(Received for publication, February 6, 1995; and in revised form, July 12, 1995)
From the
Glucose transport was investigated in rat liver microsomes in
relation to glucose 6-phosphatase (Glu-6-Pase) activity using a fast
sampling, rapid filtration apparatus. 1) The rapid phase in tracer
uptake and the burst phase in glucose 6-phosphate (Glu-6-P) hydrolysis
appear synchronous, while the slow phase of glucose accumulation occurs
during the steady-state phase of glucose production. 2)
[C]Glucose efflux from preloaded microsomes can
be observed upon addition of either cold Glu-6-P or Glu-6-Pase
inhibitors, but not cold glucose. 3) Similar steady-state levels of
intramicrosomal glucose are observed under symmetrical conditions of
Glu-6-P or vanadate concentrations during influx and efflux
experiments, and those levels are directly proportional to Glu-6-Pase
activity. 4) The rates of both glucose influx and efflux are
characterized by t values that are independent of Glu-6-P
concentrations. 5) Glucose efflux in the presence of saturating
concentrations of vanadate was not blocked by 1 mM phloretin,
and the initial rates of efflux appear directly proportional to
intravesicular glucose concentrations. 6) It is concluded that glucose
influx into microsomes is tightly linked to Glu-6-Pase activity, while
glucose efflux may occur independent of hydrolysis, so that microsomal
glucose transport appears unidirectional even though it can be
accounted for by diffusion only over the accessible range of sugar
concentrations.
Given the peculiar localization of glucose 6-phosphatase
(Glu-6-Pase) ()(EC 3.1.3.9) into the endoplasmic reticulum,
the membrane topology of this enzyme has been controversial for many
years. While most authors agree that the enzyme is an integral protein
tightly associated with the hydrophobic part of the native membrane, as
confirmed recently by the amino acid sequence inferred from the
isolated cDNA encoding human (1) and mouse (2) Glu-6-Pases, there is no consensus to date as to whether
the catalytic site is oriented toward the lumen of the endoplasmic
reticulum or freely accessible from the cytoplasm. Two major but
opposite models have thus emerged during the last 20 years.
In the substrate transport model(3, 4, 5, 6, 7, 8) , it is thought that glucose 6-phosphate (Glu-6-P) is hydrolyzed to glucose and phosphate in the lumen of the endoplasmic reticulum. The enzyme is thus viewed as a multicomponent system involving at least five polypeptides subunits (4) among which T1, T2, and T3 would represent specific transporters for Glu-6-P, phosphate, and glucose, respectively. In support of the substrate-transport model are reports on the successful purification of T3 (6) as well as cloning of a cDNA encoding a 52-kDa glucose-transport protein (GLUT7) that has been assimilated to T3(7) . Since GLUT7 has been expressed in COS7 cells(7) , a cell line that does not normally contain significant amount of Glu-6-Pase activity(8) , those studies do not resolve the functional relationships between glucose transport and Glu-6-P hydrolysis. In the combined conformational flexibility-substrate transport model(9, 10, 11) , Glu-6-Pase traverses the microsomal membrane as a channel-forming protein embedded within the hydrophobic matrix of the bilayer, a concept that is indeed supported by the cloned sequences of the human (1) and murine (2) genes that predict up to six transmembrane segments in the secondary structure of the enzyme. In that model, the catalytic site is assumed to be located within a water-filled proteinaceous pore accessible to the substrate from the cytoplasmic surface of the membrane(11) .
In agreement with the conformational model,
our recent kinetic studies using a fast sampling, rapid filtration
apparatus (FSRFA, U. S. patent 07/697,769) have brought evidence for a
tight-coupling between Glu-6-P transport and Glu-6-Pase activity (12) but failed to demonstrate any significant transport of D-glucose into microsomes under zero-trans uptake conditions (13) . ()In the presence of
[
C]Glu-6-P in the incubation medium, however, we
also showed that tracer glucose accumulates within the intramicrosomal
space against a concentration gradient(12) . In the present
studies, we thus report on the kinetics of glucose transport into
microsomes in comparison with those of Glu-6-P hydrolysis by
Glu-6-Pase. Our results demonstrate that intramicrosomal glucose
accumulation is directly proportional to Glu-6-Pase activity, while
glucose efflux may occur independent of hydrolysis, so that microsomal
glucose transport appears unidirectional.
Figure 3:
Effect of vanadate on the kinetics of
glucose influx into, and efflux from microsomes. Influx and efflux
experiments were performed as described in the text using 0.2 mM Glu-6-P and either 0 (,
), 5, 10, 20 (
,
), 50, or 100 µM vanadate (
,
).
