PI 3-kinase inhibitor, strongly inhibited both glycogen synthesis and GSa activity, irrespective of the presence of amino acids. Inhibition of glycogen synthesis by LY294002 could be ascribed in part to increased glycogenolysis and glycolysis. It is concluded that, in hepatocytes, activation of PI 3-kinase may not be responsible for the stimulation of glycogen synthesis by amino acids; LY294002 inhibits glycogen synthesis and stimulates glycogen breakdown by a mechanism that is unrelated to its action as an inhibitor of PI 3-kinase.

Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane

2004 ◽  
Vol 82 (6) ◽  
pp. 708-718 ◽  
Author(s):  
John P Vessey ◽  
Chanjuan Shi ◽  
Christine AB Jollimore ◽  
Kelly T Stevens ◽  
Miguel Coca-Prados ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Kinase Inhibitor ◽  
Regulatory Volume Decrease ◽  
Membrane Dynamics ◽  
Cell Swelling ◽  
Cl Channels ◽  
Phosphoinositide 3 Kinase ◽  
Nonpigmented Ciliary Epithelial ◽  
Volume Decrease

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl– conductance (ICl,swell), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the ICl,swell following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl– current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as ICl,swell. Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of ICl,swell. Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to ICl,swell following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.Key words: ClC-3, NPE, cell swelling, membrane trafficking, ciliary body epithelium.

scholarly journals Glutamine is a good substrate for glycogen synthesis in isolated hepatocytes from 72 h-starved rats, but not from 24 h- or 48 h-starved rats

1992 ◽  
Vol 288 (3) ◽  
pp. 795-799 ◽  
Author(s):  
O Mouterde ◽  
S Claeyssens ◽  
A Chedeville ◽  
A Lavoinne
Keyword(s):  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Isolated Hepatocytes ◽  
Cell Swelling ◽  
Starvation Period ◽  
Glutamate Concentration ◽  
Glutamine Transport ◽  
Gluconeogenic Substrate

In isolated hepatocytes from 24 h-starved rats, no glycogen synthesis was observed in the presence of glutamine. By contrast, glutamine was the best gluconeogenic substrate to induce glycogen synthesis in isolated hepatocytes from 72 h-starved rats. The effect of glutamine on glycogen synthesis was not accompanied by parallel changes in glucose or lactate production. Glutamine activated glycogen synthase independently of the starvation period; however, the extent of synthase activation was 2-fold higher in isolated hepatocytes from 72 h-starved rats than in hepatocytes from 24 h-starved rats. This increase in synthase activation was associated with increased cell swelling. The rate of glutamine transport was not significantly different in hepatocytes from 24 h- and 72 h-starved rats. By contrast, the intracellular glutamate concentration was 1.5-fold higher after 3 days of starvation in hepatocytes incubated with 5 mM-glutamine. We propose that glutamine may play a key role in the glycogen synthesis observed in vivo after 3 days of starvation.

scholarly journals Insulin-Like Growth Factor 2 and the Insulin Receptor, But Not Insulin, Regulate Fetal Hepatic Glycogen Synthesis

Endocrinology ◽  
2010 ◽  
Vol 151 (2) ◽  
pp. 741-747 ◽  
Author(s):  
Li Liang ◽  
Wei Hui Guo ◽  
Diego R. Esquiliano ◽  
Masato Asai ◽  
Susana Rodriguez ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Kinase Inhibitor ◽  
Glycogen Synthase ◽  
Fetal Liver ◽  
Glycogen Synthesis ◽  
Mouse Strains ◽  
Hepatic Glycogen ◽  
Insulin Receptor Isoforms ◽  
Phosphoinositide 3 Kinase

Whether insulin or IGFs regulate glycogen synthesis in the fetal liver remains to be determined. In this study, we used several knockout mouse strains, including those lacking Pdx-1 (pancreatic duodenal homeobox-1), Insr (insulin receptor), and Igf2 (IGF-II) to determine the role of these genes in the regulation of fetal hepatic glycogen synthesis. Our data show that insulin deficiency does not alter hepatic glycogen stores, whereas Insr and Igf2 deficiency do. We found that both insulin receptor isoforms (IR-A and IR-B) are present in the fetal liver, and their expression is gestationally regulated. IR-B is highly expressed in the fetal liver; nonetheless, the percentage of hepatic IR-A isoform, which binds Igf2, was significantly higher in the fetus than the adult. In vitro experiments demonstrate that Igf2 increases phosphorylation of hepatic Insr, insulin receptor substrate-2, and Akt proteins and also the activity of glycogen synthase. Igf2 ultimately increased glycogen synthesis in fetal hepatocytes. This increase could be blocked by the phosphoinositide 3-kinase inhibitor LY294008. Taken together, we propose Igf2 as a major regulator of fetal hepatic glycogen metabolism, the insulin receptor as its target receptor, and phosphoinositide 3-kinase as the signaling pathway leading to glycogen formation in the fetal liver.

