Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland
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ABSTRACT |
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We previously reported that glucose can be released from GLUT2-null hepatocytes through a membrane traffic-based pathway issued from the endoplasmic reticulum. Here, we further characterized this glucose release mechanism using biosynthetic labeling protocols. In continuous pulse-labeling experiments, we determined that glucose secretion proceeded linearly and with the same kinetics in control and GLUT2-null hepatocytes. In GLUT2-deficient hepatocytes, however, a fraction of newly synthesized glucose accumulated intracellularly. The linear accumulation of glucose in the medium was inhibited in mutant, but not in control, hepatocytes by progesterone and low temperature, as previously reported, but, importantly, also by microtubule disruption. The intracellular pool of glucose was shown to be present in the cytosol, and, in pulse-chase experiments, it was shown to be released at a relatively slow rate. Release was not inhibited by S-4048 (an inhibitor of glucose-6-phosphate translocase), cytochalasin B, or progesterone. It was inhibited by phloretin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and low temperature. We conclude that the major release pathway segregates glucose away from the cytosol by use of a membrane traffic-based, microtubule-dependent mechanism and that the release of the cytosolic pool of newly synthesized glucose, through an as yet unidentified plasma membrane transport system, cannot account for the bulk of glucose release.
gluconeogenesis; hepatic glucose output; intracellular traffic; glucose-6-phosphatase
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INTRODUCTION |
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GLUCOSE RELEASE FROM HEPATOCYTES is an essential physiological function that is activated in the fasted state to prevent development of hypoglycemia. In diabetes, this function becomes progressively insensitive to inhibition by insulin and therefore contributes to increased hyperglycemia. Hepatic glucose production can result from activation of two metabolic pathways, glycogenolysis or gluconeogenesis, that converge at the level of glucose 6-phosphate (G-6-P) production. Conversion to glucose then requires G-6-P to enter the endoplasmic reticulum (ER), a process catalyzed by a glucose-6-phosphate translocase (4), followed by hydrolysis to glucose and phosphate by the membrane-associated glucose-6-phosphatase, whose catalytic site is located inside the ER lumen (9, 13). Release of glucose outside the cells has been classically viewed as involving diffusion of glucose back into the cytosol and transport across the plasma membrane by the glucose transporter GLUT2 (15). Recently, we reported that, in the absence of this transporter, glucose could, however, still be released at a normal rate, even though facilitated diffusion of 3-O-methylglucose (3-MG) across the plasma membrane was reduced by >95% (5). We presented evidence that glucose release in the absence of GLUT2 could rely on a membrane traffic mechanism issued from the ER. This pathway was characterized by its sensitivity to low temperature and to the acute effect of progesterone. It was, however, insensitive to cytochalasin B or inhibitors of the classical intracellular transport pathway, brefeldin A and monensin (see Fig. 9). Importantly, this membrane traffic pathway appeared to coexist with the GLUT2-dependent pathway in normal hepatocytes.
The conclusions about the existence of a membrane traffic pathway were thus inferred from the observation that release of neosynthesized glucose could be inhibited by low temperature and progesterone and on the parallel demonstration that facilitated diffusion of 3-MG across the plasma membrane was strongly reduced. However, the formal possibility remained that glucose could still be released by a plasma membrane carrier specific for glucose and unable to transport 3-MG (5).
In the present study, we investigated the mechanisms of glucose release
from control and GLUT2-deficient [GLUT2(/
)] hepatocytes by means
of a glucose biosynthetic labeling protocol. We demonstrate that, in
the absence of GLUT2, there is an intracellular accumulation of glucose
even though there is a constant release of glucose into the culture
medium. We show that the rate of glucose release during a continuous
labeling experiment can be impaired by microtubule disruption in
addition to inhibition by progesterone and low temperature. We further
show that the intracellular pool of glucose is located in the cytosol
and is released from the cells with much slower kinetics than the bulk
of newly synthesized glucose. The release in the culture medium of
cytosolic glucose does not require return to the ER but is by a
mechanism that requires ATP production and could be inhibited by
phloretin. There are therefore two pathways for glucose release from
GLUT2(
/
) hepatocytes, a major one that segregates glucose away from
the cytosol and a minor one that probably involves glucose diffusion
across the plasma membrane by a low-affinity transport mechanism but
which cannot account for the rapid rate of glucose output. These data
therefore further support the proposal that hepatic glucose release is
mostly by a membrane traffic-based pathway.
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MATERIAL AND METHODS |
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Materials.
