(Received for publication, July 8, 1996, and in revised form, April 1, 1997)
From the Institut National de la Santé et de la
Recherche Médicale U.129, Institut Cochin de
Génétique Moléculaire, Université René
Descartes, 75014 Paris, France, the
Centre de Recherche sur
l'Endocrinologie Moléculaire et le Développement, CNRS, 9 rue Jules Hetzel, 92190 Meudon, France, and the ** Institut National de
la Santé et de la Recherche Médicale U.246, Institut
Fédératif de Recherche, Faculté de Médecine
Xavier Bichat, B.P.416, 75018 Paris, France
Twenty-six different hepatoma cell lines
established from cancer-prone transgenic mice exhibited a close
correlation between expression of the GLUT 2 glucose transporter and
activation of the L-type pyruvate kinase (L-PK) gene by glucose,
as judged by Northern blot analyses and transient transfection assays.
The L-PK gene and a transfected L-PK construct were silent in GLUT 2(+)
cells and active in GLUT 2() cells cultured in glucose-free medium.
Transfection of GLUT 2(
) cells with a GLUT 2 expression vector
restored the inducibility of the L-PK promoter by glucose, mainly by
suppressing the glucose-independent activity of this promoter. Culture
of GLUT 2(
) cells, in which the L-PK gene is constitutively
expressed, in a culture medium using fructose as fuel selected GLUT
2(+) clones in which the L-PK gene responded to glucose.
The expression of the L-PK gene in GLUT 2() cells cultured in the
absence of glucose was correlated with a high intracellular glucose
6-phosphate (Glu-6-P) concentration while under similar culture
conditions Glu-6-P concentration was very low in GLUT 2(+) cells.
Consequently, a role of GLUT 2 in the glucose responsiveness of
glucose-sensitive genes in cultured hepatoma cells could be to allow
for Glu-6-P depletion under gluconeogenic culture conditions. In the
absence of GLUT 2, glucose endogeneously produced might be unable to be
exported from the cells and would be phosphorylated again to Glu-6-P by
constitutively expressed hexokinase isoforms, continuously generating
the glycolytic intermediates active on the L-PK gene transcription.
Glucose is an important regulator of gene transcription in most
prokaryotic and eukaryotic species. In vertebrates, it modulates the
expression of genes for enzymes involved in metabolic regulation in the
liver and adipose tissue and of insulin in cells of the endocrine
pancreas (1). In these three glucose-sensitive tissues, glucose
transport and phosphorylation are performed by various isoforms of
glucose transporters and hexokinases, respectively. Glucose
phosphorylation to glucose 6-phosphate, required for glucose action on
the transcriptional machinery (2-6), is mainly mediated by hexokinase
(HK)1 II in adipocytes and by hexokinase IV
(or glucokinase) in the liver and
cells; expression of the
glucokinase gene is constitutive in the pancreas and
insulin-dependent in the liver due to the existence of two
different tissue-specific alternative promoters (7). However,
glucokinase is replaced by other insulin-independent hexokinase
isoforms (mainly HK I) in cultured hepatoma cell lines (2, 8, 9).
Tissues whose function is regulated by glucose also possess particular
glucose transporters, GLUT 4 in adipocytes and GLUT 2 in tissues
secreting glucose into the blood (liver, small intestine, and proximal
tubular cells of the kidney) as well as GLUT 2 in the
cells of the
islets of Langerhans (10, 11). In pancreas
cells, it has been
suggested that GLUT 2 and glucokinase are essential components of the
"glucose sensor" (12, 13). However, the mechanisms of the GLUT
2-specific role in the regulation of insulin secretion by glucose
remain unclear (13, 14).
In most hepatoma cell lines in culture, glucose responsiveness of glucose-dependent genes is lost (15) even when these cells remain well differentiated (2). In fact, expression of glucose-responsive genes is generally constitutive in these cells in which glucokinase is replaced by HK I and GLUT 2 is replaced by GLUT 1, another glucose transporter isoform expressed in most cells not specialized in metabolism regulation, e.g. in cancerous cells (8, 16).
