Compartmentalization of transport and phosphorylation of glucose in a hepatoma cell line.
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ABSTRACT:
SUBMITTER: Whitesell RR
PROVIDER: S-EPMC1134788 | biostudies-other | 2005 Mar
REPOSITORIES: biostudies-other
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Project description:In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24 h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426-1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and D-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15+/-4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.
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Project description:The rat insulinoma-derived RINm5F cell line retains many differentiated functions of islet beta-cells. However, it fails to recognize glucose as an insulin secretagogue in the physiological concentration range. With this cell line, glucose-transport kinetics were investigated, by using a double-label technique with the non-metabolizable glucose analogue 3-O-methylglucose (OMG). RINm5F cells possess a passive glucose-transport system with high capacity and low affinity. Equilibration across the plasma membrane of extracellular OMG concentrations up to at least 20 mM is achieved within 2 min at 37 degrees C. The half-saturation of OMG uptake occurs at 32 mM. At lower temperatures OMG uptake is markedly retarded, with a temperature coefficient (Q10) of 2.9. As indicated by efflux measurements, transport is symmetrical. Cytochalasin B at micromolar concentrations and phlorrhizin in millimolar concentrations are potent inhibitors of OMG uptake. Neutralization of the secreted insulin with antibodies does not alter OMG uptake kinetics. The glucose metabolism of RINm5F cells is much exaggerated compared with that of islet beta-cells. Nonetheless, when measured in parallel to uptake, transport exceeds by far the rate of metabolism at glucose concentrations above 3 mM. Measurements of intracellular D-glucose reveal a lower intracellular glucose concentration relative to the extracellular in RINm5F cells. This seems to be due to abnormalities in the subsequent steps of glucose metabolism, rather than to abnormalities in hexose uptake. The loss of glucose-induced insulin release in RINm5F cells cannot be explained by alterations in hexose transport.
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Project description:Protein phosphorylation is a fundamental element of cell signaling. First discovered as a biochemical switch in glycogen metabolism, we now know that this posttranslational modification permeates all aspects of cellular behavior. In humans, over 540 protein kinases attach phosphate to acceptor amino acids, whereas around 160 phosphoprotein phosphatases remove phosphate to terminate signaling. Aberrant phosphorylation underlies disease, and kinase inhibitor drugs are increasingly used clinically as targeted therapies. Specificity in protein phosphorylation is achieved in part because kinases and phosphatases are spatially organized inside cells. A prototypic example is compartmentalization of the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase A through association with A-kinase anchoring proteins. This configuration creates autonomous signaling islands where the anchored kinase is constrained in proximity to activators, effectors, and selected substates. This article primarily focuses on A kinase anchoring protein (AKAP) signaling in the heart with an emphasis on anchoring proteins that spatiotemporally coordinate excitation-contraction coupling and hypertrophic responses.
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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.
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Project description:The aim of the project was to obtain murine hepatoma MH22a [1] cell subline, resistant to anticancer agent 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ), and examine the changes of the protein expression as compared with parental cells, with the main emphasis on the changes of redox proteins. MH22a cells obtained from the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg, Russia), were grown at 37°C in DMEM medium, supplemented with 10% fetal bovine serum and antibiotics in T25 flasks until reaching 70-80% confluence. The medium is then changed to an analogous medium with 5.0 μM TPZ, and, depending on cell viability, 50% of the medium with TPZ is changed 1-2 times a week, repeating that procedure for 3.5 months to form a stable monolayer. The resulting monolayer is inoculated 1:2 and further grown by changing 50% of the culture medium twice a week until re-inoculation. 20 passages were made over 2.5 months in total. We were unable to obtain a cell subline in a stepwise fashion by growing the cells in increasing concentrations of TPZ, because its concentration increase up to 10 μM caused cell death after 1 month. For analysis, one flask per each sample, 3 samples of parental cell line and 3 samples of TPZ-resistant cell line (between 15th and 20th passages), were selected. Cells were rinsed three times with PBS, detached with TrypLE Express (ThermoFisher Scientific, Waltham, MA, USA) and cell concentration and viability was determined with Trypan blue and Countess 2 (ThermoFisher Scientific, Waltham, MA, USA) automated cell counter. Cells were rinsed by centrifuging 500xg for 5 min and suspending in PBS three times. Finally, cells were flash frozen in liquid nitrogen and transferred to -86°C freezer for storage. [1] European Collection of Authenticated Cell Cultures (ECACC), Catalogue No. 96121721.
2023-05-10 | PXD039876 | Pride
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