The glucose-6-phosphate translocase inhibitor S-4048 was a generous
gift of Dr. A. W. Herling (Aventis, Frankfurt, Germany). Progesterone was purchased from ICN (Eschwege, Germany). Calyculin A
was purchased from Calbiochem (Darmstadt, Germany). Jasplakinolide was
purchased from Molecular Probes (Eugene, OR). Phloretin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP),
cytochalasin B, streptolysin O, colchicine, methyl--cyclodextrin,
and nocodazole were purchased from Sigma. [14C]pyruvate
was purchased from New England Nuclear (Boston, MA). All other
chemicals were of reagent grade.
Animals.
RIPGLUT1 × GLUT2(/
) mice were from our own colony (6,
16). As control animals, we used wild-type C57BL/6J mice
purchased from BRL (Basel, Switzerland).
Hepatocyte preparation. Livers of 8-wk-old mice were perfused through the inferior vena cava with a buffer consisting of (in mM) 140 NaCl, 2.6 KCl, 0.28 Na2HPO4, 5 glucose, and 10 HEPES (pH 7.4). The perfusion was first for 5 min with the buffer supplemented with 0.1 mM EGTA and then for 15 min with the buffer containing 5 mM CaCl2 and 0.2 mg/ml collagenase type 2 (Worthington, Lakewood, NJ). All of the solutions were prewarmed at 37°C and gassed with a mixture of 95% O2-5% CO2, resulting in a pH of 7.4. The isolated hepatocytes were filtered on nylon mesh (0.75 µm in diameter), washed two times with the above-mentioned buffer without collagenase, and suspended in a small volume of DMEM (GIBCO, Rockville, MD) without glucose or pyruvate and counted. The viability of hepatocytes was measured by Trypan blue staining. The preparations with viability <90% were discarded.
Biosynthetic labeling. For pulse-labeling experiments, hepatocytes (7.5 × 105) were incubated at 37 or 12°C in 0.5 ml of DMEM containing 1 mM pyruvate, 0.24 mM 3-isobutyl-1-methylxanthine (IBMX), and 0.05 µCi of [14C]pyruvate in the presence of either various inhibitors or vehicle. Incubations were stopped by placing the cells on ice followed by centrifugation at 4°C for 60 s at a speed of 1,000 rpm. The supernatant was removed, and the cells were lysed in 0.2% sodium deoxycholate. An aliquot was kept for protein determination (bicinchoninic acid kit; Pierce, Rockville, IL), and the rest was used for determination of radioactivity.
For pulse-chase experiments, hepatocytes were pulse labeled for 15 or 30 min as described above. They were then washed two times using 1-min centrifugations and were returned to 0.5 ml of DMEM containing 1 mM pyruvate and 0.24 mM IBMX in the presence of various inhibitors or vehicle, but without [14C]pyruvate, and kept for the indicated periods of time at 37°C.[14C]glucose measurement and analysis. [14C]glucose was separated from charged metabolites by passage of lysates or supernatants on anion and cation exchangers (Dowex AG1-X8 and 50W-X8, respectively; Bio-Rad, Hercules, CA) exactly as described (5). Radioactivity was measured by liquid scintillation counting (Tri-Carb 2100 TR; Packard Bioscience).
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RESULTS |
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We previously showed that the kinetics of release of newly
synthesized glucose was similar in control and GLUT2-null hepatocytes (5). Here, to more precisely characterize the release
pathways, we performed biosynthetic labeling experiments. Freshly
isolated hepatocytes obtained from 24-h-fasted mice were incubated in
the presence of [14C]pyruvate for different periods of
time, and the newly synthesized [14C]glucose secreted by
the cells or remaining intracellular was then quantitated. Figure
1A shows that, in control
hepatocytes, the newly synthesized glucose is released in the cell
supernatant at a constant rate (~6 nmol · mg
protein1 · h
1) and that there is no
intracellular accumulation of glucose. In contrast, in GLUT2(
/
)
mice (Fig. 1B), there is an accumulation of glucose inside
the cells, which reaches a plateau after ~30 min. Release of glucose
in the culture medium, however, increases linearly over the time of the
experiment at a rate that is similar to that observed in control
hepatocytes. Figure 1C shows the same data expressed as a
percentage of total glucose produced at 2 h.
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We next attempted to interfere with the secretion of glucose in the
GLUT2(/
) hepatocytes by using drugs known to interfere with
vesicular trafficking. In continuous 1-h pulse-labeling experiments, and as previously reported, progesterone reduced glucose release by
~50% and incubation of the cells at low temperature (12°C) by 70%
(Table 1). In the
presence of progesterone, glucose release from control hepatocytes was
not affected (data not shown and Ref. 5).