However, we recently succeeded in isolating various well differentiated cell lines that have conserved sensitivity of their transcriptional machinery to glucose (2). Here, we show that glucose responsiveness of the L-PK gene in these cells requires synthesis of GLUT 2, and we suggest that GLUT 2, but not GLUT 1, allows for efflux of glucose endogenously produced by the gluconeogenic pathway when cells are cultured without glucose. Therefore, in the absence of GLUT 2, glucose-responsive genes would be permanently stimulated by glycolytic intermediates of endogeneously produced glucose.
mhAT and mhPKT hepatoma cell lines were derived from transgenic mice synthesizing the SV40 large T and small t antigens in the liver under the direction of either the antithrombin III gene or L-PK gene regulatory sequences (17, 18). Hepatocytes were obtained from transgenic animals of different ages with either normal, fetal (mhPKTf3), or tumoral (mhPKT, mhAT1, and mhAT3) livers and established in culture as described previously (19-21). About 30 mhAT1- and mhAT3-derived subclones were isolated using different selective media, particularly containing fructose as the only carbohydrate source (19, 20). Lines mhPKTf3F1 and F2 were subcloned from mhPKTf3 cells by using such a fructose medium. All hepatoma cell lines were usually grown in Ham's F12/Dulbecco's modified Eagle's medium (v/v), Glutamax medium (Life Technologies, Inc., Chagrin Falls, OH) supplemented with penicillin, streptomycin, 0.1 µM insulin, 1 µM dexamethasone, 1 µM triiodothyronine, and 5% (v/v) fetal calf serum.
Glucose Induction StudiesFor the glucose induction
studies, cells were cultured in a serum-free medium supplemented with
10 µg/ml transferrin and 100 µg/ml albumin. In general, cells were
cultured for 24 h before induction in a glucose-free, serum-free
medium supplemented with 1 µM triiodothyronine, 1 µM dexamethasone, and 10 mM lactate
("lactate" conditions). Induction was then performed by replacing
lactate with 17 mM glucose for 24 h (or less when
mentioned). Induction of the L-PK and GLUT 2 mRNA levels was also
analyzed in the presence of various concentrations of glucose for
24 h or in the presence of 17 mM glucose and protein
inhibitors, such as 10 mM cycloheximide or 25 mM anisomycin, for the times given in the legend to Fig. 5.
Northern Blot Analysis
Total RNAs were isolated from cell lines by lysis in 7 M guanidinium HCl followed by phenol extraction (22). RNAs were denatured with methylmercury (II) hydroxide, were electrophoretically separated on formaldehyde, 1.2% agarose gels, and then were transferred and UV cross-linked to nylon membranes (Hybond N, Amersham Corp.). Prehybridization (1 h with 100 µg/ml salmon sperm DNA) and hybridization were carried out as described previously (19) except that the temperature was 58 °C when rat or human probes were used. Quantitation of the radioactive bands on the nylon membranes was performed using a PhosphorImager (Molecular Dynamics).
The probes for rat L-PK and human poly(A)-binding protein (PABP) sequences have been described previously (19). The specific 341-base pair probe for mouse L-PK cDNA was obtained by reverse transcriptase-polymerase chain reaction of mouse mRNA in the laboratory.2 The probes for rat GLUT 2 (10) and human GLUT 1 (23) sequences were obtained from corresponding expression vectors kindly given by Bernard Thorens (Lausanne, Switzerland). The probe for rat hexokinase I sequence was from the laboratory of D. Granner (24).
