From the U465 INSERM, Centre Biomédical des Cordeliers, 15, rue de l'Ecole de Médecine, F-75270 Paris cedex 06, France and the § Molecular Medicine Group, Medical Research Council Clinical Sciences Center, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12 0NN, United Kingdom
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ABSTRACT |
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Although it is now clearly established that a number of genes involved in glucose and lipid metabolism are up-regulated by high glucose concentrations in both liver and adipose tissue, the signaling pathway arising from glucose to the transcriptional machinery is still poorly understood. We have analyzed the regulation of fatty acid synthase gene expression by glucose in cultured rat hepatocytes. Glucose (25 mM) induces an activation of the transcription of the fatty acid synthase gene, and this effect is markedly reduced by incubation of the cells with okadaic acid, an inhibitor of protein phosphatases 1 and 2A. A similar reduction in glucose-activated fatty acid synthase gene expression is obtained by incubation with 5-amino-imidazolecarboxamide riboside, a cell-permeable activator of the AMP-activated protein kinase. Taken together, these results indicate that the glucose-induced expression of the fatty acid synthase gene involves a phosphorylation/dephosphorylation mechanism and suggest that the AMP-activated protein kinase plays an important role in this process. This is the first evidence that implicates the AMP-activated protein kinase in the regulation of gene expression. AMP-activated protein kinase is the mammalian analog of SNF1, a kinase involved in yeast in the transcriptional regulation of genes by glucose.
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INTRODUCTION |
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Although it is now clearly established that a number of genes involved in glucose and lipid metabolism are up-regulated by high glucose concentrations in both liver and adipose tissue (for review, see Refs. 1 and 2), the signaling pathway arising from glucose to the transcriptional machinery is still poorly understood. There is general agreement on the fact that glucose has to be metabolized to stimulate the transcription of lipogenic-related genes such as L-pyruvate kinase, fatty acid synthase (FAS),1 acetyl-CoA carboxylase (ACC), and S14 (3-5). In previous papers, we have proposed that glucose-6-phosphate could be the signal metabolite for the glucose-induced FAS transcription (3, 5, 6) and therefore for other genes belonging to the same class. This hypothesis was challenged by Doiron et al. (7) who proposed that the signal metabolite was xylulose-5-phosphate. However, whatever the metabolite, the link between the glucose signal and the activation of gene transcription remains unknown.
Phosphorylation/dephosphorylation processes are one of the major mechanisms involved in the regulation of glucose and lipid metabolism both at the cellular and molecular levels in eukaryotic cells. It is now well established that many transcription factors have their activity regulated by phosphorylation through a modification of their DNA binding activity, their transactivating capacity, or their subcellular localization (8).
In cultured hepatocytes, it has been shown that okadaic acid, an inhibitor of phosphatases 1 and 2A led to the inhibition of glucose stimulation of S14 gene transcription (9). Further experiments with calyculin, a much more potent inhibitor of protein phosphatase 1 than okadaic acid, suggest that protein phosphatase 2A may play the major role in the glucose effect. Finally, since the calcium ionophore A23187, an activator of Ca2+/calmodulin-dependent protein kinase (CaM kinase) inhibited the glucose effect, it was concluded that CaM kinase and protein phosphatase 2A were implicated in the glucose regulation of S14 gene transcription (9).
In the present work, we show that AMP-activated protein kinase (AMPK) is involved in the regulation by glucose of FAS gene expression in cultured hepatocytes. It must be pointed out that AMPK is the mammalian equivalent of SNF1, a protein kinase complex essential for glucose-regulated gene expression in yeast through the modulation of the transcriptional activity of nuclear factors.
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EXPERIMENTAL PROCEDURES |
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Animals-- Animal studies were conducted according to the French Guidelines for the Care and Use of Experimental Animals. Female Wistar rats (200-300 g body weight) from Iffa-Credo, (L'Arbresle, France) were used. They were housed in plastic cages at a constant temperature (22 °C) with light from 0700 h to 1900 h for at least one week before the experiments.
Isolation and Primary Culture of Hepatocytes--
Hepatocytes
were isolated by the collagenase method (10). Cell viability was
assessed by the Trypan Blue exclusion test and was always higher than
85%. Hepatocytes were seeded at a density of 8 × 106
cells/dish in 100-mm Petri dishes in medium M199 with Earle's salts
(Life Technologies, Inc., Paisley, UK) supplemented with 100 units/ml
penicillin, 100 µg/ml streptomycin, 0.1% (w/v) bovine serum albumin,
2% (v/v) Ultroser G (IBF, Villeneuve la Garenne, France), 100 nM dexamethasone (Sigma), 1 nM insulin
(Actrapid, Novo-Nordisk, Copenhagen, Denmark), 100 nM
triiodothyronine (T3) (Sigma). After cell attachment (4 h),
the hepatocytes were cultured for 16-18 h in the presence of 5 mM glucose in a medium similar to the seeding medium but
free of Ultroser and albumin and containing 100 nM insulin.
