Dataset Information

Acute regulation of glucose transport in a monocyte-macrophage cell line: Glut-3 affinity for glucose is enhanced during the respiratory burst.

ABSTRACT: Activation of the respiratory burst imposes acute metabolic demands on phagocytic cells. These are met by mobilizing internal energy stores and by increasing the utilization of exogenous energy, including glucose in the circulation. To determine whether the increased glucose uptake that is known to be associated with the respiratory burst involves the regulation of glucose transporter molecules, the intrinsic transport properties of glucose transporters on the macrophage cell line RAW 264.7 were determined after activation with PMA, N-formyl-methionine-leucine-phenylalanine (fMLP) and the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3). Treatment with PMA resulted in a 2-fold increase in respiratory burst activity within 10 min; this was associated with a 30-50% increase in 2-deoxyglucose uptake and a 4-fold increase in transporter affinity for glucose. Similarly, fMLP, GM-CSF and IL-3 treatments stimulated 2-deoxyglucose uptake that was associated with a 3-4-fold increase in transporter affinity for glucose. To determine whether the changes observed in 2-deoxyglucose uptake in response to PMA, fMLP and growth factors were influenced by phosphorylation of the sugar, 3-O-methylglucose, which is not phosphorylated, was used. Increased 3-O-methylglucose uptake and increased transporter affinity for glucose were also observed after PMA, fMLP and GM-CSF treatments. Whereas both fMLP and GM-CSF stimulated superoxide production, IL-3 failed to activate respiratory burst activity. The protein kinase inhibitors genistein and staurosporine inhibited the increase in 2-deoxyglucose uptake observed with fMLP and GM-CSF, and partly reversed the affinity increase towards that of untreated control cells. In contrast, the phosphatidylinositol 3-kinase inhibitor wortmannin had little effect on 2-deoxyglucose uptake in response to these activators. Western blotting with subtype-specific antisera showed that Glut-3 was the predominant transporter on RAW 264.7 cells. These studies demonstrate that acute regulation of glucose transporters occurs in response to activators that promote respiratory burst activity, and show that this regulation involves both tyrosine kinases and protein kinase C activity.

SUBMITTER: Ahmed N

PROVIDER: S-EPMC1218803 | biostudies-other | 1997 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundCatharanthus roseus is an important Ayurvedic medication in traditional medicine. It is potentially used in countries like India, South Africa, China and Malaysia for the healing of diabetes mellitus. Although, the molecular mechanisms behind this effect are yet to be exclusively explored. Due to the great antidiabetic and hyperlipidemic potential of c. roseus, we hypothesized that the insulin mimetic effect of ethanolic extract of c. roseus might add to glucose uptake through improvement in the expression of genes of the glucose transporter (GLUT) family messenger RNA (mRNA) in liver.MethodsSTZ-induced diabetic rats treated by ethanolic extract of c. roseus 100 mg/kg and 200 mg/kg; and one group treated with Metformin (100 mg/kg). After final administration of treatment of 4 weeks, blood samples were collected under fasting conditions, and the body weights (BWs) were measured. Total RNA from liver was extracted with the Qiagen RNEasy Micro kit (GERMANY) as described in the manufacturer's instructions. First-strand complementary DNA (cDNA) was synthesized at 40 °C by priming with oligo-dT12-18 (Invitrogen, USA) and using Super ScriptII reverse transcriptase according to the protocol provided by the manufacturer (Invitrogen, USA). Real-time polymerase chain reaction (PCR) amplifications for GLUT-4 (gene ID: 25139) were conducted using Light-Cycler 480 (Roche, USA) with the SyBr® I nucleic acid stain (Invitrogen, USA) according to the manufacturer's instructions. Polymerase chain reaction products of β-actin primer gene were used as an internal standard.ResultsThe proposed study was framed to look at the antidiabetic efficacy of ethanolic extract of c. roseus and an expression of GLUT-2 and GLUT-4 gene in streptozotocin induced diabetic wistar rats. The doses were administered orally at a rate of 100 and 200 mg/kg and detrain the glucose transport system in liver for 4 weeks. The observed results showed a good positive correlation between intracellular calcium and insulin release levels in isolated islets of Langerhans. The supplementation of ethanolic extract of c. roseus significantly amplified the expression of GLUT gene mRNA by Real Time PCR in liver of diabetic rats.ConclusionsWe conclude that the observed antidiabetic effect of c. roseus on STZ induced diabetes was a result of complex mechanisms of GLUT gene mRNA expression. The findings are very encouraging and greatly advocate its candidature for the design of a novel herbal drug to cure deadly diabetes.