Transfection ExperimentsThe L4L3-119 L-PK/CAT and -119 L-PK/CAT constructs have been described previously (25). Transfection was performed by lipofection using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP, Boehringer-Mannheim, Mannheim, Germany) according to the manufacturer instructions in cells cultured in glucose-free, serum-free medium for 3-4 h. Five µg of CAT constructs and 2 µg of pRSV-luciferase (or 1 µg of pCMV-GLUT) were cotransfected in 60-mm plastic dishes (Falcon, Oxnard, CA) when cells were 60-80% confluent. After 16 h, the medium containing the liposome-DNA complex was removed and replaced with a 17 mM glucose or a 10 mM lactate medium for 24 h. CAT and luciferase activity assays were performed as described (25). CAT activity was calculated as the percentage of diacetylated form versus nonmetabolized chloramphenicol. Results were expressed as a ratio of CAT activity to luciferase activity to correct for the variable transfectability.
When transfections were performed with pCMV expression vectors, pRSV-luciferase was omitted because of the risk of titration of ubiquitous transcription factors. In such a case, CAT activities were measured in the entire extract of each dish to compensate for the differential growth of the cells (i.e. dilution of the CAT activity) when cultured in glucose versus lactate medium for induction studies. The GlRE-mediated glucose responsiveness of the L-PK promoter was assessed by standardizing the activities obtained with the L4L3-119L-PK/CAT construct according to those obtained with the control -119L-PK/CAT construct.
Immunofluorescence StudiesmhPKTf3 cells were attached to
coverslips and treated as described previously by Hughes et
al. (26) excepted that permeabilization was done with 0.2% Triton
X-100 for 5. Immunofluorescent staining of GLUT 1 and GLUT 2 was
carried out using a 1:100 dilution of antisera kindly provided by B. Thorens (Lausanne, Switzerland) that recognized GLUT1 from human, rat,
and mouse and GLUT 2 from rat. Staining of recombinant or endogenous
GLUT 2 was also confirmed with other antisera kindly provided by S. W. Cushman (Bethesda, MD) and Luc Pénicaud (Toulouse, France) (data
not shown).
Glucose 6-phosphate in cultured hepatoma cells was assayed enzymatically according to Slein (27) as described previously (9).
StatisticsResults are given as means + S.D. Statistical significance of the differences between treatment groups was determined by Student's t test.
Different lines of hepatoma cells were derived from
transgenic mice synthesizing the SV40 large T and small t antigens in the liver under the direction of either antithrombin III gene or L-PK
gene regulatory sequences (17, 18). Hepatocytes were obtained from
fetal or adult transgenic animals with either normal (mhPKTf3) or
tumoral livers (mhPKT, mhAT1, and mhAT3 cell lines) and established in
culture as described previously (19, 20). Each line was derived from a
single clone. Fig. 1A shows that induction of
L-PK mRNA accumulation by 17 mM glucose in 26 independent clones was proportional (r = 0.92 by linear
regression analysis) to their GLUT 2 mRNA content at the same time.
In addition, all these clones also contain more or less GLUT 1 mRNA
roughly in reverse proportion to GLUT 2 mRNA (not shown). The
results obtained for three clones, which synthesized different amounts
of GLUT 2 mRNA when cultured in the presence of 17 mM
glucose, are presented in Fig. 1B. In cells with low GLUT 2 mRNA levels, L-PK mRNA accumulation was practically
constitutive, independent of glucose concentration in the medium. In
contrast, in GLUT 2-expressing cells, the amplitude of the glucose
responsiveness of the L-PK mRNA expression was correlated with the
GLUT 2 mRNA level. It should be observed that GLUT 2 mRNA
accumulation is itself dependent on glucose, as previously demonstrated
in hepatocytes and mhAT3F cells (3, 28, 29), whereas abundance of GLUT
1 mRNA was independent of glucose (Fig. 5).