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It has been reported that capacitative Ca2+ entry after
depletion of the ER Ca2+ stores may involve a direct
connection between the ER and the plasma membrane. This was
demonstrated, in particular, by showing that actin overpolymerization
by jasplakinolide or calyculin A could block this Ca2+
entry by forming a tight microfilament network below the plasma membrane (10, 11). We therefore tested the effect of both jasplakinolide and calyculin A on glucose release. Even though we could
confirm the action of these molecules on induction of actin
polymerization by phalloidin-Alexa red staining, no inhibition of
glucose release could be observed (Table 1). We also evaluated whether
depleting the cells in cholesterol could reduce glucose release. This
was based on the premise that transport of newly synthesized
cholesterol from the ER to the plasma membrane may proceed through the
same pathway as glucose release, since transport of both cholesterol
and glucose is reduced by low temperature and progesterone treatment
(5, 14, 17). Thus cholesterol could have been involved in
this vesicular traffic pathway. Incubation of the cells in the presence
of methyl--cyclodextrin at concentrations that markedly reduce
cellular cholesterol content, (12) however, did not reduce
the rate of glucose release. Finally, experiments were carried out to
evaluate the role of microtubules in glucose release. As shown in Table
1, colchicine reduced the rate of secretion by ~15% and nocodazole
by ~30%.
To evaluate whether the membrane traffic-based and GLUT2-dependent
pathways could be evidenced in control hepatocytes, pulse-labeling experiments were carried out for 30 min in the presence of nocodazole and/or cytochalasin B. Figure 2 shows
that the release of [14C]glucose from control hepatocytes
could be reduced significantly by nocodazole (~15% reduction) and
the GLUT2 inhibitor cytochalasin B (~28% reduction). Importantly, a
combination of cytochalasin B and nocodazole produced an additive
inhibitory effect (~48% reduction), suggesting that both pathways
indeed coexist in normal hepatocytes.
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Next, we studied the rate of release of the pool of glucose that
accumulates inside the GLUT2(/
) hepatocytes and whether we could
interfere pharmacologically with this release. For these experiments,
freshly isolated hepatocytes were pulse labeled with [14C]pyruvate for 30 min, washed, and then returned to a
culture medium containing an excess of cold pyruvate. Intracellular and secreted [14C]glucose was then quantitated. Figure
3 shows that the rate of glucose release
is relatively slow, with a half-time of ~30 min and an absolute rate
of ~0.25 nmol · mg
protein
1 · h
1. The glucose leaving
the cells during this experiment was quantitatively recovered in the
supernatant (Fig. 3), indicating that the decrease in intracellular
glucose was the result of secretion and not of metabolism. This rate of
release is slower than that measured during continuous pulse labeling
(6 nmol · mg
protein
1 · h
1). This therefore
suggested that the intracellularly accumulated [14C]glucose is not in an intermediate compartment of the
major glucose release pathway.
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On the basis of the model presented in Fig. 9, the intracellularly
accumulated glucose could be either in a membrane compartment in
transit between the ER to the plasma membrane or in the cytosol. If
this glucose were in a closed vesicular compartment, permeabilization of the plasma membrane at the end of the pulse labeling with
streptolysin O would preserve the retention of sugar in the cells. In
contrast, if this glucose were in the cytosol, it would diffuse out of
the cells rapidly. We therefore pulse labeled the cells for 15 min, exposed them to streptolysin O for 10 min at 37°C, and determined the
amount of [14C]glucose retained intracellularly and
present in the supernatant. The concentration of streptolysin O
used was the minimal concentration that led to a complete
permeabilization of the cells, as measured by Trypan blue staining and
by measuring the release of lactate dehydrogenase (data not shown).
Figure 4 shows that streptolysin O led to
the release of ~90% of intracellular [14C]glucose. The
same cellular depletion of glucose was observed when the cells were
permeabilized with Triton X-100 (0.1%), which should also permeabilize
the intracellular membrane. We take this as an indication that the
glucose that accumulates during this period of pulse labeling is
present in the cytosol.
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To evaluate whether intracellular glucose release required
phosphorylation into G-6-P, entry into the ER, and exit by a
membrane traffic pathway, we pulse-labeled the cells for 30 min and
chased them in the presence of S-4048, an inhibitor of the
glucose-6-phosphate translocase (1, 7). The data presented
in Fig. 5 show that there was no
inhibition of glucose release induced by S-4048. To check that the
concentration of inhibitor was effective in blocking the
glucose-6-phosphate translocase, we separately showed that
it completely inhibited [14C]glucose formation from
[14C]pyruvate (data not shown). If the ER-plasma membrane
pathway was used, it should also have been inhibited by progesterone
treatment. Figure 6 shows that release
was not affected by incubation with progesterone. These data therefore
suggested that the intracellularly accumulated glucose was released by
a mechanism that did not require phosphorylation and entry into the ER.
