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Determination of the rates of appearance and loss of glucose transporters at the cell surface of rat adipose cells.

ABSTRACT: We have used an impermeant bis-mannose compound (2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos+ ++- 4-yloxy)-2- propylamine; ATB-BMPA) to photolabel the glucose transporter isoforms GLUT4 and GLUT1 that are present in rat adipose cells. Plasma-membrane fractions and light-microsome membrane fractions were both labelled by ATB-BMPA. The labelling of GLUT4 in the plasma membrane fraction from insulin-treated cells was approximately 3-fold higher than that of basal cells and corresponded with a decrease in the labelling of the light-microsome fraction. In contrast with this, the cell-surface labelling of GLUT4 from insulin-treated intact adipose cells was increased approximately 15-fold above basal levels. In these adipose cell preparations, insulin stimulated glucose transport activity approximately 30-fold. Thus the cell-surface labelling, but not the labelling of membrane fractions, closely corresponded with the stimulation of transport. The remaining discrepancy may be due to an approx. 2-fold activation of GLUT4 intrinsic transport activity. We have studied the kinetics of trafficking of transporters and found the following. (1) Lowering the temperature to 18 degrees C increased basal glucose transport and levels of cell-surface glucose transporters by approximately 3-fold. This net increase in transporters probably occurs because the process of recruitment of transporters is less temperature-sensitive than the process involved in internalization of cell-surface transporters. (2) The time course for insulin stimulation of glucose transport activity occurred with a slight lag period of 47 s and a t 1/2 3.2 min. The time course of GLUT4 and GLUT1 appearance at the cell surface showed no lag and a t 1/2 of approximately 2.3 min for both isoforms. Thus at early times after insulin stimulation there was a discrepancy between transporter abundance and transport activity. The lag period in the stimulation of transport activity may represent the time required for the approximately 2-fold stimulation of transporter intrinsic activity. (3) The decrease in transport activity after insulin removal occurred with a very high activation energy of 159 kJ.mol-1. There was thus no significant decrease in transport or less of cell-surface transporters over 60 min at 18 degrees C. The decrease in transport activity occurred with a t1/2 of 9-11 min at 37 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

SUBMITTER: Clark AE

PROVIDER: S-EPMC1151473 | biostudies-other | 1991 Aug

REPOSITORIES: biostudies-other

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Project description:Insulin-stimulated glucose transport activity in rat adipocytes is inhibited by isoprenaline and enhanced by adenosine. Both of these effects occur without corresponding changes in the subcellular distribution of the GLUT4 glucose transporter isoform. In this paper, we have utilized the impermeant, exofacial bis-mannose glucose transporter-specific photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) [Clark & Holman (1990) Biochem. J. 269, 615-622], to examine the cell surface accessibility of GLUT4 glucose transporters under these conditions. Compared with cells treated with insulin alone, adenosine in the presence of insulin increased the accessibility of GLUT4 to the extracellular photolabel by approximately 25%, consistent with its enhancement of insulin-stimulated glucose transport activity; the plasma membrane concentration of GLUT4 as assessed by Western blotting was unchanged. Conversely, isoprenaline, in the absence of adenosine, promoted a time-dependent (t1/2 approximately 2 min) decrease in the accessibility of insulin-stimulated cell surface GLUT4 of > 50%, which directly correlated with the observed inhibition of transport activity; the plasma membrane concentration of GLUT4 decreased by 0-15%. Photolabelling the corresponding plasma membranes revealed that these alterations in the ability of the photolabel to bind to GLUT4 are transient, as the levels of both photolabel incorporation and plasma membrane glucose transport activity were consistent with the observed GLUT4 concentration. These data suggest that insulin-stimulated GLUT4 glucose transporters can exist in two distinct states within the adipocyte plasma membrane, one which is functional and accessible to extracellular substrate, and one which is non-functional and unable to bind extracellular substrate. These effects are only observed in the intact adipocyte and are not retained in plasma membranes isolated from these cells when analysed for their ability to transport glucose or bind photolabel.

Project description:BackgroundThe trematode parasite, Schistosoma mansoni, has evolved to switch from oxidative phosphorylation to glycolysis in the presence of glucose immediately after invading the human host. This metabolic switch is dependent on extracellular glucose concentration. Four glucose transporters are encoded in the genome of S. mansoni, however, only two were shown to facilitate glucose diffusion.ResultsBy modeling the phase of human host infection, we showed that transporter transcript expression profiles of recently transformed schistosomula have two opposing responses to increased glucose concentrations. Concurring with the transcription profiles, our phylogenetic analyses revealed that S. mansoni glucose transporters belong to two separate clusters, one associated with class I glucose transporters from vertebrates and insects, and the other specific to parasitic Platyhelminthes. To study the evolutionary paths of both groups and their functional implications, we determined evolutionary rates, relative divergence times, genomic organization and performed structural analyses with the protein sequences. We finally used the modelled structures of the S. mansoni glucose transporters to biophysically (i) analyze the dynamics of key residues during glucose binding, (ii) test glucose stability within the active site, and (iii) demonstrate glucose diffusion. The two S. mansoni Platyhelminthes-specific glucose transporters, which seem to be younger than the other two, exhibit slower rates of molecular evolution, are encoded by intron-poor genes, and transport glucose. Interestingly, our molecular dynamic analyses suggest that S. mansoni class I glucose transporters are not able to transport glucose.ConclusionsThe glucose transporter family in S. mansoni exhibit different evolutionary histories. Our results suggested that S. mansoni class I glucose transporters lost their capacity to transport glucose and that this function evolved independently in the Platyhelminthes-specific glucose transporters. Finally, taking into account the differences in the dynamics of glucose transport of the Platyhelminthes-specific transporters of S. mansoni compared to that of humans, we conclude that S. mansoni glucose transporters may be targets for rationally designed drugs against schistosomiasis.

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Determination of the rates of appearance and loss of glucose transporters at the cell surface of rat adipose cells.

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