To confirm whether the positive correlation between GLUT 2 expression
and response of the L-PK gene to glucose was mediated by the L-PK
glucose response element (GlRE), we chose 8 clones among the 26 presented in Fig. 1 in which we tested glucose responsiveness of the
L4L3-119L-PK/CAT construct (25). It was previously demonstrated that
the responsiveness of this construct to glucose was due to the GlRE
located from 168 to
144 base pairs with respect to the cap site
(25). Fig. 2A shows that induction of the CAT
activity in transiently transfected cells was also dependent on GLUT 2 mRNA level. Activity of the transfected L-PK promoter was almost constitutive in GLUT 2(
) cells (mhAT1G8) and inducible in GLUT 2(+)
cells (mhAT3F) (Fig. 2B). It is noteworthy that 2 mM fructose was also able to activate the L-PK promoter in
GLUT 2(+) cells, in agreement with the known efficiency of GLUT 2 on
fructose transport (30).
Forced Expression of GLUT 2 but Not of GLUT 1 Glucose Transporter in Cells Lacking GLUT 2 Restores Some Glucose Responsiveness to the L-PK Promoter
To study the effect of GLUT2 (or GLUT1) on the glucose responsiveness of the L-PK promoter, two hepatoma cell lines lacking GLUT 2, i.e. mhPKTf3 previously established3 and HepG2, were transiently transfected with L-PK CAT constructs and CMV-expression vectors for GLUT 1 or GLUT 2.
Immunofluorescence studies were performed to provide evidence of the
GLUT 1 and GLUT 2 expression induced by transient transfection of the
relevant vectors (Fig. 3A).
In the mhPKTf3 cell line, GLUT 1 immunofluorescence was easily detectable at the plasma membrane of the cells while no membrane-associated GLUT 2 immunofluorescence was observed. When mhPKTf3 cells were transfected with CMV-GLUT 1 or CMV-GLUT 2 expression vectors, 4-5% of the cells exhibited greatest immunofluorescence at the plasma membrane level, confirming the correct localization of the forced expressed transporters.
Response of the L4L3-L-PK promoter to glucose was calculated by dividing the CAT activities with the glucose-sensitive L4L3-119L-PK/CAT construct by those of with glucose insensitive -119L-PK/CAT construct (Fig. 3 B). Glucose responsiveness was nil in mhPKTf3 cells and very weak (× 1.58) in HepG2 cells. Co-transfection with the GLUT 2 expression vector resulted in a clear and significant L-PK glucose responsiveness in both cell lines (3.0- and 3.8-fold increase between lactate (Fig. 3B, LO) and glucose (Fig. 3B, G17) medium in mhPKTf3 and HepG2 cells, respectively). In HepG2 cells, 17 mM fructose stimulated the L-PK promoter almost as efficiently as glucose, which confirms that GLUT 2 synthesized by transfected cells was active on fructose entry into the cells (30). Interestingly, the response to glucose in HepG2 cells resulted in both decreased activity of the L-PK promoter under lactate conditions (× 0.70) and increased activity under glucose condition (× 1.70). In contrast, forced expression of GLUT 1 in these cells, which already synthesize GLUT 1 (Fig. 3A), did not significantly change glucose responsiveness of the L-PK promoter.
Induction of GLUT2 Expression by Selection of Clones in a Fructose Medium Parallels the Appearance of Glucose Responsiveness of the L-PK GeneWe observed that most of the clones isolated in our laboratory that exhibit a high GLUT 2 expression and, thus, a good glucose responsiveness had been selected for "liver-specific functions" by culture in a medium in which glucose was replaced by 2 mM fructose (2). Initially, in designing these conditions, we wanted to select cells able to use fructose efficiently through their endogenous fructokinase and aldolsase B, two enzymes specific to liver, kidney, and small intestine (31). In fact, we also selected for the expression of GLUT 2, a transporter permissive for fructose transport (30). Accordingly, we subjected mhPKTf3 cells, which had never been selected by culture with fructose before, to fructose selection.