scholarly journals Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

2019 ◽  
Vol 295 (3) ◽  
pp. 673-689 ◽  
Author(s):  
Friederike Hans ◽  
Hanna Glasebach ◽  
Philipp J. Kahle
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Glycogen Synthase ◽  
Osmotic Shock ◽  
Protein Kinase R ◽  
Extracellular Signal ◽  
Rapid Dissolution ◽  
The Central Nervous System

Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal–regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.

Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

2010 ◽  
Vol 299 (4) ◽  
pp. C844-C853 ◽  
Author(s):  
Signe Skyum Kirkegaard ◽  
Ian Henry Lambert ◽  
Steen Gammeltoft ◽  
Else Kay Hoffmann
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Regulatory Volume Decrease ◽  
Janus Kinase ◽  
Cell Swelling ◽  
Hek 293 ◽  
Set Point ◽  
Phosphatase Inhibitor

The swelling-activated K+ currents ( IK,vol) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain (K2p), TWIK-related acid-sensitive K+ channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by IK,vol indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower cell volume. Swelling-activated K+ efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K+ efflux 1.3 times. To exclude K+ efflux via a KCl cotransporter, cellular Cl− was substituted with NO3−. Also under these conditions K+ efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive K+ channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the swelling-induced phosphorylation and activation of TASK-2 in EATC.

scholarly journals Swelling of rat hepatocytes activates acetyl-CoA carboxylase in parallel to glycogen synthase

1991 ◽  
Vol 278 (3) ◽  
pp. 887-890 ◽  
Author(s):  
A Baquet ◽  
L Maisin ◽  
L Hue
Keyword(s):  
Amino Acids ◽  
Glycogen Synthase ◽  
Rat Hepatocytes ◽  
Cell Swelling ◽  
Common Mechanism ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Parallel Activation

Incubation of hepatocytes in conditions known to increase their volume, i.e. with amino acids or in hypo-osmotic media, resulted in the parallel activation of glycogen synthase and acetyl-CoA carboxylase. The activation of both enzymes by glutamine was antagonized by the addition of raffinose to prevent cell swelling, or by glucagon and microcystin. The findings are consistent with the involvement of a common mechanism for the activation of the two enzymes.

Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells

1995 ◽  
Vol 73 (7-8) ◽  
pp. 535-544 ◽  
Author(s):  
C. Lo ◽  
J. Ferrier ◽  
H. C. Tenenbaum ◽  
C. A. G. McCulloch
Keyword(s):  
Cell Volume ◽  
Intracellular Ph ◽  
Binding Proteins ◽  
Kinase Inhibitor ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Osteosarcoma Cells ◽  
Extracellular Medium ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of ~30% in suspended (i.e., round) cells and ~60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with B APTA–AM and by depletion of Cl−, indicating the role of calcium-regulated K+ and Cl− efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (~0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na+-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl−-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl− that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.Key words: cell volume, pH, osteoblast, G proteins, actin.

scholarly journals Definition of the binding mode of a new class of phosphoinositide 3-kinase α-selective inhibitors using in vitro mutagenesis of non-conserved amino acids and kinetic analysis

2012 ◽  
Vol 444 (3) ◽  
pp. 529-535 ◽  
Author(s):  
Zhaohua Zheng ◽  
Syazwani I. Amran ◽  
Jiuxiang Zhu ◽  
Oleg Schmidt-Kittler ◽  
Kenneth W. Kinzler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rational Design ◽  
Kinase Inhibitors ◽  
In Vitro Mutagenesis ◽  
Selective Inhibitors ◽  
New Class ◽  
Phosphoinositide 3 Kinase ◽  
Conserved Amino Acids

The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110β isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser773, A-66S bound the region 1 non-conserved amino acid p110α Gln859, and J-32 binding had an indirect interaction with Lys776 and Ile771. The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.

