Abstract
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.
Highlights
To investigatethe mechanism responsible for the Glucose transport in muscle and fat cells is subject to rapid inhibition of glucose transport by dibutyryl cAMP modulation by a varietyof factors including insulin, exercise
Changing Ser4” to an Ala abolished phosphorylation of GLUT4; the inhibitoryeffect targeting of GLUT4 is necessary for the large stimulation of glucose transport in adipocytes and muscle that occurs with insulin, because insulin-stimulated transport involves the redistribution of GLUT4 from within the cell to the plasma membrane [4,5]
Ser4=, which is located in this domain, was gest that the inhibitory effects of BtzcAMP could be phosphorylated when vesicles containing GLUT4 were incumediated by direct binding of a nucleotide to GLUT4 bated with CAMP-dependentprotein kinase [12]
Summary
Estimation of Glucose Transport Actiuity-CHO cells were seeded into 24-well culture dishes and infected with various recombinant Sindbis virus stocks as described [14].After incubation in serum-free DMEM, cells were washed with solution A (135 mM NaC1, 5.4 mM KC1, and 10 mM sodium phosphate, pH 7.4) containing 0.1% bovine serum albumin, 1 mM MgS04, 1 mM CaCI, and were incubated at MATERIAL SA N DM ETHODS. The XbaI/SacII fragment Inmunoelectron Microscopy-CHO cells, cultured in 150-mm tisfrom the polymerase chain reaction product was used to replace the sue culture plates, were grown to 80% confluence and infected with corresponding region of the wild type GLUT4. Stable Expression of Glucose Transporters in L6 Myoblasts-Wild type GLUT4 cDNA and cDNAs encoding GLUT4 Ser to Ala mutations were excised with EcoRI and subcloned into the EcoRI site of pSFFVneo [19]. (Bellco).Clones expressing the highest levels of GLUT4 and mutants and GLUT4 was immunoprecipitated using 1F8 asdescribed above
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