eicosapentaenoic acid In spite of the similarities of caffei
In spite of the similarities of caffeine's effects in L929 cells and erythrocytes, a major difference can be identified in the magnitude of inhibition (35% versus 90%). It initially seems reasonable to conclude from this finding that the reduced magnitude of caffeine inhibition in L929 cells is simply a function of the lower concentration of GLUT1. It is reasonable to expect that as the concentration of GLUT1 in the membrane increases, the close proximity of the transporters would produce more GLUT1 tetramers, and therefore, be more responsive to inhibition by caffeine. This is in fact what is observed in erythrocytes. These eicosapentaenoic acid have high GLUT1 concentrations and aggregate as tetramers, and as a result, are highly responsive to inhibition by caffeine , . In addition, increasing the expression of GLUT1 in kidney cells, leads to a dose dependent aggregation of GLUT1 . Therefore, to test if GLUT1 concentration is important, we measured caffeine's effects in HCLE and HK2 cells, both of which express significantly more GLUT1 than L929 cells . As expected, both HCLE and HK2 cells also have higher basal rates of 2DG uptake. In line with these observations, we found that both cell types are much more responsive to caffeine, with maximal inhibition at about 70%, which approaches the 90% inhibition observed in erythrocytes.
While the data in HK2 and HCLE cells supports a role for GLUT1 abundance in cellular response to caffeine, our parallel study with “activated” L929 cells demonstrates that the maximal effect of caffeine on 2DG transport is not simply a function of cellular GLUT1 content. L929 cells deprived of glucose for 20 min display 2DG uptakes greater than 3-fold that of basal cells, but show no change in cellular GLUT1 content. Interestingly, the stimulated uptake rate in L929 cells is similar in magnitude to the basal uptake rates in HCLE and HK2 cells, and in this context L929 cells are similarly inhibited by caffeine (Fig. 4A). As such, the inhibitory effect of caffeine appears to be more sensitive to the activity state of GLUT1 than to the total GLUT1 content.
If caffeine binds only to GLUT1 tetramers in L929 cells—as has been reported to be the case in erythrocytes—it seems likely that glucose deprivation stimulates tetramer formation by GLUT1 in L929 cells. Though this supposition has yet to be proven formally by biochemical isolation of tetramers in L929 cells, it is consistent with a model in which acute activation of glucose uptake is driven by dynamic changes in the thiol chemistry of GLUT1, shifting its quaternary structure to favor the tetrameric structure. Previous work by our lab has shown that the activation of 2DG uptake in L929 cells depends on thiol chemistry , , , , . For example, nitroxyl (HNO), which promotes disulfide bond formation especially in hydrophobic environments , , , stimulates 2DG uptake 5-fold within 2 min and this activation is blocked by thioreactive compounds such as pretreatment with iodoacetamide . This is consistent with the notion that nitroxyl chemically promotes a disulfide formation within GLUT1 that leads to stabilization of the tetramer structure. However, future work, beyond the scope of this study, needs to be done to directly detect formation of GLUT1 tetramers within the membrane if this mechanism is to be confirmed.
Acknowledgements This research was supported by a NIH R15 grant (DK08193-1A1). Special thanks go to the Ubels lab for supplying the HCLE cells.
Materials and Methods
Conclusions We extended the steady-state Michaelis-Menten theory to be applicable to the case in which two competing molecular species bind to and cross the cell membrane via the same carrier protein. This new theory was used to determine the values of the kinetic parameters for three different monofluorinated glucoses (FDG-2, FDG-3, and FDG-4; Fig. 1) as substrates of GLUT1.