Influx experiments have been performed following 1-min preincubation
with the respective concentrations of vanadate. Arrow indicates the time of vanadate additions for efflux experiments.
For the sake of clarity, only three out of the seven uptake curves and
two out of the six efflux curves are shown with their respective
regression lines. Each point is the mean of four determinations using
the same microsomal preparation. The error structure followed a
proportional distribution with mean S.D. of 5.8 ±
2.0%.
As shown previously(12, 16) , total glucose production (P) is characterized by a burst phase over an initial period of about 10 s followed by a steady-state phase that proceeds linearly for up to 120 s. Such data can be fitted to
in which V and V
are the steady-state and initial burst rates of glucose
formation, respectively,
is the inverse of the time constant for
the hysteretic transition, and P
represents the background in radioactivity. As validated in
a previous report(12) , V
can be
determined independently in parallel experiments using microsomes
permeabilized with 0.4% deoxycholate, and its value was fixed into during the fitting process in order to get more reliable
estimations of
at varying Glu-6-P concentrations.
As discussed and justified in a previous report(12) , tracer accumulation (A) into microsomes is best described by the double exponential
in which A represents the
background in radioactivity while A
and A
stand for the steady-state levels of tracer
accumulation reached with first-order rate constants k
and k
, respectively. Since k
is 1 order of magnitude larger than k
(12) , the uptake time courses were first
fitted over the 10-240-s time range using
in order to determine the parameters A, A
, and k
. Those values were
then used as prompted constants into when analyzing the
whole time range. Amplitudes and initial rates of accumulation during
the fast (AMP
, V
) and slow
(AMP
, V
) phases were calculated as
shown in and .
The kinetic parameters pertaining to the rate and amplitude data (y) were determined from the analysis of the respective displacement curves of tracer (T, 0.2 mM tracer Glu-6-P taken as a reference) by cold substrate (S) according to
which applies to a saturable phenomenon working in parallel with
a nonspecific component(s)(17, 18) . In that equation, C represents the background value (undisplaceable tracer),
while Y and K
stand for the maximum rate (V
) or
amplitude (AMP
) and the Michaelis-Menten constant of the
saturable process, respectively.
The inhibition constants (K) for vanadate (I) were
estimated using
which assumes competitive inhibition relative to Glu-6-P (S, 0.2 mM in those experiments)(19) , and
where y and y stand for the
inhibited and uninhibited rates or amplitudes, respectively, while K
has the same meaning as above and can
be fixed to the values determined in the corresponding
data(20) .
Efflux (E) data were analyzed according to Equations 8 and 9, which assume either single or double exponential decay, respectively,
and where E represents the
starting level while E
and E
stand for the steady-state levels of intramicrosomal tracer
reached with first-order rate constants k
and k
, respectively. The initial efflux rates (V
) shown in Fig. 5B were
calculated from using .
Figure 5:
Efflux kinetics as a function of
intramicrosomal glucose concentrations. As in Fig. 1(closedsymbols) for influx experiments, microsomes were
preincubated for 2 min at various [C]Glu-6-P
concentrations before the addition of 200 µM vanadate. The
time courses of efflux were monitored for an additional 2 min. The
intramicrosomal glucose concentrations, and both the t for
efflux (A) and the initial rates of efflux (B) were
determined as described in the text. Lines shown correspond to
the linear regression analysis of the data points ±
S.E.R.
Figure 1:
Time course of
[C]glucose accumulation into and efflux from
microsomes. Influx and efflux experiments were performed as described
in the text in the presence of final concentrations of 0.2 (
),
0.5, 1, 2 (
,
), 5, and 10 mM Glu-6-P (
,
). Alternatively, 50 mM cold glucose (
) was added
under efflux conditions. Arrow indicates the time of effector
additions for efflux experiments. For the sake of clarity, only three
out of the six uptake curves and two out of the five efflux curves are
shown with their respective regression lines. Each point represents the
mean of five determinations using the same microsomal preparation. The
error structure followed a proportional distribution with mean S.D. of
7.9 ± 1.3%.
In this paper, we have chosen the parameter t, which represents the time at which 50% of a process has been completed, to characterize the rates of influx into, and efflux from microsomes. This parameter was calculated using .
All kinetic analyses were performed using the Enzfitter software (Elsevier-Biosoft) and nonlinear regression analyses to pertinent equations above were performed using the robust weighting routine in conjunction with the explicit weighting routine when appropriate. Accordingly, the errors associated with the kinetic parameters as given in the text and figures represent the standard errors of regression (S.E.R.) on these parameters.