It rather involved diffusion through the plasma membrane.
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To determine whether the release of accumulated
[14C]glucose involved passive diffusion across the plasma
membrane or diffusion through a membrane transporter with low affinity
for glucose, we tested the effect of transport inhibitors.
Pulse-labeled hepatocytes were therefore incubated during the chase
period with cytochalasin B, a specific inhibitor of glucose
transporters, or phloretin, an inhibitor of several types of
transporters, including glucose and monocarboxylate transporters.
Figure 7, A and B,
shows that cytochalasin B did not affect the rate of glucose efflux,
whereas phloretin significantly slowed down this release.
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To evaluate whether energy was required for this release, we inhibited
oxidative phosphorylation by the uncoupler FCCP during the chase period
and measured the rate of glucose release. Figure 8A shows that FCCP
significantly reduced the rate of glucose release. Finally, we
evaluated the effect of low temperature incubation (12°C) on the rate
of glucose release. Figure 8B shows that glucose release was
strongly suppressed at this temperature.
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DISCUSSION |
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This study presents novel evidence that glucose release from
hepatocytes in the absence of GLUT2 proceeds differently than in its
presence. Our pulse-labeling experiments show that neosynthesized glucose can be secreted at a constant high rate but that there is also
formation of an intracellular pool of glucose. Extracellular release of
this intracellular glucose pool proceeds at a slower rate than release
of the bulk of glucose and is sensitive to different pharmacological
interference. Our data are compatible with the existence of a major
pathway for glucose release that compartmentalizes glucose away from
the cytosol and that is based on a membrane traffic mechanism.
Separately, a smaller pool of cytosolic glucose is formed that can be
released slowly from the cells, probably by a facilitated diffusion
process through the plasma membrane, but that is unable to account for
the rapid rate of glucose output from the hepatocytes. This is shown in
Fig. 9.
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A major difference in the release of glucose from control and
GLUT2(/
) hepatocytes is revealed by our continuous pulse-labeling experiment. Although no accumulation of glucose in the cytosol can be
observed in the control cells, in the absence of GLUT2, the
neosynthesized glucose is found predominantly inside the cell for ~30
min, and then the intracellular pool remains constant for the rest of
the pulse labeling. At the same time, however, a linear accumulation of
glucose is measured in the supernatant. Because there is no delay in
appearance of glucose in the culture medium, this suggests that the
intracellular pool of glucose does not represent the filling of an
obligatory intermediate compartment in the normal glucose secretory
pathway; it may be a side compartment not involved in the major release pathway.
We previously presented evidence that the major pathway for glucose
release in GLUT2(/
) hepatocytes was through membrane traffic issued
from the ER and reaching the plasma membrane without transiting through
the Golgi complex (5). We showed that this pathway was
sensitive to low temperature (12°C) and to the acute effect of
progesterone, two conditions that also slow down the appearance of
newly synthesized cholesterol to the plasma membrane (14,
17). Here, to get further evidence for the involvement of a
membrane traffic mechanism, we evaluated the effect of substances interfering with actin and microtubule polymerization. The
polymerization of the microfilament was stimulated by jasplakinolide or
the phosphatase inhibitor calyculin A. However, in contrast to the
interference of this treatment with capacitative Ca2+ entry
(10, 11), no impairment of glucose release could be observed. This may suggest that there is no direct interaction between
the ER and the plasma membrane. Alternatively, this may be because of a
relatively lower level of actin expression in hepatocytes compared with
the fibroblasts studied in the mentioned reports and, therefore, to an
insufficient density of the subplasma membrane microfilament mesh to
prevent an ER-plasma membrane interaction.
The effect of depolymerizing microtubules was, however, very
significant, reaching ~30% with nocodazole. Microtubule disruption has been shown in many situations to block membrane traffic and is
therefore an additional evidence that glucose release in GLUT2-(/
) hepatocytes is through a vesicular pathway. Similar treatment of
control hepatocytes with nocodazole reduced the rate of neosynthesized glucose release by ~15%. Inhibition of glucose transporter by cytochalasin B reduced glucose release by ~28%, and a combination of
both the membrane traffic and facilitated diffusion inhibitors reduced
release by ~48%. This therefore indicates that, in control hepatocytes, both pathways participate in glucose release.
