From INSERM, By using a rapid procedure of isolation of
microsomes, we have shown that the liver glucose-6-phosphatase activity
was lowered by about 30% (p < 0.001) after refeeding
for 360 min rats previously unfed for 48 h, whereas the amount of
glucose-6-phosphatase protein was not lowered during the same time. The
amount of the regulatory subunit (p85) and the catalytic activity of
phosphatidylinositol 3-kinase (PI3K) were higher by a factor of 2.6 and
2.4, respectively (p < 0.01), in microsomes from
refed as compared with fasted rats. This resulted from a translocation
process because the total amount of p85 was the same in the whole liver
homogenates from fasted and refed rats. The amount of insulin receptor
substrate 1 (IRS1) was also higher by a factor of 2.6 in microsomes
from refed rats (p < 0.01). Microsome-bound IRS1 was
only detected in p85 immunoprecipitates. These results strongly suggest
that an insulin-triggered mechanism of translocation of PI3K onto
microsomes occurs in the liver of rats during refeeding. This process,
via the lipid products of PI3K, which are potent inhibitors of
glucose-6-phosphatase (Mithieux, G., Danièle, N., Payrastre, B.,
and Zitoun, C. (1998) J. Biol. Chem. 273, 17-19), may
account for the inhibition of the enzyme and participate to the
inhibition of hepatic glucose production occurring in this situation.
Glucose-6 phosphatase
(Glc6Pase)1 is a crucial
enzyme in systemic glucose homeostasis. By catalyzing the
dephosphorylation of glucose 6-phosphate (Glc6P), e.g. the
last biochemical reaction of glycogenolysis and gluconeogenesis, it
allows the gluconeogenic tissues (liver and kidney) to release glucose
in blood (1). Numerous data have been recently provided suggesting that
Glc6Pase is an important regulatory factor of glucose production by the liver and the kidney in various physiological situations through mechanisms involving either gene expression (2-5) or inhibitions of
its enzymatic activity (Refs. 6-9, recently reviewed in Ref. 10).
The inhibition of the Glc6Pase activity during the postprandial period
was suggested more than 10 years ago (11). This inhibition has remained
uncertain for a long time because the biochemical evidence for the
inhibition of the enzyme activity in the test tube was lacking. A
significant breakthrough has been recently achieved, as we have shown
that the Glc6Pase activity, assayed in the homogenates from rat livers
freeze-clamped in situ, is inhibited for a few h after
refeeding (9). Other indirect data have strongly suggested that
hyperinsulinemia, associated or not to hyperglycemia, should be the key
signaling factor in the inhibition mechanism (10, 12, 13). However, the
inhibition was not observed in the liver homogenates from rats perfused
with insulin, whereas euglycemia was maintained by glucose perfusion
(9). This might be explained because the latter conditions dramatically differ from the physiological ones. We have therefore chosen to study
the mechanism of inhibition of Glc6Pase under physiological conditions
of hyperinsulinemia, e.g. during the refeeding of rats previously unfed for 48 h (9). A key feature of this mechanism is
its lability because the inhibition was not retained in isolated microsomes in any study (6, 9, 10, 13). This was a serious hindrance to
the elucidation of the mechanism at the molecular level. Our first aim
has, thus, been to design a purification procedure of microsomes
retaining the inhibited state of the enzyme.
In parallel, we have wished to assess the hypothesis that the
inhibition of Glc6Pase could be dependent on the activity of phosphatidylinositol 3-kinase (PI3K). There is indeed growing evidence
suggesting that PI3K is a crucial enzyme in the signal transduction of
insulin to glucose metabolism in the liver and in peripheral tissues
(see Refs. 14 and 15 as reviews). Suggesting a role for PI3K in the
control of Glc6Pase activity, we have recently shown that the enzyme is
specifically inhibited in vitro in the presence of
µM amounts of the lipid products of PI3K (16). In adipocytes, it has been strongly suggested that the specificity of
insulin to stimulate glucose transport is dependent on the intracellular targeting of PI3K to low density microsomes (17-19). Given the intracellular location of Glc6Pase in the liver endoplasmic reticulum (which is not explained at the present time), the hypothesis that a similar PI3K translocation process could be involved in the
Glc6Pase inhibition seems therefore especially attractive.
Refeeding Experiments--
Male Sprague-Dawley rats (IFFA CREDO,
L'Arbresle, France) weighing 230-240 g were housed for 3 days with
free access to water and rat chow (50% starch, 23.5% proteins, 5%
lipids, 4% cellulose, 5.5% mineral salts, 12% water (weight basis),
Unité d'Alimentation Rationnelle, Epinay/Orge, France). Fasted
rats were then deprived of food for 48 h with free access to
water. After the same fasting time, refed rats were given free access
to rat chow (see above) for 360 min (9). The results were obtained from
several sets of experiments. Each set involved one control group of
three fasted rats and one group of three refed rats processed the same day.
Rapid Liver Subcellular Fractionation--
Unfed and refed rats
were anesthetized by a single injection of pentobarbital (7 mg/100 g of
body weight). Once they were asleep, the abdomen was incised to expose
the liver. A small liver lobe (about 2 g) was cut and immediately
homogenized at 4 °C in 18 ml of 10 mM Hepes, 0.25%
sucrose, pH 7.3, using a Teflon glass homogenizer. The homogenate was
rapidly centrifuged for 10 min at 4 °C at 30,000 × g to pellet all dense intracellular vesicles (20).
