Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulin-stimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.
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PMID:Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol. 176 64

Twelve loci have been assigned to rat chromosome 5: aldolase B (ALDOB), atrial natriuretic factor (ANF = pronatriodilatin, PND), D4RP1, DSI1, galactosyltransferase (GGTB2), glucose transporter (GLUT1), interferon alpha 1 and related interferon alpha (INFA), interferon beta (INFB), lymphocyte-specific protein-tyrosine kinase (LCK), oncogene MOS, alpha 2U-globulin (major urinary protein, MUP), and orosomucoid (ORM, also called alpha 1-acid glycoprotein, AGP). Among these, the interferon alpha and beta genes map in the q22-23 region, which also contains a transformation suppressor gene (SAI1). The other loci reside outside this region. This study also indicated that the rat genome contains 2 LCK genes, unlike the human and murine genomes. These new assignments on rat chromosome 5 demonstrate that this chromosome is highly homologous to mouse chromosome 4 and carries synteny groups conserved on human chromosome 9 (interferon alpha and beta, galactosyltransferase, orosomucoid, and aldolase B genes) and on the short arm of human chromosome 1 (MYCL, glucose transporter, protein kinase LCK, and atrial natriuretic factor genes).
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PMID:Assignment of 12 loci to rat chromosome 5: evidence that this chromosome is homologous to mouse chromosome 4 and to human chromosomes 9 and 1 (1p arm). 234 Nov 57

This study characterizes the actions of insulin and parathyroid hormone (PTH) on the glucose transport system in the rat osteogenic sarcoma cell line UMR 106-01, which expresses a number of features of the osteoblast phenotype. Using [1,2-3H]2-deoxyglucose (2-DOG) as a label, UMR 106-01 cells were shown to possess a glucose transport system which was enhanced by insulin. In contrast, PTH influenced glucose transport in a biphasic manner with a stimulatory effect at 1 h and a more potent inhibitory effect at 16 h on basal and insulin-stimulated 2-DOG transport. To explore the mechanism of PTH action, a direct agonist of cAMP-dependent protein kinase (PKA) was tested. 8-Bromo-cAMP had no acute stimulatory effect but inhibited basal and insulin-stimulated 2-DOG transport at 16 h. This result suggested that the prolonged, but not the acute, effect of PTH was mediated by the generation of cAMP. Further studies with the cell line UMR 4-7, a UMR 106-01 clone stably transfected with an inducible mutant inactive regulatory subunit of PKA, confirmed that the inhibitory but not the stimulatory effect of PTH was mediated by the PKA pathway. Northern blot data indicated that the prolonged inhibitory effects of PTH and 8-bromo-cAMP on glucose transport were likely to be mediated in part by reduction in the levels of GLUT1 (HepG2/brain glucose transporter) mRNA.
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PMID:Modulation of glucose transport by parathyroid hormone and insulin in UMR 106-01, a clonal rat osteogenic sarcoma cell line. 761 14

Downstream mediators of insulin signaling are thought to include multiple cytoplasmic serine/threonine kinases such as the product of the cellular proto-oncogene c-raf-1. To investigate a role for the Raf-1 protein kinase in insulin-stimulated glucose transport, a gene encoding an oncogenically activated Raf-1 mutant was introduced into 3T3-L1 fibroblasts by retroviral gene transfer. Expression of activated Raf-1 in differentiated 3T3-L1 adipocytes markedly increases hexose uptake compared to control adipocytes or those infected with the retroviral vector. Basal 2-deoxyglucose uptake in adipocytes expressing activated Raf-1 is approximately 40-fold higher than in parental adipocytes, and insulin further increases uptake about 1.2-fold. As determined by the plasma membrane "sheet" assay, Raf-1-expressing adipocytes contain greatly elevated levels of the ubiquitous glucose transporter (GLUT1) on the cell surface in the absence or presence of insulin. Total cellular GLUT1 protein is increased about 5-fold. In contrast, activated Raf-1 affects neither the expression of the "insulin-responsive" glucose transporter (GLUT4) nor its cellular distribution; GLUT4 is virtually undetectable on the plasma membrane in the absence of insulin and translocates normally following the addition of hormone. These data suggest that activation of Raf-1 mediates the chronic effect of insulin on hexose uptake but is not sufficient for the rapid translocation of GLUT4. Moreover, the differential effects of activated Raf-1 expression on the two transporter isoforms define divergent signaling pathways by which insulin regulates glucose transport in cultured adipocytes.
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PMID:A role for Raf-1 in the divergent signaling pathways mediating insulin-stimulated glucose transport. 814 13

