Hypoxia and CYP1A1 induction-dependent regulation of proteins
involved in glucose utilization in Caco-2 cells
Véronique
Carrière1,
Annie
Rodolosse1,
Michel
Lacasa1,2,
Danièle
Cambier1,
Alain
Zweibaum1, and
Monique
Rousset1
1 Unité de Recherches sur
la Différenciation Cellulaire Intestinale, Institut National
de la Santé et de la Recherche Médicale U178, 94807 Villejuif Cedex; and
2 Université Pierre et Marie
Curie, 75252 Paris Cedex 05, France
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ABSTRACT |
Although
induction of cytochrome P-450 1A1
(CYP1A1) in the Caco-2 clone TC7 alters glucose utilization and
modifies the expression of sucrase-isomaltase (SI) and hexose
transporters, nothing is known of the events that control these
effects. In this study, we analyzed the effects of
-naphthoflavone (
-NF) and hypoxia on these parameters
and expression of key enzymes of glucose metabolism. Both
-NF and
hypoxia induce similar changes: 1)
induction of CYP1A1 mRNA; 2)
increased glucose consumption and lactic acid production and lower
glycogen content; 3) downregulation
of SI and upregulation of GLUT1 mRNAs;
4) downregulation of
fructose-1,6-bisphosphatase and pyruvate kinase mRNAs and upregulation
of phosphoenolpyruvate carboxykinase,
pyruvate dehydrogenase, lactate dehydrogenase, and phosphofructokinase
mRNAs; and 5) upregulation of
c-fos and c-jun mRNAs. Although addition of
inhibitors of CYP1A1 catalytic activity to
-NF-treated cells totally
inhibits the enzyme activity, it does not modify CYP1A1 mRNA response
and associated effects, thus excluding a direct role for the enzyme per
se. These results point to a possible physiological implication of the
signal-transduction pathway responsible for CYP1A1 induction.
-naphthoflavone; cobalt chloride; sucrase-isomaltase; GLUT1; glucose metabolism
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INTRODUCTION |
EXPRESSION OF cytochrome
P-450 1A1 (CYP1A1), a monooxygenase
involved in the metabolism of xenobiotics, is highly inducible by
polycyclic aromatic hydrocarbons such as
2,3,7,8-tetrachlorodibenzo-p-dioxin,
-naphthoflavone (
-NF) (27), or imidazole derivatives (9). Although most studies on the induction of CYP1A1 have concerned the
liver or animal models, recent studies have shown that CYP1A1 is also
inducible in the human small intestine (26) and in the enterocytic
human cell line Caco-2 (4, 30).
Among clonal populations isolated in our laboratory from the Caco-2
cell line (7), one clone, TC7, has been shown to be even closer to the
small intestinal enterocytes than the parental population as to the
level of expression of differentiation-associated proteins (7, 23). We
have previously reported that the treatment of TC7 cells with CYP1A1
inducers results in increases of glucose consumption and lactic acid
production and in marked modifications, at the mRNA and protein levels,
of the expression of membrane proteins involved in the uptake and
transport of hexoses (6). These modifications include a downregulation
of the brush-border hydrolase sucrase-isomaltase (SI) and of hexose
transporters GLUT2, GLUT5, and SGLT1 and an upregulation of hexose
transporters GLUT1 and GLUT3 (6). These results raised the question of
whether CYP1A1 per se or the signal-transduction system that controls its induction could be involved in these regulations.
CYP1A1 induction requires the recruitment of two members of the basic
helix-loop-helix-PAS (bHLH-PAS) family of DNA-binding proteins (1): the aryl hydrocarbon receptor (AHR), associated in the
cytoplasm with the heat-shock protein 90 (HSP90), and the aryl receptor
nuclear translocator (ARNT) proteins. The binding of the ligand to AHR
results in the nuclear translocation of the complex followed by the
release of HSP90 and dimerization with the ARNT protein. Then the
heterodimer AHR-ARNT interacts specifically with DNA on the
xenobiotic-responsive element (XRE) and enhances the transcription of
CYP1A1 and other specific genes (see Ref. 32 for review). To date no
endogenous ligand has been found and the biological role of
Ahr and
Arnt genes is under investigation. Recently, the establishment of Ahr
null mice has indicated that AHR may play a role in the development of
the liver and in the function of the immune system (see Ref. 32 for
review). Concerning the physiological function of ARNT, a recent study
has demonstrated that ARNT is identical to the
-subunit of the
hypoxia-inducible factor 1 (HIF-1
) (39). Subsequently, several
experiments have shown that ARNT/HIF-1
is able to dimerize with
HIF-1
subunit and that this heterodimer binds to the
hypoxia-responsive elements (HRE) and modulates the expression of
oxygen-regulated genes (see Ref. 33 for review). Among them are the
genes coding for erythropoietin (EPO) (34), the vascular endothelial
growth factor VEGF (21), the hexose transporter GLUT1 (22), and several
glycolytic enzymes such as phosphoglycerate kinase 1, lactate
dehydrogenase A (LDH A), aldolase A, phosphofructokinase L (PFK L), and
enolase 1 (13, 33), and pyruvate kinase M (PK M) (12). In addition, a
recent report of targeted inactivation of the
Arnt gene in mice indicates that ARNT,
through its involvement in the cellular response to hypoxia, regulates
angiogenesis and vasoformation during mammalian embryonic development
(24). Finally, the cellular response to hypoxia was also reported to be
characterized by an increased glycolysis (22, 28) and by modifications
of the expression of genes without any identified HRE such as the
glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (17) and the
hexose transporters GLUT2 and GLUT3 (12).
Because of the striking similarities between some of these findings and
our own data, and to decipher the mechanisms responsible for the
changes observed when TC7 cells are exposed to CYP1A1 inducers, the
purpose of the present work was to further investigate which
physiological modifications are associated with the observed effects.
Three approaches were used. First, we analyzed whether CYP1A1 inducers
such as
-NF would modify the expression of key enzymes of glycolysis
or gluconeogenesis. Second, we compared the effects of
-NF with
those of cellular hypoxia on 1)
glucose utilization, 2) expression
of some key enzymes of glucose metabolism, and
3) expression of the
CYP1A1 gene compared with that of two membrane-associated proteins chosen for their inverse variations, i.e.,
SI and GLUT1 (6). Third, we analyzed whether the observed effects were
dependent on CYP1A1 activity.
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MATERIALS AND METHODS |
Cell culture.
TC7 cells (7) were seeded at 0.2 × 106 cells/25
cm2 T flask (Corning Glassworks,
Corning, NY) and cultured in DMEM (25 mM glucose) supplemented with 1%
nonessential amino acids (GIBCO, Paisley, UK) and 20% heat-inactivated
(30 min at 56°C) fetal bovine serum (Boehringer, Mannheim,
Germany). Aliquots of concentrated solution (50 mM in DMSO) of
-NF
(Sigma, Saint-Quentin-Fallavier, France) and
-NF (Sigma) were added
to the culture medium at a final concentration of 20 µM (6, 41).
Aliquots of concentrated solution (200 mM in chloroform) of
8-methoxypsoralen (MOP) (Sigma) were added to the culture medium at a
final concentration of 20 or 200 µM (37). At these final
concentrations, DMSO (0.04%) and chloroform (0.01% and 0.1%) have no
effect on any parameter of this study.
CoCl2 (Sigma) was added to the
culture medium at a final concentration of 75 µM, a concentration
currently used in various cellular systems (24, 39). TC7 cells were
cultured under hypoxic conditions in an atmosphere of 1%
O2, 10%
CO2, and 89%
N2. In all culture conditions the
medium was changed 48 h after seeding and daily thereafter.
Glucose consumption, intracellular glycogen, cAMP concentrations,
and protein assays.
Glucose consumption was determined daily by measuring the amount of
glucose remaining in the medium 16-18 h after the medium changes,
unless otherwise indicated, with the use of a Beckman Glucose Analyzer
2. Glycogen content was measured with anthrone as previously reported
(7). The concentration of intracellular cAMP was measured using a cAMP
enzyme immunoassay system (Amersham). Protein content was measured with
bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).
Microsomal fraction preparation.
TC7 cells were washed twice with
Ca2+-Mg2+-free
PBS, detached from their support by scraping, and immediately frozen
and stored in liquid nitrogen. The cells were slowly thawed at 4°C
in a 2 mM Tris · HCl (pH 7) buffer containing 50 mM
mannitol and supplemented with protease inhibitors (100 µM
phenylmethylsulfonyl fluoride and 25 µg/ml benzamidine). The cell
suspension was sonicated at 4°C, and an aliquot of the homogenate
was stored in liquid nitrogen for determination of the sucrase and
dipeptidyl-peptidase IV (DPP-IV) activities. The homogenate was
centrifuged at 9,000 g for 20 min to
remove nuclear and cell debris. The supernatant was then centrifuged at
100,000 g for 60 min. The pellet,
corresponding to the microsomal fraction, was resuspended in a small
volume of 100 mM NaPO4 (pH 7.4)
buffer containing 10 mM MgCl2 and
20% glycerol, supplemented with protease inhibitors, and stored in
liquid nitrogen until use.
Enzymatic activities.
Sucrase and DPP-IV activities were measured in the cell homogenates as
previously reported (7), using 1.5 mM
glycyl-L-proline-4-nitroanilide as substrate for DPP-IV. Results are expressed as milliunits per milligram of protein; one unit is defined as the activity that hydrolyzes 1 micromole of substrate per minute at 37°C. The
7-ethoxyresorufin-O-deethylase activity was measured using the direct fluorimetric assay described by
Burke and Mayer (5). Microsomal fractions (0.1-0.3 mg/ml of
proteins) were incubated at 37°C in buffer containing 50 mM Tris · HCl (pH 7.5) and 25 mM
MgCl2 in the presence of 125 µM of reduced NADP (Boehringer Mannheim) and 2 µM of
7-ethoxyresorufin (Sigma). At the end of the assay, 10 pmol of
resorufin (Sigma) were added to calibrate the amount of resorufin
produced. Parameters for fluorescence detection were 522 nm for the
excitation wavelength and 586 nm for the emission wavelength,
determined with the use of a Jobin Yvon spectrofluorimeter JYD3.
Results are expressed as picomoles of resorufin produced per milligram
of microsomal protein per minute.
RNA extraction and Northern blot analysis.
Total RNA was extracted by the guanidinium isothiocyanate method (8).
Twenty micrograms of total RNA denatured in 1 M glyoxal for 60 min at
50°C were applied to a 1% agarose gel. After the run in 10 mM
sodium phosphate (pH 6.5) buffer, the RNA was transferred to nylon
(Hybond N; Amersham) with 20× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). Prehybridization was performed overnight at 42°C in the presence of 50% formamide, and
hybridization was performed at 42°C in the presence of 40%
formamide and 10% dextran sulfate. The probes were
32P labeled using a megaprime DNA
labeling kit (Amersham). After hybridization, the blots were washed
twice in 2× SSC-0.1% SDS for 10 min at room temperature, once in
0.1× SSC-0.1% SDS for 15 min at 50°C, and once in 0.1×
SSC-0.1% SDS for 15 min at 65°C. For control of RNA loading,
filters were dehybridized and stained with methylene blue (25).
Quantitation was achieved with a densitometric scanner (Mark III CS;
Joyce, Loebl, Gatehead, UK).
Probes.
The probes used for the detection of CYP1A1, SI, DPP-IV, and GLUT1
mRNAs were the same as previously reported (6). AHR was detected with
phuAHR (11) and ARNT with phuARNT (36) obtained from C. Bradfield (Northwestern University Medical School, Chicago, IL).
Hexokinase II (HK II) was detected with a rat cDNA probe (38) obtained
from J. E. Wilson (Michigan State University, East Lansing, MI). The
c-fos probe was generated by reverse
transcription-PCR from mouse mRNA (as described in Ref. 2), and the
c-jun probe (3) was obtained from B. Binetruy (Institut Fédératif sur le Cancer, Villejuif,
France). The other cDNA probes for human glucose metabolic enzymes were
obtained by reverse transcription-PCR of total RNA isolated from TC7
postconfluent cells. Reverse transcription was performed at 42°C
for 2 h, using a first-strand cDNA synthesis system kit (Amersham) with
6 µg of total RNA, 20 U of RNasin, 1 mM each of deoxynucleotide
triphosphates (dNTPs), 1.6 µg of oligo(dT) primers, and
40 U of RT in 20 µl of final reactional volume. PCR was
performed on one-fifth of the reverse-transcription product in 25 µl
of Tfl polymerase buffer (Tebu, Houdan, France) containing 0.5 µM of
oligonucleotide primers, 125 µM each of dNTPs, and 25 U of Tfl
polymerase. The sequences of the sense and antisense oligonucleotide
primers were respectively 5'-AGCACCCAGCAGCCAAACTG-3' and
5'-GAGGTCGGCATTGACTTGAT-3' for
phosphoenolpyruvate carboxykinase (PEPCK), 5'-CAGGCAATGAAGGAGATGCA-3' and
5'-CTCCAGGATGAGGTCGTCAC-3' for pyruvate carboxylase
(PyC), 5'-GCTGCCATCATTGTGCTGAC-3' and 5'-ATCACCAGGT
CTCCAACACG-3' for PK,
5'-TGAGGCTGCAGTTCCATGAT-3' and
5'-CATAAATATAGGGGATGGGC-3' for glucose-6-phosphate
dehydrogenase (G-6-PDH),
5'-TGGTGGACAAGGATGTGAAG-3' and
5'-TGGGCAGAGTGCTTCTCATA-3' for
fructose-1,6-bisphosphatase
[Fru(1,6)P2ase],
5'-GTATGTACCTGGGTGAAATC-3' and 5'-TCTGGAGAAGTGTGG
ATGAA-3' for hexokinase I (HK I),
5'-GAGGATGGGCTCAAATAC-3' and
5'-GTAGTAATCAGTGCTGGC-3' for the
-subunit of pyruvate
dehydrogenase (PDH), 5'-TGTGGCAGAGCTGAAGAAGC-3' and
5'-AGCATGAGACACACTCCACA-3' for PFK 1, and
5'-CACTGTCTAGGCTACAACAGG-3' and
5'-ACTGGATCCCAGGATGTGACT-3' for LDH.
Statistical analysis.
The level of significance was tested with Student's unpaired
t-test. The minimum level of
significance accepted was P < 0.05.
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RESULTS |
Permanent exposure of TC7 cells to
-NF results in
modifications of the parameters of glucose utilization and the pattern
of expression of glucose metabolism enzymes.
Permanent exposure of TC7 cells to
-NF results, as previously
reported (6), in a high level of CYP1A1 and, compared with untreated
cells, a permanently higher level of GLUT1 and a much lower expression
of SI in postconfluent differentiated cells (Fig. 1A).
These changes were associated with increased rates of glucose consumption and lactic acid production (Table
1). Interestingly, the ratio between the
amount of glucose consumed and the amount of lactic acid produced
indicates that in
-NF-treated cells >90% of the glucose consumed
is metabolized into lactic acid, vs. 55% in control cells. Although
the intracellular cAMP concentration was unchanged in both treated and
untreated cells, the intracellular glycogen content was much lower in
-NF-treated than in control cells (Table 1), indicating that the
variations observed are not related to cAMP-dependent glycogenolysis.

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Fig. 1.
Analysis of mRNA levels of cytochrome
P-450 1A1 (CYP1A1), sucrase-isomaltase
(SI), and GLUT1 (A) and some enzymes
of glucose metabolism (B) during the
time course in culture of control and -naphthoflavone
( -NF)-treated TC7 cells. TC7 cells were cultured in absence of drug
[untreated (UT)] or under permanent exposure to 20 µM
-NF [ -NF(p)]. Cells were harvested on day of culture
indicated. Same amount of total RNA (20 µg) was loaded in each lane.
After hybridization with indicated probes, filters were stained with
methylene blue. HK I, hexokinase I; G6PDH, glucose-6-phosphate
dehydrogenase; Py C, pyruvate carboxylase; PK, pyruvate kinase; F-1,6
bPase, fructose-1,6-bisphosphatase; PEPCK,
phosphoenolpyruvate carboxykinase;
PDH, pyruvate dehydrogenase.
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To determine whether these effects of
-NF occur via perturbations of
glucose metabolism, we have analyzed the mRNA levels of key enzymes of
the glycolytic and gluconeogenic pathways during the time course of
cell proliferation and differentiation, i.e., from seeding to late
postconfluence. In control cells, as shown in Fig.
1B, PK, PyC,
Fru(1,6)P2ase,
and PEPCK increase, whereas HK I, PDH, and G-6-PDH decrease during the
time course of the culture. The same pattern was observed in
-NF-treated cells, but quantitative changes are characterized by a
marked decrease of
Fru(1,6)P2ase and
PK mRNA levels, a moderate increase of PDH, and a dramatic increase in
PEPCK mRNA levels compared with control cells (Fig.
1B). No changes were observed for
PyC, HK I, and G-6-PDH. No HK II mRNA could be detected in either
control or
-NF-treated cells.
The inhibitory effect of
-NF on SI expression does
not require CYP1A1 enzymatic activity.
To analyze whether the effect of
-NF on the expression of SI, chosen
as a marker of the effect, is dependent on CYP1A1 catalytic activity
per se, TC7 cells were exposed between days
7 and 9 of culture to
enzymatic inhibitors of CYP1A1 activity in the absence or presence of
-NF. This period between days 7 and
9 was chosen not only because it
coincides with the onset of the process of differentiation (7) but also
because major changes occur in control cells with regard to glucose
utilization, namely, a dramatic decrease of the rates of glucose
consumption and lactic acid production (6, 7) and a marked increase of
glycogen accumulation (31). As shown in Fig.
2,
-NF and MOP increase the level of
CYP1A1 mRNA and enhance the induction of CYP1A1 mRNA produced by
exposure to
-NF (Fig. 2A).
However, although
-NF and MOP inhibit the enzymatic activity of
CYP1A1 (Fig. 2B), they do not
suppress the inhibitory effect of
-NF on the expresion of SI (Fig.
2A).

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Fig. 2.
Effect of inhibitors of CYP1A1 activity on expression of SI and CYP1A1.
TC7 were analyzed on day 9 of culture after a 48-h treatment
with -NF (20 µM) or 8-methoxypsoralen (MOP; 20 µM and 200 µM)
in presence or absence of -NF (20 µM).
A: Northern blot analysis of CYP1A1
and SI mRNAs. Same amount of total RNA (20 µg) was loaded in each
lane. After hybridization with indicated probes, filters were stained
with methylene blue. B: CYP1A1
activity toward 7-ethoxyresorufin. nd, Not detectable.
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Exposure of TC7 cells to CoCl2 results in
the same alterations of glucose utilization as observed with
-NF.
In contrast to
-NF, which has no effect on cell growth and viability
(6), exposure of the cells to
CoCl2 [which mimics cellular
hypoxia (20)] from the beginning of culture, i.e., during the
phase of cell proliferation, resulted in a high rate of mortality, thus
excluding a protocol of permanent exposure. We turned therefore to the
same short-term treatment of 48 h as reported above, starting on the
day of confluence, i.e., day
7 under our conditions of seeding. We
first established that, at this time of culture,
CoCl2 has no deleterious effect on
cell viability. As shown in Table 2, both
-NF and CoCl2 induced similar alterations of glucose utilization, namely, a smaller decrease of the
rates of glucose consumption and lactic acid production associated with
a stabilization of the ratio of lactic acid production to glucose
consumption and a smaller increase of glycogen
concentration.
Similar effects of
-NF and
CoCl2 on the level of expression of SI,
GLUT1, and glucose metabolism enzyme mRNAs.
Northern blot analysis of total RNA isolated from cells exposed for 48 h, between days
7 and
9, to
-NF or
CoCl2 is shown in Fig.
3. Analysis of these results shows that
1) short-term treatment with
-NF
and permanent exposure to the drug have the same effect on the genes
studied here (see Fig. 1), and 2)
CoCl2 induces the same changes as
-NF, i.e., a smaller increase of the level of SI and increased
expression of GLUT1, as well as a smaller increase of
F(1,6)P2ase and
PK and an increase of PEPCK, PFK, and LDH. The decrease of SI gene
expression after
-NF or CoCl2
treatment was confirmed by enzyme activity assays (Fig. 4). In contrast, DPP-IV, analyzed here as a
control of differentiation not linked to glucose utilization, was found
to be unchanged at both mRNA (Fig. 3) and activity (Fig. 4) levels.

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Fig. 3.
Effect of a short treatment of TC7 cells with -NF or
CoCl2 on mRNA levels of SI, GLUT1,
and indicated glucose metabolism enzymes. TC7 cells were analyzed on
day 7 and after 48 h of culture (day 9) in the absence (UT) or presence
of 20 µM -NF [ -NF(s)] or 75 µM
CoCl2. Same amount of total RNA
(20 µg) was loaded in each lane. Filter was hybridized with indicated
probes and then dehybridized and stained with methylene blue. PFK,
phosphofructokinase; LDH, lactate dehydrogenase; DPP-IV,
dipeptidyl-peptidase IV.
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Fig. 4.
Determination of sucrase (hatched bars) and DPP-IV (open bars)
activities in TC7 cells harvested on
day 9 of culture after 48-h treatment with 20 µM -NF or 75 µM
CoCl2. Results are means ± SD
of 3 different cultures.
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To analyze whether CoCl2 would
modify the effects of
-NF on the genes studied,
CoCl2 was added for 48 h between
days
7 and 9 to cells permanently exposed to
-NF from the beginning of the culture. No additional effect was
observed for most of the genes studied, except for a lower level of SI
and Fru(1,6)P2ase
mRNA (Fig. 5).

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Fig. 5.
Scanning analysis of a Northern blot of SI (hatched bars) and
fructose-1,6-bisphosphatase (open bars) mRNAs extracted from TC7 cells
on day 9 of culture under indicated culture
conditions, i.e., in the absence of treatment, presence of
CoCl2 for the last 48 h, and permanent exposure to -NF
[ -NF(p)] without or with the addition of
CoCl2 during the last 48 h.
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CoCl2 upregulates CYP1A1 at the mRNA
level but inhibits its activity.
The expression of CYP1A1 was analyzed at both mRNA and enzyme activity
levels in cells treated with
CoCl2,
-NF, or a combination of
the two drugs. As shown in Fig.
6A, a 48-h
exposure of TC7 cells to CoCl2
between days
7 and
9 resulted in a threefold increase of
CYP1A1 mRNA, an increase that is, however, lower than the 80-fold increase observed with
-NF during the same period. Interestingly, CoCl2, when added for 48 h to
cells permanently treated with
-NF, potentiates the effect of
-NF
on the increase of CYP1A1 mRNA levels (Fig.
6B). These effects on CYP1A1 occur
without modifications of AHR and ARNT mRNA levels (Fig.
6A). Contrasting with the high activity of CYP1A1 observed in cells exposed to a short or a permanent treatment with
-NF, and despite the increase of CYP1A1 mRNA, not
only was CYP1A1 activity undetectable in
CoCl2-treated cells but the drug
completely inhibited the activity of CYP1A1 when added to
-NF-treated cells (Fig. 6C).

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Fig. 6.
Effects of -NF and CoCl2 on
mRNA levels of CYP1A1, aryl hydrocarbon receptor (AHR), and aryl
receptor nuclear translocator (ARNT) and on CYP1A1 activity.
A: Northern blot analysis of CYP1A1,
AHR, and ARNT mRNAs in TC7 cells analyzed on
days 7 and
9 of culture under same treatment
conditions as in Fig. 3. B:
quantification of CYP1A1 mRNA as deduced from a scanning of a Northern
blot of TC7 cells analyzed on day 9 of culture, with same treatment
conditions as in Fig. 5. C: activity
of CYP1A1 toward hydroxylation of 7-ethoxyresorufin in -NF and
CoCl2-treated TC7 cells. CYP1A1
activity was measured in control TC7 cells (UT), after 48-h exposure to
-NF [ -NF(s)], or after permanent exposure to -NF
[ -NF(p)] with or without a short treatment with
CoCl2 during the last 48 h. All
cells were analyzed on day 9. Results are means ± SD of 3 different cultures.
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-NF and CoCl2 upregulate
c-jun and
c-fos.
The short treatment with CoCl2
also induced an increase of c-jun and
c-fos mRNA levels, an increase that,
in cells treated permanently with
-NF, occurred only after
confluence, i.e., on day 9 (Fig.
7). It must be noted that a 48-h exposure
to CoCl2 of cells treated
permanently with
-NF resulted in a marked increase in
c-jun but not of
c-fos mRNA compared with treatment
with
-NF alone (Fig. 7).

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Fig. 7.
Effect of -NF and CoCl2 on
levels of c-jun and
c-fos mRNAs. Control TC7 cells and
cells permanently exposed to -NF and either not treated or treated
with CoCl2 for 48 h, starting on
day 7 of the culture, were harvested on indicated days. Same amount of total
RNA (20 µg) was loaded in each lane. After hybridization the filter
was stained with methylene blue.
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Hypoxia induces the same changes as
-NF or
CoCl2 treatment on the rate of glucose
consumption and the expression of CYP1A1 and of proteins involved in
glucose utilization.
The culture of TC7 cells under hypoxic conditions for 48 h between
days
7 and
9 results in an increase in the rate
of glucose consumption (53 ± 6 µg · mg
1 · h
1
protein compared with 40 ± 3 µg · mg
1 · h
1
protein in control cells), an upregulation of CYP1A1 and GLUT1 mRNAs,
and decreased levels of SI and
Fru(1,6)P2ase
mRNAs (Fig. 8). Exposure of the cells to
both
-NF and hypoxic conditions enhances the inhibitory effects of
-NF on SI and
Fru(1,6)P2ase expression (Fig. 8), while totally inhibiting CYP1A1 activity (data not
shown).

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Fig. 8.
Effect of -NF and hypoxia on CYP1A1, SI, GLUT1, and
fructose-1,6-bisphosphatase mRNA levels. TC7 cells were analyzed on
day 9, after 48 h of culture under
indicated conditions. Same amount of total RNA (20 µg) was loaded in
each lane. Filter was hybridized with indicated probes and then
dehybridized and stained with methylene blue.
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 |
DISCUSSION |
The present results show that treatment of TC7 cells with
CoCl2 results in the same
modifications as those previously reported with inducers of CYP1A1 (6),
namely, increased glucose utilization (i.e., increased rates of glucose
consumption and lactic acid production and lower glycogen content) and
alterations of the expression of membrane proteins involved in glucose
uptake and transport, i.e., a downregulation of SI and an upregulation
of GLUT1. These results also show that both
CoCl2 and
-NF, utilized here as
an inducer of CYP1A1, produce marked changes in the mRNA levels of
enzymes involved in glucose metabolism, namely, a downregulation of
Fru(1,6)P2ase and
PK and an upregulation of PEPCK and, to a lesser extent, LDH, PFK, and
PDH. CoCl2 also induces an
increase in CYP1A1 mRNA, similar to
-NF but to a lesser extent.
Culture of TC7 cells in hypoxic conditions results in an induction of CYP1A1 mRNA, increased glucose consumption, and the same modifications of expression of SI,
Fru(1,6)P2ase,
and GLUT1 as observed when the cells were exposed to
-NF and
CoCl2. The unexpected finding that
CoCl2 and hypoxia induce an
increased level of CYP1A1 mRNA in TC7 cells can be explained by the
induction of c-jun and
c-fos, as previously reported in other
cell systems (40). Indeed, c-jun and
c-fos could be responsible for the
increase in CYP1A1 mRNA levels via the AP-1 site located in the
promoter region of the CYP1A1 gene (19, 35).
Among the modifications associated with the increased glucose
utilization observed in TC7 cells exposed to
-NF and
CoCl2, the increase in PFK, LDH,
and PDH and the decrease in
Fru(1,6)P2ase mRNAs are consistent with the increase of glycolysis observed in these
cells. However, it must also be noted that the glucose-dependent regulation of PEPCK and PK is the inverse of that observed in the liver
(10, 16, 20) or in skeletal muscle cells (28) and does not correlate
with an increase of glycolysis. Further studies are needed to determine
whether this different behavior is a characteristic of intestinal cells
or depends on the neoplastic status of TC7 cells.
That the observed effects are associated with the induction of CYP1A1
mRNA but do not depend on the catalytic activity of the enzyme can be
concluded from the results obtained with the inhibitors of CYP1A1
activity, CoCl2 or hypoxia. This
raises the question of whether the signal-transduction pathway
responsible for CYP1A1 induction is directly or indirectly involved in
the modifications of the parameters of glucose utilization observed in
the cells.
The molecular mechanisms that control CYP1A1 induction and, more
particularly, the involvement of the AHR and ARNT protein are now well
documented (32). It has also been recently reported that ARNT is
involved in the cellular response to hypoxia (39, 42). However, nothing
is known of an involvement of its transduction system in any of the
metabolic effects and changes observed in TC7 cells.
One hypothesis is that these metabolic effects could be the consequence
of a cascade of events triggered by a signal that remains to be
determined, which results in an increase of glycolysis, which in turn
modifies the expression of genes shown to be glucose dependent (7, 29).
The increase of glycolysis may result from a direct role of ARNT on the
regulation of genes of glycolytic and gluconeogenic pathways via its
dimerization with HIF-1
and binding to HRE. Such HRE have been
described in GLUT1, LDH, and PFK (33). ARNT can also dimerize with AHR
and interact with XRE sites after treatment with xenobiotics (see Ref.
32 for review). Such XRE sites have been reported to be implicated in the response to hypoxia as well (14). When both hypoxia and treatment
with xenobiotics are combined, AHR and HIF-
may compete for the
recruitment of ARNT. Such a competition has been recently described in
Hep G2 cells. Pretreatment of the cells with
CoCl2 for 16 h before
administration of 2,3,7,8-tetrachlorodibenzofuran (TCDF) for an
additional 8 h reduced the TCDF-dependent induction of CYP1A1 (15).
There was no such competition in our experiments; we observed an
amplification of the effects not only on CYP1A1 but also on SI and
Fru(1,6)P2ase
expression when cells permanently treated with
-NF were exposed to
CoCl2. Interestingly, we did not
find any XRE or HRE sites in the region of the promoter of the SI gene
responsible for its glucose-dependent expression or in the promoter
region of the
Fru(1,6)P2ase
gene. Modification of expression of these genes by
-NF treatment or
hypoxic conditions could therefore be a consequence of a preliminary
increase of glycolysis.
Some of the modifications observed could also be a consequence of the
action of the drugs on mitochondria. Indeed, the increase of glycolysis
in CoCl2-treated cells may be
explained by the fact that CoCl2
inhibits the functions of the mitochondrial respiratory chain by its
action on heme synthesis and degradation (18, 20), and therefore the
hypoxic cells increase glycolysis to compensate for the loss of ATP
production that occurs when the mitochondria are not functional. The
effects of CYP1A1 inducers on mitochondria are not documented. However,
if such an effect exists, the mitochondrial target must differ between
-NF and CoCl2, since cell
viability differs between both conditions according to the duration of
the treatment.
At present, in TC7 cells treated with
-NF or hypoxia, the mechanism
responsible for the increase in glucose utilization and for the
coordinate variations of expression of CYP1A1 mRNA and genes associated
with glucose utilization remains to be elucidated. A
possible implication of AHR or ARNT cannot be excluded.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by Université Paris XI and
contract 1393 from the Association pour la Recherche sur le Cancer. V. Carrière is supported by a fellowship from the Association de
Secours des Amis des Sciences, and A. Rodolosse is supported by a
fellowship from the Ministère de l'Education Nationale de la
Recherche et de la Technologie.
 |
FOOTNOTES |
Address for reprint requests: V. Carrière, INSERM U178, 16 Ave.
Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France.
Received 15 October 1997; accepted in final form 2 March 1998.
 |
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