Figure 2:
Effect of Glu-6-P on the kinetic
parameters of Glu-6-P hydrolysis and glucose uptake into microsomes. A, t values were calculated according to from the first order rate constants of the data shown in Fig. 1for the fast () and slow (
) phases of glucose
uptake, as well as for efflux (
). For the initial burst phase in
glucose production (
), rate constants were determined on the same
microsomal preparation using as described in the text. At
10 mM Glu-6-P, the low signal to noise ratio precluded the
determination of the t of the rapid phases of Glu-6-P
hydrolysis and glucose uptake. B, AMP
, the
steady-state level of intramicrosomal glucose reached during the slow
phase of tracer uptake (
) was determined from the data of Fig. 1as described in the text. Steady-state rates of tracer
Glu-6-P hydrolysis (
) were determined as described in the text
using the same microsomal preparation. Lines shown are the best-fit
lines to in the text that reflects the apparent
competitive inhibition by carrier
dilution(17, 18, 20) . In both A and B, the errorbars represent S.E.R. values
and, when not shown, were smaller than the symbol
sizes.
The uptake time curves shown in Fig. 1(closedsymbols) clearly indicate, however, that the steady-state
levels of intramicrosomal tracer accumulation are affected by unlabeled
Glu-6-P concentrations. A close link between the steady-state rate of
tracer Glu-6-P hydrolysis (V) and the amplitude
of the slow phase of intramicrosomal tracer accumulation
(AMP
) is indeed suggested by the results of Fig. 2B showing similar K
values for both processes (1.37 ± 0.05 and 1.22
± 0.12 mM, respectively). Those results indicate, as
also argued previously (12) but demonstrated in the companion
paper(21) , that glucose is the main labeled species to be
found inside of the microsomes. In this respect, it is worth pointing
out that we failed to demonstrate directly tracer uptake from
[
C]Glu-6-P when incubated under zero-trans
conditions with liver microsomes isolated from a type 1a glycogen
storage disease patient(22) .
Figure 4:
Effect
of vanadate on the kinetic parameters of Glu-6-Pase activity and
glucose transport into microsomes. A, t values for
influx into () and efflux from (
) microsomes were calculated
according to from the first order rate constants of the
data shown in Fig. 3. t values for influx in the
absence of preincubation with vanadate (
) have been determined
from similar experiments performed in parallel on the same membrane
preparation. B, AMP
, the steady-state level of
intramicrosomal glucose accumulation reached during the slow phase of
tracer uptake (
) was determined from the data of Fig. 3as
described in the text. V
, the steady-state rates
of Glu-6-P hydrolysis (
), were determined as described in the
text using the same microsomal preparation. Lines shown are the
best-fit lines to equations discussed in the text. The errorbars represent S.E.R. values and, when not shown, were
smaller than the symbol sizes.
Next, [C]glucose-loaded microsomes were
exposed to vanadate concentrations varying from 0 to 100 µM in a 50 mM TRIS-HCl buffer (pH 7.3) containing 0.25 M sucrose. As shown in Fig. 3, buffer addition alone (opencircles) had no effect on the steady-state
level of intramicrosomal glucose accumulation. In contrast, vanadate
addition (opensquares and triangles) caused
glucose efflux from microsomes down to the same steady-state levels
that were achieved under the symmetrical zero-trans uptake experiments.
The efflux data can be fitted to only over the whole range
of inhibitor concentrations, and Fig. 4A (opencircles) demonstrates that the t values of
glucose efflux decreased from 197 ± 26 down to 47.5 ± 1.4
s for vanadate concentrations increasing from 10 up to 100
µM. The higher t values obtained after the
addition of low concentrations of vanadate (5-20 µM)
may be related to the relative inaccessibility of the inhibitor to the
catalytic site in intact microsomes(23) . In agreement with
that idea, the apparent t for glucose accumulation into
microsomes decreased from 35.2 ± 3.9 s in the absence of
inhibitor down to 8.5 ± 2.2 s at 100 µM vanadate in
zero-trans uptake experiments where the preincubation step with
vanadate was omitted (Fig. 4A, closedtriangles). Under these conditions, however, the
steady-state levels of intramicrosomal glucose accumulation and their
dependence on inhibitor concentrations were similar to those presented
in Fig. 4B for preincubated microsomes.