To investigate the mechanism responsible for the inhibition of glucose transport by dibutyryl cAMP (Bt2cAMP), two different transporter isoforms (GLUT1 and GLUT4) and several GLUT1/4 chimeric transporters were expressed in Chinese hamster ovary (CHO) cells by using a Sindbis virus expression system. Bt2cAMP inhibited GLUT4-mediated 2-deoxy[3H]glucose (2DOG) uptake by 50% but was without effect on GLUT1-mediated uptake. When the subcellular distribution of GLUT4 was assessed by quantitative immunocytochemistry, neither the overall concentration of GLUT4 nor the regional distribution of GLUT-4 within the plasma membrane was found to be altered by Bt2cAMP. Thus, inhibition of 2DOG uptake by Bt2cAMP appears to be due to a decrease in transporter activity rather than a decrease in the number of transporters exposed at the plasma membrane. By using chimeric transporters, a region of GLUT4 necessary for the inhibitory effect of Bt2cAMP was localized to the last 29 amino acids in the COOH terminus. This intracellular region contains the site (Ser488) phosphorylated in vitro by cAMP-dependent protein kinase (cAdPK). Changing Ser488 to an Ala abolished phosphorylation of GLUT4; however, the inhibitory effect of Bt2cAMP on glucose transport was not diminished by this mutation. Therefore, phosphorylation of GLUT4 was not required for the inhibition. The effects of other nucleotides on GLUT4 transport activity were assessed to investigate the role of cAdPK. Uptake of 2DOG by GLUT4 was inhibited by 8-bromo-AMP, but not by 8-bromo-cAMP, suggesting that the inhibitory effect did not involve activation of cAdPK. Results consistent with this interpretation were obtained with CHO cells (line 10248), which express a cAdPK that is resistant to activation by cAMP. No difference in the concentrations of Bt2cAMP required to inhibit GLUT4-mediated transport was observed in normal CHO cells and 10248 cells. The results presented suggest that the inhibitory effects of Bt2cAMP could be mediated by direct binding of a nucleotide to GLUT4 at a site involving the intracellular COOH terminus of the transporter.
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PMID:GLUT4 phosphorylation and inhibition of glucose transport by dibutyryl cAMP. 839 69

The effect of insulin on protein kinase activity and plasma membrane translocation of the glucose transporter GLUT 4 has been studied in adipocytes permeabilized by Streptolysin-O. Insulin increased protein kinase activity, and this was completely inhibited by the PKC pseudosubstrate inhibitor peptide (PKC19-36). Insulin-mediated translocation of GLUT 4 was also inhibited by the PKC inhibitor peptide. Both these insulin effects were blocked by a PKCbeta neutralizing antibody. Our results are consistent with the hypothesis that insulin activates PKCbeta activity in adipocytes in situ, and that this PKC activation is a component of the system whereby insulin regulates translocation of GLUT 4 to the plasma membrane.
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PMID:The protein kinase C pseudosubstrate peptide (PKC19-36) inhibits insulin-stimulated protein kinase activity and insulin-mediated translocation of the glucose transporter glut 4 in streptolysin-O permeabilized adipocytes. 928 34

We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 microM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80-100% losses of all DAG-dependent PKCs (alpha, beta1, beta2, delta, epsilon) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-zeta, as well as insulin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCbeta2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate glucose transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubstrate inhibited insulin-stimulated glucose transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-zeta pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-beta (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants.
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PMID:Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. 934 99

