(Received for publication, November 6, 1995; and in revised form, January 9, 1996)
From the
Glucose catabolism induces the expression of the L-type pyruvate kinase (L-PK) gene through the glucose response element (GIRE). The metabolic pathway used by glucose after its phosphorylation to glucose 6-phosphate by glucokinase to induce L-PK gene expression in hepatocytes remains unknown. The sugar alcohol xylitol is metabolized to xylulose 5-phosphate, an intermediate of the nonoxidative branch of the pentose phosphate pathway. In this study, we demonstrated that xylitol at low concentration (O.5 mM) induced the expression of the L-PK/CAT construct in glucose-responsive mhAT3F hepatoma cells at the same level as 20 mM glucose, while it did not affect intracellular concentration of glucose 6-phosphate significantly. The effect of xylitol on the induction of the L-PK gene expression was noncumulative with that of glucose since 20 mM glucose plus 5 mM xylitol induced the expression of the L-PK/CAT construct similarly to 20 mM glucose alone. In hepatocytes in primary culture, 5 mM xylitol induced accumulation of the L-PK mRNA even in the absence of insulin. Furthermore, the response to xylitol as well as glucose required the presence of a functional GIRE. It can be assumed from these results that glucose induces the expression of the L-PK gene through the nonoxidative branch of the pentose phosphate pathway. The effect of xylitol at low concentration suggests that the glucose signal to the transcriptional machinery is mediated by xylulose 5-phosphate.
Glucose, a major fuel of mammalian tissues, induces the
transcription of several glycolytic and lipogenic genes in hepatocytes
and adipocytes(1, 2, 3, 4) . In
particular, it induces the expression of the L-type pyruvate kinase
(L-PK) ()gene in the liver through the glucose response
element (GIRE) located at position -168 to -144 bp with
respect to the cap site(5, 6, 7) . This GIRE
consists of two palindromic binding sites for upstream stimulating
factor (USF) proteins separated by 5 base
pairs(5, 8, 9) . Similar elements, also
termed carbohydrate response element, have been identified in the
regulatory regions of several glucose-responsive genes, i.e. the spot 14 gene (10) and the fatty acid synthase
gene(11, 12) . We have shown that activation of the
L-PK promoter through its GIRE requires phosphorylation of glucose to
glucose 6-phosphate, mediated by insulin-dependent glucokinase
induction in hepatocytes(13) . However, insulin can be replaced
in hepatocytes by transfection of a glucokinase expression vector and
by low concentrations of fructose acting through fructose
1-phosphate-dependent activation of residual glucokinase (13) .
Furthermore, insulin is not necessary in the glucose-responsive
hepatoma cell lines in which glucokinase is replaced by other isoforms
of insulin-independent hexokinases(14) . However, the pathway
by which glucose 6-phosphate activates transcription of the L-PK gene
and other glucose-responsive genes remains unknown. Glucose 6-phosphate
is an important compound at the junction of several metabolic pathways
(glycolysis, gluconeogenesis, pentose phosphate pathway, glycogenesis,
and glycogenolysis). In adipocytes, the glucose analogue 2-deoxyglucose
(transported in the cell, phosphorylated into 2-deoxyglucose
6-phosphate but was not further metabolized in the Embden Meyerhoff
pathway) has been shown to stimulate expression of the fatty acid
synthase and acetyl-CoA carboxylase genes(15) . Similarly,
2-deoxyglucose can activate the L-PK promoter in the insulinoma cell
line INS-1(16) , but not in hepatocyte or hepatoma
cells(14) . However, the efficiency of 2-deoxyglucose in
mimicking the glucose effect in some cells does not signify that the
observed induction was mediated by 2-deoxyglucose 6-phosphate itself.
Indeed, although its isomerization into fructose 6-phosphate is
impossible, 2-deoxyglucose 6-phosphate is partly further metabolized
into various compounds(17, 18) . Therefore, if the
2-deoxyglucose-dependent induction of glucose-responsive genes in
adipocytes and INS-1 cells rules out the involvement of the Embden
Meyerhoff pathway, it does not rule out the involvement of
intermediates rising from 2-deoxyglucose 6-phosphate, especially
through the pentose phosphate pathway.
In this study, we show that xylitol is active at a lower concentration than glucose for stimulating the L-PK promoter in both mhAT3F hepatoma cells and hepatocytes. In mhAT3F cells, the activating xylitol concentration is too low to modify intracellular glucose 6-phosphate concentration. The xylitol acts as glucose through the GIRE. Since xylitol is transformed into xylulose 5-phosphate in the cells, we suggest that glucose acts on glucose-responsive genes in the liver, and probably adipocytes, through the nonoxidative branch of the pentose phosphate pathway. Consequently, xylulose 5-phosphate is the major metabolite candidate of the nonoxidative branch of the pentose phosphate pathway responsible for mediating transcriptional machinery induction by glucose catabolism.
The different L-PK/CAT constructs (termed -183 PK/CAT, -150 PK/CAT, -96 PK/CAT, L4mi-L3 -119 PK/CAT) have been previously described(5, 20) . The KSV2 CAT plasmid, used as a transfection control, contains the CAT gene directed by the early promoter and enhancer of simian virus 40 (SV40)(14) .
The mhAT3F hepatocyte-like cell lines were derived from tumoral liver of transgenic mice expressing the SV40 large T and small T antigens under the direction of the liver-specific antithrombin III promoter (14, 23, 24) . Cells were cultured in Ham's F-12-Dulbecco's modified Eagle's medium (v/v) (Life Technologies, Inc.) medium supplemented with penicillin, streptomycin, 20 nM insulin, 1 µM triiodothyronine, 1 µM dexamethasone, and 5% (v/v) fetal calf serum. Twenty-four h before the experiment, cells were cultured in a serum-free, glucose-free medium containing 10 mM lactate and supplemented with the same mixture of hormones as described above. Induction of the different L-PK/CAT constructs was measured in the presence of the various concentrations of glucose and xylitol.
Figure 1: Effect of xylitol on the activation of the L-PK gene promoter in mhAT3F cells. The mhAT3F cells were incubated for 24 h in the medium containing 20 nM insulin, 1 µM triiodothyronine, 1 µM dexamethasone, and 10 mM lactate and, after the induction of the L-PK/CAT construct, were measured in the presence of the various concentrations of glucose and xylitol with the same mixture of hormones as described above. The CAT activities of the -183 PK/CAT construct transfected in mhAT3F cells was determined by the percentage of chloramphenicol conversion to its acetylated forms. The CAT activities were normalized with respect to the activity of KSV2 CAT and expressed in percentage with respect to the results obtained with 20 mM glucose equal to 100%. Transfection assays were performed by lipofection with 5 µg of plasmid DNA. All values represent the means ± S.D. of at least seven independent experiments. *, significantly higher than lactate (p < 0.05).**, significantly higher than lactate (p < 0.001).
Figure 2: Effect of xylitol on the activation of the CAT activities generated by various 5`-deleted L-PK/CAT gene constructs in mhAT3F cells. A, the 5` deletions of the L-PK gene constructs were introduced by lipofection in mhAT3F cells. Broken lines between the box represent the 8 bp introduced during the preparation of the constructs. B, the cell culture condition was the same as described in Fig. 1. The CAT activities were normalized with respect to the activity of KSV2 CAT used as a transfection standard and expressed in percentage by reference to the results obtained with 20 mM glucose equal to 100%. Each value represents the mean ± S.D. of four experiments.**, significantly higher than lactate (p < 0.001).
Figure 3: Effects of xylitol on the level of L-type pyruvate kinase mRNA in cultured hepatocytes. Hepatocytes were isolated from 3-day-starved male rats and plated on 10-cm dishes in a medium supplemented with penicillin, streptomycin, and 10% (v/v) dialyzed fetal calf serum. After culture in the presence of 1 µM triiodothyronine, 1 µM dexamethasone, xylitol (5 and 10 mM) or glucose (25 mM), with or without insulin (20 nM), hepatocytes were harvested and total RNA was purified. Total RNA was purified from the hepatic cells. The L-type pyruvate kinase mRNA was quantified by scanning the autoradiograms of Northern blot analysis. The values are the means of three distinct experiments and are expressed relative to the value obtained under the lactate culture condition.
In addition, the role of a
pentose phosphate as an inducer of the signaling pathway leading to
activation of glucose-responsive genes is concordant with the effect of
2-deoxyglucose in adipocytes (11) and insulinoma INS-1
cells(16) . Indeed, it has been reported that in some cells (e.g. granulocytes, monocytes, and macrophages) the
2-deoxyglucose 6-phosphate can enter the pentose phosphate pathway and
be metabolized into a decarboxylated intermediate, most likely a
pentose phosphate, although 40-fold less efficiently than glucose
6-phosphate(17) . However, the considerable accumulation of
2-deoxyglucose 6-phosphate as compared to glucose 6-phosphate (15, 17, 18) can partly compensate for its
decreased metabolism through the pentose phosphate pathway, especially
in cells in which this pathway is very active, as in
adipocytes(36) . In contrast, the pentose phosphate pathway is
much less active in hepatocytes(37) . This, associated with a
glucose-6-phosphatase activity decreasing the accumulation of
2-deoxyglucose 6-phosphate(18) , could explain why
2-deoxyglucose is unable to stimulate glucose-responsive genes in
hepatic cells(14) . Accordingly, we found that 2-deoxyglucose
6-phosphate concentration enzymatically determined after incubation for
20 min with 10 mM 2-deoxyglucose, was 4-fold lower in mhAT3F
cells than in rat and hamster insulinoma cell lines (R1N and H1T cells,
respectively). ()
In conclusion, we have demonstrated that a pentose entering the nonoxidative branch of the pentose phosphate pathway is active at very low concentrations for stimulating the L-PK gene through its glucose response elements. We suggest that xylulose 5-phosphate, capable of activating a protein phosphatase activity(31, 32) , could trigger a phosphorylation/dephosphorylation cascade modulating the activity of the glucose response complex assembled on the glucose-responsive elements in hepatocytes and adipocytes.