Destabilizing effects of fructose-1,6-bisphosphate on membrane bilayers.

Fructose-1,6-bisphosphate (FBP) is a high-energy glycolytic intermediate that decreases the effects of ischemia; it has been used successfully in organ perfusion and preservation. How the cells utilize external FBP to increase energy production and the mechanism by which the molecule crosses the membrane bilayer are unclear. This study examined the effects ofFBP on membrane bilayer permeability, membrane fluidity, phospholipid packing, and membrane potential to determine how FBP crosses the membrane bilayer. Large unilamellar vesicles composed of egg phosphatidylcholine (Egg PC) were made and incubated with 50 mM FBP spiked with 14C-FBP at 30 degrees C. Uptake of FBP was significant (P < 0.05) and dependent on the lipid concentration, suggesting that FBP affects membrane bilayer permeability. With added calcium (10 mM), FBP uptake by lipid vesicles decreased significantly (P < 0.05). Addition of either 5 or 50 mM FBP led to a significant increase (P < 0.05) in Egg PC carboxyfluorescein leakage. We hypothesized that the membrane-permeabilizing effects of FBP may be due to a destabilization of the membrane bilayer. Small unilamellar vesicles composed of dipalmitoyl pC (DPPC) were made containing either diphenyl-1,3,5-hexatriene (DPH) or trimethylammmonia-DPH (TMA-DPH) and the effects of FBP on the fluorescence anisotropy (FA) of the fluorescent labels examined. FBP caused a significant decrease in the FA of DPH in the liquid crystalline state of DPPC (P < 0.05), had no effect on FA of TMA-DPH in the liquid crystalline state of DPPC, but increased the FA of TMA-DPH in the gel state of DPPC. From phase transition measurements with DPPC/DPH or TMA-DPH, we calculated the slope of the phase transition as an indicator of the cooperativity of the DPPC molecules. FBP significantly decreased the slope, suggesting a decrease in fatty acyl chain interaction (P< 0.05). The addition of 50 mM FBP caused a significant decrease (P< 0.05) in the liquid crystalline/gel state fluorescence ratio of merocyanine 540, indicating increased head-group packing. To determine what effects these changes would have on cellular membranes, we labeled human endothelial cells with the membrane potential probe 3,3'-dipropylthiacarbocyanine iodide (DiSC3) and then added FBP. FBP caused a significant, dose-dependent decrease in DiSC3 fluorescence, indicating membrane depolarization. We suggest that FBP destabilizes membrane bilayers by decreasing fatty acyl chain interaction, leading to significant increases in membrane permeability that allow FBP to diffuse into the cell where it can be used as a glycolytic intermediate.