Fig. 4, A and B, shows that the
amount of endogenous L-PK mRNA in mhPKTf3 GLUT 2() cells was
rather independent of glucose (× 1.8) before selection and became
significantly glucose-responsive in fructose-selected mhPKTf3F1 (× 3, 7) and mhPKTf3F2 (× 11) clones, concomitantly with appearance of GLUT
2 expression. Fig. 4C confirms that the recovery of GLUT 2 expression in mhPKTf3F1 clone is parallel with restoration of the L-PK
promoter activation by glucose (× 2.5) or by fructose (× 2.4).
In GLUT 2(+) cells, L-PK and GLUT 2 Genes Are Similarly Regulated by Glucose
The GLUT 2 gene is known to be regulated by glucose (3, 28, 29). We raised the question of whether the kinetics of glucose responsiveness of the GLUT 2 and L-PK genes were similar or different in mhAT3F cells. Fig. 5 shows that time course (Fig. 5B) and concentration dependence of the response of the endogenous GLUT 2 and L-PK genes to glucose (Fig. 5C) were practically similar, both requiring active protein synthesis and, therefore, being blocked by translation inhibitors such as cycloheximide and anisomycin (Fig. 5A). However, these results did not preclude that a major cause of the delay of L-PK gene response to glucose (and perhaps of its dependence on active protein synthesis) could be the need for active GLUT 2 protein synthesis. In fact, the same requirement could exert itself on GLUT 2 mRNA accumulation, with GLUT 2 gene induction by glucose being subjected, in such a case, to a positive auto-regulatory loop. Fig. 5A also shows that mhAT3F cells expressed the GLUT 1 and HK 1 genes regardless of the presence of glucose in the medium and with a low sensitivity to translation inhibitors. In contrast, these cells, like all the lines used in this study or reported in the literature (2, 8, 9), were devoid of glucokinase gene expression (not shown).
GLUT 2 Production from an Expression Vector Neutralizes the Latency of L-PK Gene Transcription by Glucose in mhAT3F CellsTo further
test the hypothesis that the need for previous GLUT 2 synthesis could
be involved in the delayed response of the L-PK gene to glucose in
cells deprived of glucose for more than 30 h before induction, we
forced the expression of GLUT 2 in mhAT3F cells cultured in lactate for
64 h (endogenous GLUT 2 mRNA being absent) and then analyzed
the time course of the L4L3-119L-PK/CAT construct activation by
glucose (Fig. 6B). As recalled in Fig. 6A, when mhAT3F cells were deprived of glucose for more than
30 h, activation of the L-PK promoter was very low until the
14th h (2) while induction was more rapid after glucose
deprivation of only 15 h. In cells that accumulated exogenous GLUT
2 for 24 h before glucose switch, the L-PK promoter was already
activated 2.5- and 3.8-fold after 2 and 6 h of glucose refeeding,
respectively. GLUT 2 overexpression resulted in both decreased activity
under lactate conditions and increased activity under high glucose
conditions (Fig. 6C). In contrast, overexpression of GLUT 1 tended rather to decrease the response to glucose (Fig. 6B),
particularly by increasing activity of the L-PK promoter in cells
cultured in the presence of lactate (Fig. 6C), as also
observed in mhPKTf3 and HepG2 cells (Fig. 3).
GLUT 2 Expression Is Associated with Glu-6-P Depletion in GLUT 2(+) Hepatoma Cells When Cultured in Gluconeogenic Conditions
Activation of the L-PK promoter by glucose requires
Glu-6-P accumulation (9). The permanent transcription of the L-PK gene in GLUT 2(-) cells cultured in lactate suggested that Glu-6-P produced
by the gluconeogenic pathway might remain at a high concentration in
these cells while it may be low in GLUT 2(+) cells. To test this
hypothesis, GLUT 2() lines (mhPKTf3 and HepG2 cells) and GLUT 2(+)
lines (mhPKTf3F1 and mhAT3F cells) were cultured for 30-40 h in a
lactate-containing, glucose-free medium, and intracellular Glu-6-P was
assayed. Its value was compared with that observed after 1 h of
culture in the presence of 17 mM glucose. Table
I shows that Glu-6-P was practically undetectable in
mhAT3F and mhPKTf3F1 cells cultured for 30 h in lactate and
slightly increased at the 40th h. In contrast, it was about 80% of the
values obtained after 1 h of culture in a high glucose medium in
mhPKTf3 and HepG2 cells. This high Glu-6-P concentration in GLUT 2(
)
hepatoma cells cultured without glucose easily explains the
glucose-independent activity of the L-PK promoter and gene in these
cells.
