Addition of G6P to digitonin-permeabilized hepatocytes results in a time- and dose-dependent hydrolysis of G6P and generation of glucose and inorganic phosphate (Pi) into the incubation system [16
]. In the present study, G6P concentrations ranging from 1 to 10mM were added to digitoning-permeabilized hepatocytes, and the hydrolysis rate measured as the amount of Pi generated into the incubation system at various time points. Maximal hydrolysis was observed upon addition of 5mM G6P, not increasing significantly at higher G6P concentrations (not shown
). Hence, this concentration of G6P was used in the majority of the experiments reported in this study.
As illustrated in , varying Mg2+ concentrations in the incubation system had a bi-phasic effect on G6P hydrolysis. Glucose 6-phosphate hydrolysis and inorganic phosphate generation increased at an external Mg2+ concentration of 0.25mM as compared to 0 mM [Mg2+]o, but decreased in the presence of larger extra-reticular Mg2+ concentrations (i.e. 0.5 or 1mM). In keeping with the physiological range of cytoplasmic Mg2+ concentration within mammalian cells, concentrations of Mg2+ larger than 1 mM were not tested. Radioisotope distribution of G6P indicated that over 45 min of incubation, digitonin-permeabilized hepatocytes incubated in the presence of 0.25mM Mg2+ accumulated 30±3% more G6P than hepatocytes incubated in the presence of nominal 0mM Mg2+ (4.90±0.27 vs. 3.76±0.19 μmol G6P/mg protein/45 min, respectively, n=4 for both experimental conditions, p<0.001) whereas hepatocytes incubated in the presence of 0.5 or 1mM Mg2+ accumulated ~10% more G6P (e.g. 4.19±0.21 μmol G6P/mg protein/45 min, n=4, for cells incubated in the presence of 1mM Mg2+) as compared to hepatocytes incubated in the presence of nominal 0mM Mg2+.
G6P hydrolysis in the presence of varying Mg2+ (Fig. 1A) and G6P (Fig. 1B) concentrations
The effect on G6P hydrolysis was Mg2+ specific, as it was not observed when permeabilized hepatocytes were incubated in the presence of equivalent concentrations of other divalent cations (). The enhanced hydrolysis in the presence of 0.25mM [Mg2+]o was observed at all the concentrations of G6P tested ().
Effect of Different Cations on G6P hydrolysis
The biphasic effect of Mg2+ appears to be specific for G6P. In experiments in which 1mM PPi was used as the hydrolytic substrate instead of G6P, the amount of Pi generated increased over time in a manner directly proportional to the Mg2+ concentration present in the incubation system (). Increasing the concentration of PPi beyond 1mM did not result in a larger hydrolytic activity (not shown).
PPi hydrolysis in the presence of varying Mg2+ concentrations
To confirm that the effect of Mg2+
on G6P and PPi was specific, EDTA was added at a selected time to chelate Mg2+
from the system. Experimental samples containing either nominal 0 (i.e. no Mg2+
added) or 1mM Mg2+
were split into two samples after 15 min incubation. While one sample retained the indicated Mg2+
concentration, the other received a concentration of EDTA calculated to decrease the level of Mg2+
to 0mM [21
]. As shown in , chelating Mg2+
by EDTA resulted in a decreased G6P hydrolysis rate as compared to the EDTA-free counterpart. The net change in G6P hydrolyzed, reported in the inset of , indicates a 20–25% increase in hydrolysis rate in the presence of 0.25mM Mg2+
vs. 0 mM Mg2+
at 30 and 45 min. Following EDTA addition, G6P hydrolysis was marked attenuated, reaching levels similar to those observed at 0mM Mg2+
in the absence of EDTA (Fig, 3A
). Similar experiments were performed using 1mM PPi as substrate, in the presence of 1mM Mg2+
, as PPi was maximal at this Mg2+
concentration. Also in this case, the addition of EDTA at a concentration sufficient to chelate Mg2+
level to 0mM resulted in a complete inhibition of PPi hydrolysis (, and relative inset). Similar inhibition of G6P and PPi hydrolysis were obtained adding EDTA at t = 0 min after quickly splitting the sample (not shown
G6P (Fig. 3A) and PPi (Fig. 3B) hydrolysis in the presence of varying Mg2+ concentration, in the absence or in the presence of EDTA
To exclude that the effect of Mg2+ on G6P or PPi hydrolysis was due to a leak of Mg2+ across the ER membrane over time, the amount of Mg2+ retained within the ER of digitonin permeabilized hepatocytes was measured by AAS. The values, reported in , indicate that Mg2+ content did not change significantly during our incubation procedure irrespective of the absence or the presence or EDTA or the amount of Mg2+ added to the system.
Changes in the amount of Mg2+ retained within the hepatocytes following addition of digitonin, EDTA, taurocholate, histone 2A, alamethicin, or A23187.
The next series of experiments was designed to determine whether Mg2+ effect was at the level of G6P transport mechanism, or intra-ER Mg2+ concentration was involved in the process. Taurocholate was used to permeabilize the ER membrane and allow G6P to access the hydrolytic site bypassing the G6P transport mechanism. Following addition of taurocholate a loss of Mg2+ from the ER lumen larger than that observed in digitonin-only treated cells was observed (). Under these conditions, the effect of 0.25mM extra-reticular Mg2+ on G6P hydrolysis was completely abolished (). Similar results were obtained when 5mM mannose 6-phosphate (M6P) instead of G6P () were used as alternative substrates.
G6P (Fig. 4A) and M6P (Fig. 4) hydrolysis in the presence of varying Mg2+ concentrations
Histone 2A [19
] and alamethicin [9
] have been used in alternative to taurocholate or deoxycholate to enhance permeability of ER membrane to G6P bypassing the transport mechanism. The effect of either of these agents on G6P hydrolysis is reported in . As the figure illustrates, in the presence of alamethicin (3μg/ml) or histone 2A (250μg/ml or isodose vs. the protein content in the incubation system, [9
]) G6P hydrolysis in the presence of 0.25mM extra-reticular Mg2+
was significantly lower than that observed in cells permeabilized only with digitonin. Following the addition of alamethicin or histone 2A, a similar amount of Mg2+
was lost from the ER (). No additional Mg2+
loss was observed when concentrations of histone 2A or alamethicin larger than those indicated above were used. When the Mg2+
ionophore A23187 was used to decrease intra-luminal Mg2+
] without altering ER permeability to G6P, the amount of Mg2+
retained within the hepatocyte was comparable to that measured following administration of histone 2A or alamethicin (). Under these conditions, G6P hydrolysis was only partially stimulated (plus 13%) by the addition of 0.25mM extra-reticular Mg2+
as compared to the hydrolysis occurring in the presence of 0mM Mg2+
(). This trend did not reach statistical significance and was further abolished in the presence of EDTA to levels similar to those observed with alamethicin or histone 2A ().
G6P hydrolysis in the presence of varying histone 2A or alamethicin
Perturbation of endo-luminal Mg2+ content by taurocholate, alamethicin or A23187 also abolished the effect of extra-reticular Mg2+ (1mM) on PPi hydrolysis (not shown).