Elevation of intracellular glucose within retinal vascular cells is believed to be an important causal factor in the development of diabetic retinopathy. The intracellular glucose concentration is regulated by both the rate of glucose metabolism and glucose transport. Because retinal hypoxia often precedes proliferative diabetic retinopathy, we have studied the regulation of the glucose transport system by hypoxia in cultured bovine retinal endothelial cells (BRECs). Because retinal ischemia is known to increase intracellular adenosine levels, which subsequently regulate hypoxia-inducible genes, such as vascular endothelial growth factor and erythropoietin, the role of adenosine and its receptor-mediated pathways has also been evaluated. Hypoxia (0.5% O2, 5% CO2, and 94.5% N2) stimulated GLUT1 mRNA expression in BRECs in a time-dependent manner with an 8.9 +/- 1.5-fold (P < 0.01) increase observed after 12 h. GLUT1 mRNA expression returned to baseline (1.4 +/- 0.3-fold of control) within 12 h after reinstitution of normoxia. N6-Cyclopentyl adenosine (adenosine A1 receptor agonist, Kd = 1 nmol/l) did not affect GLUT1 mRNA expression at concentrations up to 1 micromol/l, while 2-p-(2-carboxyethyl)-phenethyl-amino-5'-N-ethylcarboxamidoadenosine and 5'-(N-ethylcalboxamido)-adenosine (adenosine A2 receptor [A2R] agonists, Kd = 15 and 16 nmol/l, respectively) increased mRNA levels at concentrations as low as 10 nmol/l. Maximal stimulation was 2.3 +/- 0.2- and 2.1 +/- 0.2-fold, respectively (P < 0.01). The adenosine A2a receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) (Kd = 100 nmol/l for A2R) inhibited hypoxia-stimulated GLUT1 mRNA expression by 40 +/- 8% at 100 nmo/l. Hypoxia upregulated GLUT1 protein expression by 3.0 +/- 0.3-fold after 12 h (P < 0.01), but this response was attenuated by CSC (P < 0.05). Hypoxia increased glucose transport activity by 2.1 +/- 0.3-fold (P < 0.001) after 12 h, a response inhibited 65% by CSC (P < 0.01). A protein kinase A (PKA) inhibitor (H89, 20 micromol/l) suppressed hypoxia-induced GLUT1 mRNA expression by 42 +/- 9% (P < 0.01). These data suggest that hypoxia in BRECs upregulates glucose transport activity through an increase of GLUT1 expression that is partially mediated by adenosine, A2R, and the cAMP-PKA pathway.
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PMID:Hypoxia upregulates glucose transport activity through an adenosine-mediated increase of GLUT1 expression in retinal capillary endothelial cells. 972 38

The process linking increased glucose utilization and activation of metabolic pathways leading to end-organ damage from diabetes is not known. We have previously described rat mesangial cells that were transduced to constitutively express the facilitative glucose transporter 1 (GLUT1, MCGT1 cells) or bacterial beta-galactosidase (MCLacZ, control cells). Glucose transport was rate limiting for extracellular matrix production in the MCGT1 cells. In the present work, we investigated the effect of GLUT1 overexpression in mesangial cells on aldose reductase (AR), protein kinase Calpha (PKCalpha), and native GLUT1 transcript levels, to determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. MCGT1 cells grown in normal (8 mM) or elevated (20 mM) glucose had elevated abundance of AR, PKCalpha, and the native GLUT1 transcripts compared with control cells. AR protein levels, AR activity, sorbitol production, and PKCalpha protein content were also greater in the MCGT1 cells than in control cells grown in the same media. This is the first report of the concomitant activation of AR, PKCalpha, and GLUT1 genes by enhanced GLUT1 expression. We conclude that increased GLUT1 expression leads to a positive feedback of greater GLUT1 expression, increased AR expression and activity with polyol accumulation, and increased total and active PKCalpha protein levels, which leads to detrimental stimulation of matrix protein synthesis by diabetic mesangial cells.
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PMID:Glucose transporters control gene expression of aldose reductase, PKCalpha, and GLUT1 in mesangial cells in vitro. 1040 2

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.
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PMID:Regulation of insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes. 1050 86


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