In a last series of experiments, it was observed that other classical inhibitors of Glu-6-Pase activity that act by mechanisms different from that of vanadate like 4,4`-diisothiocyanatostilbene-2,2`-disulfonic acid, NaCl, NaF, and phlorizin(19) , were also able to induce glucose efflux from glucose-loaded microsomes (data not shown).
The above results seem
to indicate that glucose equilibration through microsomes occurs by
diffusion only over a physiological range of glucose concentrations, in
contradiction with the recent suggestion that GLUT7 might be involved
in glucose transport through microsomal membranes(7) . Since it
has been shown that phloretin inhibits GLUT7 when expressed in COS7
cells(7) , we tested the effect of the concomitant addition of
1 mM phloretin and 200 µM vanadate on glucose
efflux from glucose-loaded microsomes upon preincubation for 2 min in
the presence of 0.2 mM [C]Glu-6-P. Not
only was phloretin unable to counteract glucose efflux under these
conditions but, furthermore, phloretin addition alone could cause
glucose efflux from microsomes (results not shown), thus acting like
any of the other Glu-6-Pase inhibitors tested in our previous
experiments. Indeed, phloretin has been shown to also inhibit
Glu-6-Pase activity(24) .
Finally, the question as to
whether glucose transport in microsomes may behave as a unidirectional
process was evaluated further as follows. Microsomes were first
preincubated for 2 min at 30 °C in the presence of 0.2 mM cold Glu-6-P and then exposed to a saturating concentration of
either 200 µM vanadate or 10 mM Glu-6-P in the
presence of 0.1 mMD-[C]glucose. These experiments failed
to detect any measurable glucose uptake (results not shown) under
conditions shown herein to reveal maximum rates of glucose efflux.
Glucose uptake into microsomes can be readily monitored in
the presence of zero-trans concentrations of
[C]Glu-6-P (Fig. 1, closedsymbols), and can be inhibited by vanadate (Fig. 3, closedsymbols) or saturating
concentrations of phlorizin(12) . Similarly, glucose efflux
from [
C]glucose-loaded microsomes can be
demonstrated following isotopic dilution by cold Glu-6-P (Fig. 1, opensymbols), inhibition of
Glu-6-Pase activity by vanadate (Fig. 3, opensymbols), or a series of Glu-6-Pase inhibitors acting by
different mechanisms. It should be stressed, however, that zero-trans
uptake of radiolabeled D-glucose could not be observed under
conditions where glucose produced from Glu-6-P hydrolysis readily
exchanges between the intravesicular space and the external medium ( (13) and this study). Accordingly, glucose influx into
microsomes appears tightly linked to Glu-6-Pase activity, while glucose
efflux may occur independently of hydrolysis so that glucose transport
appears unidirectional in the absence of enzyme activity.
The close
link between glucose transport and Glu-6-P hydrolysis is further
supported by the observations that the steady-state levels of
intramicrosomal glucose accumulation 1) follow Michaelis-Menten
kinetics relative to outside Glu-6-P concentrations with a K value similar to that of total glucose
production via Glu-6-P hydrolysis (Fig. 2B) and 2)
appear to be inhibited competitively relative to outside vanadate
concentrations with a K
value in the same
range as that obtained for inhibition of Glu-6-Pase activity (Fig. 4B). Accordingly, glucose accumulation from
[U-
C]Glu-6-P is directly proportional to
Glu-6-Pase activity and limited by the maximum rate of the enzyme.
The impermeability of microsomes to glucose in the inward direction
is further demonstrated by the absence of isotopic exchange between
intra- and extramicrosomal glucose following addition of an excess cold
glucose to [C]glucose-loaded microsomes. Under
similar conditions, however, addition of an excess cold Glu-6-P does
result in glucose exchange with the intravesicular space and leads to
faster glucose efflux (Fig. 2A, opencircles, mean t = 20.1 ± 2.0 s)
than observed upon addition of 100 µM vanadate (Fig. 4A, opencircles, t = 47.5 ± 1.4 s). On the other hand, a t value in the range of that found for influx (Fig. 2A, closed circles, t = 46.4
± 5.3) is observed for efflux (Fig. 5A, t = 37.3 ± 5.5 s) when initiated by the addition of a
saturating concentration of vanadate to
[
C]glucose-loaded microsomes. To understand
these results, one should remember that, contrary to the situation with
vanadate where efflux occurs at constant specific radioactivity of
inside glucose and outside Glu-6-P, tracer glucose efflux elicited by
the addition of cold Glu-6-P takes place under conditions of net
glucose influx, i.e. under conditions where the specific
radioactivity of the intramicrosomal glucose pool is continuously
changing with time. What is observed, then, is an apparent rate of
tracer efflux, and it can be predicted that its value would be twice as
fast as that recorded under net efflux conditions when assuming that
the same process of diffusion is involved for both tracer efflux and
cold glucose influx. Indeed, the results of Fig. 2A (opencircles, no change in the t for
efflux at increasing Glu-6-P concentrations) and 5B (no
saturation of the efflux rate over the analyzable range of
intramicrosomal glucose concentrations) do support such an
interpretation.