The presence of an internal pool of glucose that reached a maximal level after ~30 min of pulse labeling provided the basis for subsequent studies to evaluate how it was released from the cells. First, we could demonstrate that this pool was most probably within the cytosol, since it could be completely released from the cells by streptolysin O permeabilization. Indeed, if it were partly in a membrane compartment en route to the plasma membrane, we would have expected it to remain associated with the cells after this treatment.
The kinetics of glucose release into the culture medium from this cytosolic pool, as studied in pulse-chase experiments, was much slower than the rate of glucose release measured in the continuous pulse. This indicates that this intracellular pool was released through a minor pathway distinct from that taken by the majority of glucose. This was further confirmed by assessing the sensitivity of its release to several compounds. First, blocking the glucose-6-phosphate translocase with S-4048 did not slow down this glucose release, indicating that there was no need for glucose reentry in the ER. The fact that progesterone also did not impair release further indicated that release did not follow the same path as the bulk of secreted glucose. Second, in contrast to the release of most of the glucose, release of the intracellular pool could be blocked by phloretin, FCCP, and low temperature but not cytochalasin B. This further indicated a differential pharmacological sensitivity of this minor release pathway. It also supported the hypothesis that release was not by nonspecific diffusion across the plasma membrane but involved some specific membrane component. The sensitivity to phloretin and to energy depletion could suggest the possible involvement of a membrane protein belonging to the class of ATP-binding cassette (ABC) transporters. Importantly, because the kinetics of glucose release from the cytosol of GLUT2-null hepatocytes are not rapid enough to account for the bulk of glucose output, this therefore excludes the possibility that glucose is released by an as yet uncharacterized plasma membrane carrier that would be specific for glucose and unable to transport 3-MG (see Introduction).
Our previous study on hepatic glucose metabolism in GLUT2(/
) mice
during the fed-to-fasted transition indicated that the abnormal control
of glycogen metabolism and paradoxical regulation of glucose-sensitive
genes was associated with, and partly caused by, persistently elevated
intracellular G-6-P levels (2). We postulated
that this G-6-P originated from continuous phosphorylation of glucose reentering the cytosol from the ER lumen and on the way back
to the ER, thereby forming a futile cycle. Our present data, however,
show that the cytosolic pool of glucose issued from hydrolysis of
G-6-P in the ER leaves the cells without reentering the ER.
This suggests that, even though part of the cytosolic glucose could be
phosphorylated back to G-6-P, it may not return to the ER
lumen. This could be possible if distinct pools of G-6-P existed with different capability to eventually enter the ER. This is
actually compatible with a previous report by Christ and Jungermann
(3), demonstrating the existence of separate pools of
G-6-P issued from gluconeogenesis and glycogenolysis.
Together these data bring important new information about the mechanism by which glucose is released from hepatocytes when GLUT2 is no longer present. Two pathways can be identified. The minor one consists of the release of a cytosolic pool of glucose by diffusion through the plasma membrane by a mechanism catalyzed by a so far unidentified carrier. This pathway is relatively inefficient, as determined by its very slow kinetics. The major pathway is through a mechanism that compartmentalizes glucose away from the cytosol. It is probably based on vesicular traffic from the ER to the plasma membrane, which is sensitive to low temperature, progesterone, and microtubule depolymerization. This pathway is sufficient for the rate of liver glucose output in the fasted state to reach the same high level in control and mutant mice. This pathway coexists in normal hepatocytes with the GLUT2-dependent pathway. The relative contribution of each pathway in hepatic glucose output is not known. However, a recent study on the kinetics of glucose synthesis from dihydroxyacetone and its secretion from rat hepatocytes showed that the accelerating effect of glucagon on glucose release was mostly suppressed at 21°C. This could not be explained by changes in the activity of the involved enzymes (8). It was suggested that, instead, the temperature-sensitive effect of glucagon was on regulating the membrane traffic-based pathway we previously described. This pathway may therefore represent an important physiological target of glucagon action.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledged Dr. Kaethi Geering for critical reading of this manuscript.
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FOOTNOTES |
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This work was supported by Swiss National Science Foundation Grant 31-46958.96 to B. Thorens.
Address for reprint requests and other correspondence: B. Thorens, Institute of Pharmacology and Toxicology, 27, rue du Bugnon, Ch-1005 Lausanne, Switzerland (E-mail: Bernard.Thorens{at}ipharm.unil.ch).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00374.2001
Received 17 August 2001; accepted in final form 10 November 2001.
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