scholarly journals Acidocalcisomes and the Contractile Vacuole Complex Are Involved in Osmoregulation inTrypanosoma cruzi

2004 ◽  
Vol 279 (50) ◽  
pp. 52270-52281 ◽  
Author(s):  
Peter Rohloff ◽  
Andrea Montalvetti ◽  
Roberto Docampo
Keyword(s):  
Amino Acids ◽  
Inorganic Phosphate ◽  
Water Movement ◽  
Regulatory Volume Decrease ◽  
Subcellular Fractionation ◽  
Etiologic Agent ◽  
Contractile Vacuole ◽  
Cell Swelling ◽  
High Concentration ◽  
Volume Decrease

Trypanosoma cruzi, the etiologic agent of Chagas disease, resists extreme fluctuations in osmolarity during its life cycle.T. cruzipossesses a robust regulatory volume decrease mechanism that completely reverses cell swelling when submitted to hypo-osmotic stress. The efflux of amino acids and K+release could account for only part for this volume reversal. In this work we demonstrate that swelling of acidocalcisomes mediated by an aquaporin and microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of this aquaporin and the resulting water movement are responsible for the volume reversal not accounted for by efflux of osmolytes. Contractile vacuole bladders were isolated by subcellular fractionation in iodixanol gradients, showed a high concentration of basic amino acids and inorganic phosphate, and were able to transport protons in the presence of ATP or pyrophosphate. Taken together, these results strongly support a role for acidocalcisomes and the contractile vacuole complex in osmoregulation and identify a functional role for aquaporin in protozoal osmoregulation.

scholarly journals Stimulation of glycogen synthesis by heat shock in L6 skeletal-muscle cells: regulatory role of site-specific phosphorylation of glycogen-associated protein phosphatase 1

2003 ◽  
Vol 371 (3) ◽  
pp. 857-866 ◽  
Author(s):  
Byoung MOON ◽  
Noreen DUDDY ◽  
Louis RAGOLIA ◽  
Najma BEGUM
Keyword(s):  
Heat Shock ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Shock Treatment ◽  
Protein Phosphatase 1 ◽  
Heat Shock Treatment ◽  
Wild Type ◽  
Gsk 3Β ◽  
Phosphoinositide 3 Kinase

Recent evidence suggests that glycogen-associated protein phosphatase 1 (PP-1G) is essential for basal and exercise-induced glycogen synthesis, which is mediated in part by dephosphorylation and activation of glycogen synthase (GS). In the present study, we examined the potential role of site-specific phosphorylation of PP-1G in heat-shock-induced glycogen synthesis. L6 rat skeletal-muscle cells were stably transfected with wild-type PP-1G or with PP-1G mutants in which site-1 (S1) Ser48 and site-2 (S2) Ser67 residues were substituted with Ala. Cells expressing wild-type and PP-1G mutants, S1, S2 and S1/S2, were examined for potential alterations in glycogen synthesis after a 60min heat shock at 45°C, followed by analysis of [14C]glucose incorporation into glycogen at 37°C. PP-1G S1 mutation caused a 90% increase in glycogen synthesis on heat-shock treatment, whereas the PP-1G S2 mutant was not sensitive to heat stress. The S1/S2 double mutant was comparable with wild-type, which showed a 30% increase over basal. Heat-shock-induced glycogen synthesis was accompanied by increased PP-1 and GS activities. The highest activation was observed in S1 mutant. Heat shock also resulted in a rapid and sustained Akt/ glycogen synthase kinase 3β (GSK-3β) phosphorylation. Wortmannin blocked heat-shock-induced Akt/GSK-3β phosphorylation, prevented 2-deoxyglucose uptake and abolished the heat-shock-induced glycogen synthesis. Muscle glycogen levels regulate GS activity and glycogen synthesis and were found to be markedly depleted in S1 mutant on heat-shock treatment, suggesting that PP-1G S1 Ser phosphorylation may inhibit glycogen degradation during thermal stimulation, as S1 mutation resulted in excessive glycogen synthesis on heat-shock treatment. In contrast, PP-1G S2 Ser phosphorylation may promote glycogen breakdown under stressful conditions. Heat-shock-induced glycogenesis appears to be mediated via phosphoinositide 3-kinase/Akt-dependent GSK-3β inactivation as well as phosphoinositide 3-kinase-independent PP-1 activation.

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