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GLUT4 phosphorylation and inhibition of glucose transport by dibutyryl cAMP
R C Piper1, D E James, J W Slot
1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
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.
Insights
Dibutyryl cAMP (Bt2cAMP) inhibits glucose transport by decreasing GLUT4 transporter activity, not by altering its cell surface levels. This inhibition is mediated by direct nucleotide binding to the transporter's C-terminus, independent of phosphorylation.
Area of Science:
- Molecular biology
- Cellular physiology
- Biochemistry
Background:
- Glucose transport is crucial for cellular energy metabolism.
- Cyclic AMP (cAMP) signaling pathways regulate glucose transporter activity, particularly GLUT4.
- The precise mechanism by which cAMP analogs inhibit glucose transport remains incompletely understood.
Purpose of the Study:
- To elucidate the mechanism of glucose transport inhibition by dibutyryl cAMP (Bt2cAMP).
- To identify the specific glucose transporter isoforms and regions involved in Bt2cAMP-mediated inhibition.
- To determine the role of phosphorylation and cAMP-dependent protein kinase (cAdPK) in this inhibitory process.
Main Methods:
- Expression of GLUT1, GLUT4, and chimeric transporters in Chinese hamster ovary (CHO) cells using a Sindbis virus system.
- Measurement of 2-deoxy[3H]glucose (2DOG) uptake to assess glucose transport activity.
- Quantitative immunocytochemistry to evaluate transporter subcellular distribution.
- Site-directed mutagenesis to investigate the role of Ser488 phosphorylation.
- Assessment of nucleotide effects using various analogs and cAMP-resistant cAdPK cell lines.
Main Results:
- Bt2cAMP significantly inhibited GLUT4-mediated glucose uptake by 50% but had no effect on GLUT1.
- Bt2cAMP did not alter the total cellular or plasma membrane levels of GLUT4, indicating regulation of transporter activity.
- A region within the last 29 amino acids of the GLUT4 C-terminus was identified as necessary for inhibition.
- Mutation of Ser488 to alanine abolished phosphorylation but did not diminish Bt2cAMP's inhibitory effect.
- Inhibition by Bt2cAMP was observed in cAMP-resistant cAdPK cells and was mimicked by 8-bromo-AMP, suggesting a non-cAdPK mediated mechanism.
Conclusions:
- The inhibition of GLUT4-mediated glucose transport by Bt2cAMP is due to a decrease in transporter activity, not altered membrane localization.
- The intracellular C-terminal region of GLUT4 is critical for mediating this inhibition.
- Phosphorylation of GLUT4 by cAdPK is not required for Bt2cAMP-induced inhibition; direct nucleotide binding to the C-terminus is suggested.