The presence of dexamethasone, T3, and insulin in the
culture medium ensures a maximal activity of glucokinase, necessary for
a full glucose effect on FAS gene expression (11, 12). After 16-18 h,
the culture medium was removed and hepatocytes were then cultured for
1, 3, or 6 h in the presence of either 5 or 25 mM
glucose, hormones, and various compounds including 10-100
nM okadaic acid (Sigma), 50 µM H7 (protein
kinase A and C inhibitor) (Sigma), 1 µM A23187 (activator
of CaM kinases) (Sigma), 1 µM KN62 (inhibitor of CaM
kinase II) (Sigma), 50-500 µM
5-aminoimidazole-4-carboxamide-1--D-ribofuranosyl (AICAR) (Sigma) (activator of AMPK).
Metabolite Concentration Assay-- Cells were scrapped into 0.5 ml of 6% (v/v) ice-cold HClO4 in less than 5 s after removing the culture medium. The concentrations of metabolites including glucose-6-phosphate, lactate, AMP, ADP, and ATP were assayed enzymatically as described previously (13).
Measurement of AMPK Activity-- Hepatocytes were directly lyzed in the culture medium by adding 1.5 ml of buffer A (final concentrations of 50 mM Tris-HCl, pH 7.5, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol) + 1% Triton X-100. The cellular debris were pelleted by centrifugation at 4000 × g for 15 min, and the resulting supernatant was removed, adjusted to 10% with PEG 6000 (Appligene, Illkirch, France), and kept on ice for 20 min. Following further centrifugation (7000 × g, 15 min), the pellet of proteins was resuspended in 400 µl of buffer A. Aliquots (5 µl) were used to assay the AMPK activity by the SAMS peptide phosphorylation assay in the presence of saturating concentrations of 5'-AMP (200 µM) as described previously (14).
Purification of AMPK--
AMPK was partially purified from rat
liver up to and including the DEAE-Sepharose ion-exchange step as
described previously (15). AMPK was further purified by
immunoprecipitation using antibodies raised against the AMPK1
subunit bound to protein A-Sepharose. The immune complex was washed
thoroughly with buffer A, and the resin was suspended in buffer A as a
50% (v/v) slurry and used for phosphorylation reactions.
Isolation of Total RNA and Northern Blot Hybridization-- Total cellular RNAs were extracted from hepatocytes using the guanidine thiocyanate method (16) and prepared for Northern blot hybridization as described previously (17). Labeling of each probe was performed by random priming (Rediprime labeling kit, Amersham Pharmacia Biotech). Autoradiograms of Northern blots were scanned and quantified using an image processor program.
FAS and PEPCK cDNAs were as described previously (17). Albumin,Nuclear Run-on Transcription Assay-- Nuclei isolation and nuclear run-on transcription experiments were performed as described by Dugail et al. (18).
Statistical Analysis-- Results are expressed as means ± S.E. Statistical analysis was performed with Student's t test for unpaired data. When quantified, FAS mRNA concentrations were normalized with respect to the 18 S hybridization signal.
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RESULTS AND DISCUSSION |
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Okadaic Acid Inhibits the Glucose Effect on FAS Gene
Expression--
To investigate whether a
phosphorylation/dephosphorylation mechanism is involved in the glucose
signaling pathway, we have used okadaic acid, a potent inhibitor of
type 1 and 2A protein phosphatases (19), the most abundant phosphatases
present in the liver (20). Okadaic acid has been shown to increase the phosphorylation state of a number of proteins including ACC or L-PK in rat hepatocytes, leading to increased glucose
output and reduced glycolysis and lipogenesis (20). A major problem
faced by studies using okadaic acid in whole cells is that, due to the widespread role of protein phosphorylation, long-term incubations could
lead to secondary effects not related to glucose signaling. To minimize
this problem in the present study, okadaic acid was added at the same
time as 25 mM glucose in cultured hepatocytes, and FAS
mRNA concentration measurements were performed after only 3 h.
Okadaic acid at a dose of 10 nM decreases glucose-induced FAS expression by 50%, with a maximal effect obtained at 50 nM (Fig. 1). Moreover, the
inhibitory effect of okadaic acid is not due to a general impairment of
transcription mechanisms since (i) the expression of a control gene,
-actin, is not affected in the same experiments (Fig. 1), and (ii)
the inhibitory effect of okadaic acid on PEPCK mRNA abundance in
hepatocytes, a phenomenon previously described (21), can still be
counteracted by glucagon (Fig. 1).
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An Activator of AMPK Opposes the Glucose Effect on FAS Gene Expression-- Having established that protein phosphatases are involved in the glucose signaling pathway, we next investigated which protein kinases were involved. Cultured hepatocytes were incubated in the presence of a number of different compounds to modulate the activity of various protein kinases (see "Experimental Procedures"). In all cases, except when Bt2cAMP was used, hepatocytes were incubated with a sub-maximal concentration of glucose (15 mM) to be able to detect both an increase or decrease in FAS gene expression by the various drugs.
We have analyzed the effects of H7, an inhibitor of both cAMP-dependent protein kinase (PKA) and protein kinase C (22), A23187, a calcium ionophore that activates CaM kinases (23) and KN62, a specific inhibitor of CaM kinase II (24). In the presence of these various compounds (Fig. 2, top and middle panels), there was no significant change in the glucose-induced FAS gene expression. In contrast, AICAR, a compound which has been shown to activate AMPK in a number of cell types, including primary rat adipocytes and hepatocytes (14, 25) and Bt2cAMP (an activator of PKA), strongly reduces the stimulating effect of glucose on FAS gene expression (Fig. 2, middle and bottom panels). The inhibitory effect of AICAR is not a general effect on gene transcription as shown by the fact that glucokinase gene expression is not modified by the compound (Fig. 2, middle panel). Similarly, the inhibitory effect of cAMP on FAS gene expression was specific since PEPCK gene expression was strongly stimulated by Bt2cAMP (Fig. 2, bottom panel). These results suggest a potential involvement of AMPK and PKA in the glucose signaling pathway.
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Activation of AMPK Decreases Glucose-induced FAS Gene Expression by a Transcriptional Mechanism-- To assess whether the decreased expression of FAS gene by the activation of AMPK was due to a transcriptional and/or a post-transcriptional mechanism, run-on experiments were performed. Nuclei were isolated from hepatocytes cultured for 1 h in the presence of 5 or 25 mM glucose and in the absence or presence of AICAR (250 µM). Glucose at the high concentration clearly increases FAS gene transcription (Fig. 5). This increase was strongly (but not totally) repressed in the presence of AICAR, whereas glucokinase transcription was neither affected by glucose nor by AICAR (Fig. 5). It is therefore clear that the decreased FAS gene expression when AMPK is activated involves a transcriptional mechanism. Nevertheless, since the inhibitory effect of AICAR on FAS gene transcription is not total whereas the effect on FAS mRNA concentration is drastic, this might indicate an effect of AMPK activation on FAS mRNA half-life. On the other hand, the partial inhibitory effect of AICAR on FAS gene transcription might suggest that other kinases are also involved in the glucose-signaling system.
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Glucose and Its Signal Metabolite Do Not Modulate the Concentration of AMP or the Activity of AMPK-- It has been suggested that SNF1 activity varies in response to glucose in yeast (35). One obvious possibility was that a high glucose concentration decreases the activity of AMPK. AMP activates AMPK through several independent mechanisms: allosteric activation of AMPK, stimulation of an AMPK kinase (AMPK is activated by phosphorylation), and inhibition of the deactivation of AMPK by PP-2C (42). A decreased AMPK activity in the presence of a high glucose concentration could be achieved (i) by decreasing the concentration of AMP, (ii) by altering the phosphorylation state of the enzyme, or (iii) by a direct allosteric interaction between the glucose signal metabolite and AMPK.
To test the first hypothesis, we have measured the concentration of AMP in the presence of 5 or 25 mM glucose. As can be seen from Table I, a high glucose concentration does not modify AMP, ADP, or ATP concentrations.
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General Conclusion-- The main stimulator of FAS gene expression in adipose tissue and in the liver is glucose with insulin having an indirect effect (3, 5). We have shown presently that phosphorylation/dephosphorylation mechanisms are involved in the control of FAS gene expression by glucose. AMPK could be among the kinases involved in this regulation. The mechanisms by which glucose and its signal metabolite modulate the system do not involve a change in AMPK activity itself but more probably the phosphorylation state of a downstream target of the kinase. Although it is well established that PKA directly modulates the transcriptional activity of nuclear factors (45), it is not possible to conclude from the present study whether that is also the case for AMPK, and this must await the clear identification of the transcription factors involved in the glucose response.
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FOOTNOTES |
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* This work was supported in part by Grant 96/3011 from the European Economic Community Fair Program.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.
Recipient of a doctoral fellowship from the Ministère de
l'Enseignement Supérieur et de la Recherche.
¶ Supported by the CNRS and to whom correspondence should be addressed. Tel.: 33-1-42-34-6922; Fax: 33-1-40-51-8586; E-mail: pferre{at}planete.net.
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The abbreviations used are: FAS, fatty acid
synthase; ACC, acetyl-CoA carboxylase; AICAR,
5-aminoimidazole-4-carboxamide-1--D-ribofuranosyl; AMPK,
AMP-activated protein kinase; CaM kinase,
Ca2+/calmodulin-dependent kinase;
L-PK, L-pyruvate kinase; PEPCK, phosphoenolpyruvate carboxykinase; PKA, protein kinase
cAMP-dependent; SAMS, the synthetic peptide substrate with
the amino acid sequence HMRSAMSGLHLVKRR; Bt2cAMP, dibutyryl
cyclic AMP; ZMP, 5-amino-4-imidazolecarboxamide ribotide.
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REFERENCES |
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