Project description:The effect of culture conditions simulating hypo- and hyper-glycaemia on glucose transport and on the subcellular localization of the glucose transporter GLUT-1 was studied in L8 myocytes. Incubation of the cells with 20 mM-glucose for 25 h decreased the rate of 2-deoxy-D-[3H]glucose (dGlc) uptake to 0.106 +/- 0.016 nmol/min per 10(6) cells compared with 0.212 +/- 0.025 in cells maintained at 2 mM-glucose (final glucose concentrations at the end of the incubation period were 16-17 mM and 0.7-1.0 mM respectively). An additional 5 h incubation of these cells with medium containing the opposite glucose concentration (i.e. change from 17 mM to 1 mM and from 1 mM to 17 mM) increased the transport rate to 0.172 +/- 0.033 nmol/min per 10(6) cells in cultures initially conditioned at high glucose, and decreased the transport to 0.125 +/- 0.029 in those conditioned at low glucose. Plasma-membrane- and microsomal-membrane-enriched fractions were prepared from these cells for [3H]cytochalasin B (CB) binding and Western-blot analysis with antibodies against GLUT-1 and GLUT-4. A decrease in glucose concentration increased the number of D-glucose-displaceable CB-binding sites and GLUT-1 protein in the plasma-membrane fraction to the same extent as the increase in dGlc transport. Under downregulatory conditions, the lower dGlc-transport capacity could be accounted for by a decreased number of transporters in the plasma membrane of the cells. No apparent modification of the intrinsic activity of the glucose transporters was observed in up- or down-regulated cells. Under downregulatory conditions, the CB-binding data indicated a large increase in the number of transporters in the intracellular membranes of the myocytes. Western blots of the same membranes also indicated an increase in GLUT-1 content. However, the interaction of the intracellular GLUT-1 protein with the polyclonal antibodies was much weaker than that of the plasma-membrane-associated GLUT-1. The GLUT-4 concentration was too low to permit quantification in membrane fractions. Our findings suggest that autoregulation of glucose transport in L8 myocytes is accompanied by parallel changes in the number of GLUT-1 transporters in the plasma membrane, and that the rate of transporter degradation may be augmented in the upregulated myocytes. These glucose-induced changes are fully reversible.

Project description:Growth factors promote cell survival and proliferation by activating signal transduction pathways that result in progression through the cell cycle and differential gene expression. Uptake of simple sugars needed for basal cell metabolism, and for macromolecular synthesis necessary for cell growth and proliferation, is thought to follow as a consequence of signal transduction to the nucleus. However, in the presence of inhibitors of DNA synthesis and respiration, growth factors can still promote cell survival responses in the short term, raising the possibility that they may also regulate critical membrane and cytosolic processes necessary for cell survival. We have tested this hypothesis directly by investigating the role of the haemopoietic growth factor, interleukin-3 (IL-3), in the regulation of glucose transport in the bone marrow-derived cell line, 32D. We show that IL-3 promotes glucose transport by actively maintaining the affinity of the plasma membrane, glucose transporter for glucose (Km 1.35 +/- 0.15 mM, n = 4). Withdrawal of IL-3 for 1 h resulted in reduced affinity for glucose (Km 2.96 +/- 0.28 mM, n = 4) without an associated change in Vmax. Furthermore, glucose transporter molecules as the cell surface, as determined by cytochalasin B binding to isolated plasma membranes, did not differ significantly between control and IL-3-treated cells. Inhibition of DNA synthesis with mitomycin C or with the respiratory poison, sodium azide, did not affect the ability of IL-3 to promote glucose transport. In contrast, the tyrosine kinase inhibitors genistein and erbstatin extensively inhibited control and IL-3-stimulated glucose transport, some preference of IL-3-stimulated glucose transport, some preference for IL-3-stimulated responses being observed at low inhibitor concentrations. The light-activated protein kinase C inhibitor, calphostin C, also inhibited control and IL-3-stimulated glucose transport but without preference for IL-3 responses. Additionally, the tyrosine phosphatase inhibitor, orthovanadate, stimulated control and IL-3-dependent glucose transport by 50-80% while the protein kinase A inhibitor, KT5720, inhibited glucose transport by about 20% at plateau values. These results indicate that IL-3 is involved in continuous maintenance of glucose transporter activity by a mechanism that involves tyrosine kinases and protein kinase C, and demonstrate that this activation is not dependent on respiration or signal transduction to the nucleus.

Dataset Information

Acute regulation of glucose transport in a monocyte-macrophage cell line: Glut-3 affinity for glucose is enhanced during the respiratory burst.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets