Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway

Masaya Hosokawa and Bernard Thorens

Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 protein-1 · 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.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Kinetics of [14C]glucose synthesis and release from hepatocytes of control or GLUT2-deficient [GLUT2(-/-)] mice. Freshly isolated hepatocytes were incubated in the presence of 1 mM pyruvate and 0.05 µCi [14C]pyruvate for the indicated periods of time, and [14C]glucose present in the supernatants (SN) and cell was quantitated. A: control hepatocytes. There is no intracellular accumulation of glucose. B: GLUT2(-/-) hepatocytes. There is an accumulation of glucose inside the cells, which reaches its maximum at ~30 min. Glucose released in the medium proceeds at a constant rate. C: data in A and B are replotted as a percentage of total glucose produced at 2 h. Data are means ± SE for n = 4 experiments.

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of various inhibitors on [14C]glucose release measured at the end of the 60-min continuous pulse labeling

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-beta -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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Glucose is released from normal hepatocytes by both the GLUT2-dependent and membrane traffic-based pathways. Freshly isolated hepatocytes were incubated in the presence of 1 mM pyruvate and 0.05 µCi [14C]pyruvate for 30 min, and [14C]glucose present in the supernatants and cell lysate was quantitated. The pulse-labeling experiments were performed in the presence of cytochalasin B (cytoB; 50 µM), nocodazole, (50 µM), or a combination of both inhibitors. The presence of both inhibitors showed an additive inhibitory effect. Data are means ± SE for n = 6.

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.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Kinetics of release of intracellularly accumulated [14C]glucose. GLUT2(-/-) hepatocytes were pulse labeled for 30 min with [14C]pyruvate, washed, and returned to a nonradioactive culture medium. The amount of [14C]glucose remaining inside the cells or secreted in the cell culture medium was then measured at the indicated time and expressed as nmol/mg protein (prot). The total amount of glucose radioactively labeled remained constant during the time of the experiment. Data are means ± SE for n = 4.

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.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4.   The intracellular pool of glucose is located in the cytosol. GLUT2(-/-) hepatocytes were labeled for 15 min with [14C]pyruvate and incubated in control (Ctr) conditions with streptolysin O (SLO, 3,300 IU/ml in the presence of 1 mM dithiothreitol) or with 0.1% Triton X-100 (Tx) for 10 min. Intracellular glucose was then determined. Permeabilization of the plasma membrane (SLO) and the intracellular membranes (Tx) leads to an ~90% release of glucose. Data indicate intracellular glucose expressed as a percentage of total synthesized glucose. Data are means ± SE for n = 4.

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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   The intracellular pool of glucose is released without reentering the endoplasmic reticulum. GLUT2(-/-) hepatocytes were pulse labeled for 30 min with [14C]pyruvate, washed, and then chased in cold medium in the presence of the glucose-6-phosphate translocase inhibitor S-4048 (10 µM) for the indicated periods of time. Blocking the translocase did not reduce the rate of glucose release. Data indicate intracellular glucose expressed as a percentage of total synthesized glucose. Data are means ± SE for n = 4.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   Release of the intracellular store of glucose is not sensitive to progesterone. GLUT2(-/-) hepatocytes were pulse labeled for 30 min with [14C]pyruvate, washed, and then chased in cold medium in the presence of progesterone (10 µg/ml). No inhibition of glucose release could be observed. Data indicate intracellular glucose expressed as a percentage of total synthesized glucose. Data are means ± SE for n = 6.

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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7.   Release of the intracellular store of glucose is not sensitive to cytochalasin B (50 µM; A) but is impaired by phloretin (0.3 mM; B). GLUT2(-/-) hepatocytes were pulse labeled for 30 min with [14C]pyruvate, washed, and then chased in cold medium in the presence of the glucose transporter inhibitor cytochalasin B or the less specific carrier inhibitor phloretin for the indicated periods of time, and intracellular [14C]glucose was quantitated. Data indicate intracellular glucose expressed as a percentage of total synthesized glucose. Data are means ± SE for n = 4.

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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8.   The rate of intracellular glucose release is reduced by mitochondrial uncoupling (A) and low temperature (B). GLUT2(-/-) hepatocytes were pulse labeled for 30 min with [14C]pyruvate, washed, and then chased in cold medium in the presence of the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 30 µM) or at low temperature (12°C) for the indicated periods of time, and intracellular [14C]glucose was quantitated. Significant inhibition of release was observed in both conditions. Data indicate intracellular glucose expressed as a percentage of total synthesized glucose. Data are means ± SE for n = 4.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 9.   Glucose (Glc) release from hepatocytes. Gluconeogenesis and glycogen degradation pathways converge at the level of G-6-P production. G-6-P enters the endoplasmic reticulum (ER) to be hydrolyzed in glucose and phosphate, a mechanism that requires the presence of a glucose-6-phosphate translocase (G6T) and of glucose-6-phosphatase (G-6-Pase). Glucose release from the cells can take two major pathways that are both present in control hepatocytes as follows: 1) return of glucose into the cytosol for its release out of the cells by facilitated diffusion through GLUT2 and 2) release by a membrane traffic pathway, issued from the ER and reaching the plasma membrane without transiting through the Golgi complex. This pathway is sensitive to progesterone, low temperature (5), and microtubule depolymerization (present study). In the absence of GLUT2, facilitated diffusion across the plasma membrane is reduced by at least 95%, but the rate of glucose release from hepatocytes is the same as from control hepatocytes, indicating that route 2 can quantitatively replace route 1. Here we show that a part of the newly formed glucose reenters the cytosol to form a pool that can slowly diffuse out the cells (route 3). This minor release pathway does not involve reentry into the ER, since the G6T inhibitor S-4048 does not block it. It is, however, inhibited by phloretin, low temperature, and mitochondrial uncoupling by FCCP.