|
Hepatocytes and cells were the only cells to express the
couple "GLUT 2-glucokinase" for transport and phosphorylation of glucose from the blood. In the hepatoma cell lines described to date,
neither GLUT 2 nor glucokinase were ever reported to be significantly
expressed, being replaced by GLUT 1 and insulin-independent hexokinase
isoforms, mainly HK 1 (8, 16). We have previously established and
characterized various new hepatoma cell lines, some of which express
GLUT 2 but not glucokinase and yet are responsive to glucose (2, 19,
20). Hepatoma cells that express mostly GLUT 1 exhibit a
glucose-independent L-PK gene expression, whereas GLUT 2(+) cell lines
express the L-PK gene poorly under glucose-free conditions and strongly
under glucose conditions. Induction of GLUT 2 synthesis in GLUT 2(
)
cells, either by selection for ability to grow in a fructose medium or
by transient transfection of a GLUT 2 expression vector, allows for
concomitant occurrence of glucose responsiveness of the L-PK
promoter.
Thus, the main question is what is the mechanism of GLUT 2 action on the response of glucose-sensitive genes to glucose in liver cells? In other words, what does GLUT 2 do that GLUT 1 is unable to do ? GLUT 1 and GLUT 2 have similar Vmax, but affinity of GLUT 1 is 2-3-fold higher than that of GLUT 2. In addition, GLUT 1 is highly asymmetrical, much more efficient for glucose influx than efflux, wheras GLUT 2 is symmetrical, which explains its specific role in organs whose function is to secrete glucose (11). Finally, GLUT 2 but not GLUT 1 is capable to transport fructose (30).
In endocrine pancreas cells, the predominant glucose-sensor has
been definitively identified as glucokinase by knock out of the gene in
mice (32, 33), and the significance of the specific GLUT 2 expression
in rodent
cells is still disputed, especially as the amount of the
GLUT 2 isoform seems to be very low in human
cells (34, 35). German
(36) has shown that
cells overexpressing GLUT 1 do not lose their
ability to sense changes in glucose concentration within the
physiological range and to respond by an appropriate stimulation of the
insulin gene promoter. In contrast, Newgard and co-workers (12, 26)
hypothesized that GLUT 2, perhaps by specific interaction with
glucokinase, could generate an intracellular signal needed for the
response of insulin secretion to glucose. This view is consistent with the report by Valera et al. that, in mice, an antisense GLUT
2 mRNA impaired the normal activation of insulin secretion by
glucose (37). Therefore, GLUT 2 could be needed to allow normal
regulation for insulin secretion by glucose in
cells, at least in
rodents, but be non-essential for glucose-dependent
regulation of insulin gene transcription.
A glucose sensor system is also essential in the liver to provide a
correct metabolic response to dietary modifications, either glucose
utilization and glycogen storage or glucose production. This sensor
seems to be composed, as in cells, of GLUT 2 and glucokinase.