The conclusions that microsomal glucose transport is
unidirectional and occurs through diffusion deserve some comments.
First, unidirectionality of transport may only be apparent since, as
argued recently by Burchell(8) , ``direct measurement of
labeled glucose uptake into microsomes is difficult because the
transport is very rapid and relatively low number of counts are taken
up into the microsomal lumen.'' We do think, however, that
zero-trans glucose transport, if occurring, should be measurable in
that preparation; 1) glucose uptake can be readily measured in other
vesicle systems with similarly small intravesicular
volumes(17) ; 2) the problem of low number of counts can be
partly solved by using pure tracer concentrations of glucose with high
specific activity (18) (the sensitivity of the transport assay
in terms of measurable cpms would be maximum under those conditions,
see when S = 0); and 3) the rapidity of
the uptake process is readily overcome in our laboratory when using the
FSRFA. In agreement with those statements, it is worth emphasizing that
zero-trans uptake of inorganic phosphate could be successfully measured
in both rat and human liver microsomes(22) . Moreover, since
phosphate was taken up into an equivalent microsomal space of 1.2
µl/mg of protein(22) , thus closely matching that of 0.8
µl/mg of protein determined in previous studies using H
O(12) , it can be concluded further
that the use of the FSRFA does not damage a major fraction of the
vesicles such as to preclude significant zero-trans uptake
measurements. Accordingly, our failure to detect glucose uptake under
similar and appropriate experimental conditions can thus be taken as
evidence that glucose does not enter into a measurable intravesicular
space at any significant rate. We do not rule out, however, the
possibility that glucose may permeate the endoplasmic reticulum
membrane through passive diffusion with extremely low rate constant.
Next, diffusion may only be apparent since, in the low range of
substrate concentrations, a low affinity carrier or channel may not
show saturation kinetics (the apparent diffusion constant in that case
is equal to the V
/K
ratio of the transport protein, see with S and TK
). Unfortunately,
due to their low glucose permeability, it is not possible to passively
load microsomes in a reliable way at fixed glucose concentrations, and
thus to extend the range of sugar concentrations over which saturation
kinetics (or the lack thereof) might be observed in plots like those
shown in Fig. 5B. Accordingly, the upper range of
analyzable intravesicular glucose concentrations is fixed to
15-20 mM when active loading from
[
C]Glu-6-P is used, a value dictated by the V
of Glu-6-P hydrolysis by Glu-6-Pase. In that
respect, the slight deviation from linearity observed at 20 mM inside glucose in the plot of Fig. 5B should not
be taken as evidence for the beginning of the development of a
hyperbolic relationship because the low signal to noise ratio that is
achieved at close to complete saturation of the enzyme does not allow
for a precise estimate of the intramicrosomal glucose concentration.
Moreover, if one assumes that a transport protein like GLUT7 is present
within the microsomal membrane, we may expect 1) inhibition of glucose
efflux from microsomes by phloretin (7) , 2) accelerated efflux
of glucose from [
C]glucose-loaded microsomes
following addition of an excess cold glucose to the incubation medium,
3) zero-trans uptake of tracer glucose with t in the range of
30-50 s since the same V
/K
ratios should
be observed under influx and efflux conditions independent of whether
the transporter is symmetrical or not due to the law of microscopic
reversibility(25) , and 4) zero-trans uptake of tracer glucose
with overshoot phenomenon (25) in microsomes loaded with cold
glucose. Since none of these expectations could be demonstrated in our
experiments, we must conclude that glucose transport is indeed
unidirectional and may not involve GLUT7. Should a specific glucose
transport protein (T3) be present in native liver microsomes, however,
we do state that its kinetic properties are different from those
demonstrated for GLUT7 in COS7 cells(7) , maybe due to a close
association with a multicomponent enzyme
complex(4, 6, 8) . In any case, it should be
stressed that the model proposed in the companion paper (21) requires the presence of neither GLUT7 nor any other
glucose transport protein but for Glu-6-Pase in order to explain the
steady-state kinetics of Glu-6-P hydrolysis and glucose exchange
through the microsomal membrane.