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.


    ACKNOWLEDGEMENTS

We gratefully acknowledged Dr. Kaethi Geering for critical reading of this manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arion, WJ, Canfield WK, Ramos FC, Su ML, Burger HJ, Hemmerle H, Schubert G, Below P, and Herling AW. Chlorogenic acid analogue S3488: a potent competitive inhibitor of the hepatic and renal glucose-6-phosphatase system. Arch Biochem Biophys 351: 279-285, 1998[ISI][Medline].

2.   Burcelin, R, Muñoz MC, Guillam MT, and Thorens B. Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway. J Biol Chem 275: 10930-10936, 2000[Abstract/Free Full Text].

3.   Christ, B, and Jungermann K. Sub-compartmentation of the "cytosolic" glucose-6-phosphate pool in cultured rat hepatocytes. FEBS Lett 221: 375-380, 1987[ISI][Medline].

4.   Gerin, I, Veiga-da-Cunha M, Achouri Y, Collet J-F, and Van Schaftingen E. Sequence of a putative glucose 6-phosphate translocase, mutated in glycogen storage disease type Ib. FEBS Lett 419: 235-238, 1997[ISI][Medline].

5.   Guillam, MT, Burcelin R, and Thorens B. Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes. Proc Natl Acad Sci USA 95: 12317-12321, 1998[Abstract/Free Full Text].

6.   Guillam, MT, Hümmler E, Schaerer E, Yeh JY, Birnbaum MJ, Beermann F, Schmidt A, Dériaz N, and Thorens B. Early diabetes and abnormal postnatal pancreatic islet development in mice lacking GLUT2. Nat Genet 17: 327-330, 1997[ISI][Medline].

7.   Hemmerle, H, Burger HJ, Below P, Schubert G, Rippel R, Schindler PW, Paulu E, and Herling AW. Chlorogenic acid and synthetic chlorogenic acid derivatives: Novel inhibitors of hepatic glucose-6-phosphate translocase. J Med Chem 40: 137-145, 1997[ISI][Medline].

8.   Ichai, C, Guignot L, El-Mir MY, Nogueira V, Guigas B, Taine E, Mithieux G, and Leverve XM. Glucose-6-phosphate hydrolysis is activated by glucagon in a low temperature-sensitive manner. J Biol Chem 276: 28126-28133, 2001[Abstract/Free Full Text].

9.   Mithieux, G. New knowledge regarding glucose-6-phosphatase gene and protein and their roles in the regulation of glucose metabolism. Eur J Endocrinol 136: 137-145, 1997[ISI][Medline].

10.   Patterson, RL, van Rossum DB, and Gill DL. Store-operated Ca++ entry: evidence for a secretion-like coupling model. Cell 98: 487-499, 1999[ISI][Medline].

11.   Putney, JW. "Kissin' cousins": intimate plasma membrane-ER interactions underlie capacitative calcium entry. Cell 99: 5-8, 1999[ISI][Medline].

12.   Rodal, SK, Skretting G, Garred O, Vilhardt F, van Deurs B, and Sandvig K. Extraction of cholesterol with methyl cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell 10: 961-974, 1999[Abstract/Free Full Text].

13.   Shelly, LL, Lei KJ, Pan CJ, Sakata SF, Ruppert S, Schutz G, and Chou JY. Isolation of the gene for murine glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a. J Biol Chem 268: 21482-21485, 1993[Abstract/Free Full Text].

14.   Smart, EJ, Ying YS, Donzell WC, and Anderson RGW A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane. J Biol Chem 271: 29427-29435, 1996[Abstract/Free Full Text].

15.   Thorens, B. Glucose transporters in the regulation of intestinal, renal and liver glucose fluxes. Am J Physiol Gastrointest Liver Physiol 270: G541-G553, 1996[Abstract/Free Full Text].

16.   Thorens, B, Guillam MT, Beermann F, Burcelin R, and Jaquet M. Transgenic reexpression of Glut1 or Glut2 in pancreatic beta  cells rescues Glut2-null mice from early death and restores normal glucose-stimulated insulin secretion. J Biol Chem 275: 23751-23758, 2000[Abstract/Free Full Text].

17.   Urbani, L, and Simoni RD. Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane. J Biol Chem 265: 1919-1923, 1990[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 282(4):E794-E801
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society