However, glucokinase could be replaced by other insulin-independent hexokinases, in vivo and ex vivo. In vivo,
knocked out mice specifically deficient in glucokinase in liver have an
almost normal glucose regulation (32), whereas deficiency in pancreas
glucokinase leads to neonatal death from severe
insulin-dependent diabetes mellitus (33). Ex
vivo, we have shown that hepatoma cells with hexokinase 1 (2, and
this paper) respond to glucose in modulating transcription of
glucose-responsive genes, e.g. the L-PK gene. Thus, in
contrast to the essential role of glucokinase in
glucose-dependent insulin secretion by
cells,
glucokinase seems to be needed in the liver mainly for phosphorylating
glucose to Glu-6-P, a role that can be performed by other hexokinase
isoforms as well. However, GLUT 2 seems to be essential for the liver
glucose sensor function, as suggested by its requirement for the normal
response of the L-PK gene to glucose in liver-derived cells.
We have found that the main effect of GLUT 2 does not seem to be to generate a positive signal needed for glucose action, but rather to allow for L-PK gene extinction in cells cultured in the absence of glucose. All the hepatoma cells used in this work are more or less capable of gluconeogenesis and, accordingly, several of them have been selected for persistence of liver-specific functions by prolonged culture in glucose-free media (18, 19). Therefore, hepatoma cells cultured under glucose-free conditions are expected to synthesize Glu-6-P and glucose, and to secrete glucose provided that Glu-6-Pase and the transporter specialized in glucose export, i.e. GLUT 2, are present.
In the absence of GLUT 2, glucose efflux from the cells might be
limited. As a consequence, the sequestered intracellular glucose could
be phosphorylated back to Glu-6-P in a sort of futile cycle by the
constitutive hexokinase isoforms replacing glucokinase, resulting in a
high Glu-6-P concentration. This hypothesis is in complete agreement
with our results that hepatoma cells cultured for 30 or 40 h in
lactate medium had a high Glu-6-P concentration when they are GLUT
2() and an undectable or low Glu-6-P concentration when they are GLUT
2(+). Intracellular Glu-6-P concentration in GLUT 2(+) cells seems to
increase between hour 30 and 40 of culture in glucose-free medium
(Table I). In fact, this result was not surprising because expression
of the GLUT 2 gene is itself glucose-dependent (3).
Consequently, prolonged culture in lactate medium should result in the
progressive disappearance of the GLUT 2 transporter. Glu-6-P is the
first glucose metabolite needed for the transcriptional response of
glucose-responsive genes to glucose (4-6). Accumulation of this
compound (and derivated glycolytic intermediates) in GLUT 2(
) cells
cultured without glucose could therefore activate L-PK gene
transcription. In contrast, GLUT 1 expression could contribute to
limited glucose export or promote rapid reentry of glucose secreted in
the extracellular medium.
In conclusion, our results provide a new insight into the correlation between expression of the GLUT 2 transporter and the physiological response to glucose observed in gluconeogenic cells. In hepatoma cell lines, the usually expressed "couple" for transport and phosphorylation of glucose is GLUT 1-HK1, whereas it is GLUT 2-glucokinase in the liver. We show in this paper that hepatoma cell lines that express the couple GLUT 2-HK1 conserve the sensitivity of metabolic genes to glucose concentration and suggest that GLUT 2 acts in this process more as a component of the gluconeogenic pathway than as an essential element for glucose influx. In vivo, GLUT 2 is present in the liver under both gluconeogenic and glycolytic conditions. Under the former condition, GLUT 2 activity would be essential for both glucose secretion and keeping the intracellular Glu-6-P concentration low and thus avoid permanent activation of glycolytic and lipogenic genes, which otherwise would result in a futile metabolic cycle and inappropriate energy dissipation.
We are very grateful to Dr. Bernard Thorens (Lausanne, Switzerland) for the kind gift of the CMV-GLUT 2 and -GLUT 1 expression vectors as well as corresponding antisera. We also thank Dr. S. W. Cushman (Bethesda, MD) and Dr.Luc Pénicaud (Toulouse, France) for the gift of other anti-GLUT2 antisera. We thank Bruno Doiron and Franck Rencurel for technical advice on Glu-6-P measurement and Michel Raymondjean and Jean A. Boutin for critically reading the manuscript.