Microsomes were further extracted from the supernatant by one
centrifugation step for 30 min at 100,000 × g at
4 °C. The microsomal pellet was resuspended in 1.8 ml of the
Hepes/sucrose buffer and was immediately used for Glc6Pase and PI3K
assays. Using this procedure, less than 1 h was spent between the
removal of the liver lobe and the enzyme assays. In some experiments, a
further fractionation of microsomal membranes in sucrose gradient was
performed according to the method described by Aronson and Touster
(21). The rest of the microsomes were frozen and kept at Enzyme Assays--
Glc6Pase was assayed after rapid
fractionation by complex formation of Pi produced from
Glc6P. Briefly, the microsome suspension was diluted 50 times in 10 mM Hepes, 0.25 M sucrose, pH 7.3, and 100 µl
(about 30 µg) of protein were added to a 500-µl (total volume)
incubation medium composed of 20 mM Tris-HCl, 1 or 20 mM Glc6P, pH 7.4. After a 10-min incubation at 30 °C,
the reaction was stopped by the addition of 2 ml of ascorbic
acid/trichloroacetic acid (2%/10%, mass/vol), and Pi was
determined as described (5, 7, 9). PI3K was assayed essentially as
described previously (22), with slight modifications. Briefly, 5 µl
of the suspension of microsomes (about 75 µg of proteins) were
diluted in a 70-µl incubation medium (total volume) composed of 50 mM Tris-HCl, 5 mM MgCl2, 0.5 mM
EDTA, 1.5 mM dithiothreitol, 100 mM NaCl, 100 µM vanadate, 50 µM ATP (20 µCi [ Western Blot Analyses--
A rabbit polyclonal antiserum against
rat Glc6Pase was obtained by injection of a synthetic peptide matching
the 14 amino acids of the C terminal end of Glc6Pase sequence
(CLARLLGQTHKKSL) coupled to keyhole limpet hemocyanin. A rabbit
polyclonal antiserum against the regulatory subunit (p85) of rat PI3K
and a rabbit immunopurified IgG fraction raised against rat insulin
receptor substrate 1 (IRS1) were obtained from Euromedex
(Souffelweyersheim, France). Microsomal proteins were subjected to
electrophoresis on 9% polyacrylamide gels in the presence of SDS and
transferred to Immobilon-P membranes (Millipore S.A., St. Quentin sur
Yvelines, France). After blocking, the membranes were incubated with
either anti-Glc6Pase antiserum (1:500 dilution), anti-PI3K antiserum (1:1000), or anti-IRS1 IgG (1:1000) followed by incubation with a pure
goat-IgG fraction directed against rabbit IgG and linked to peroxydase
(1:10000) (Sigma). The detection was performed using a
specichrom-chemiluminescence system (Speci S.A., Ste-Foy-les-Lyon, France) with ECL-hyperfilms from Amersham Pharmacia Biotech.
Other Methods--
Protein was assayed according to the
procedure of Lowry with bovine serum albumin as a standard in all
experiments (25), with the exception of membrane subfractions from
sucrose gradients, which were assayed according to the Bradford
procedure with the same standard (26). Statistical analyses were
performed using the Student's t test for unpaired data
(27).
The challenging problem in studying the molecular mechanism of
Glc6Pase inhibition after refeeding was to achieve the isolation of
microsomes with Glc6Pase retaining its inhibited state. In a first
approach, we tried to homogenize the liver and purify the microsomes in
the presence of insulin mimetics such as orthovanadate and its powerful
derived compounds, i.e. aqueous bisperoxovanadate and
bisperoxovanadium picolinate. Unfortunately, this approach could not be
used because, after homogenization of the liver in the presence of
either of the vanadate compounds, Glc6Pase activity was revealed to be
unstable and to decrease progressively with time, even if the
inhibitors were rapidly removed upon washing microsomes (not shown).
Therefore, we tried to obviate the problem of the lability of Glc6Pase
inhibition by using a rapid procedure of extraction of microsomes from
the liver homogenates (see "Materials and Methods"). Using this
approach, we demonstrated that microsomal Glc6Pase activity was
inhibited after refeeding, when it was assayed at low concentration (1 mM) of substrate (65 ± 2 versus 96.5 ± 3 nmol/min/mg of protein (33% inhibition)) and at high
concentration (20 mM) of substrate (225 ± 6 versus 306 ± 9 nmol/min/mg of protein (26% inhibition
(see Fig. 1))). This was in good agreement with our previous results
involving Glc6Pase assays in the homogenates from livers freeze-clamped
in situ in anesthetized refed rats (9). The inhibition was
because of an inhibition of the enzyme activity and not to a decrease
in the amount of protein because there were no differences regarding to
the amounts of immunoreactive 36-kDa protein (Glc6Pase), irrespective
of the isolation procedure of microsomes or of the nutritional status
of the animals (upper panel of Fig.
1). This confirmed our previous data
reporting that there was no detectable decrease in Glc6Pase activity
after refeeding when the enzyme was assayed in microsomes isolated
using the classical procedure (9).
On the basis of this result, we were able to test the hypothesis that a
mechanism of translocation of PI3K could be involved in the inhibition
mechanism of Glc6Pase. In agreement with such a hypothesis, both the
amount of immunoreactive p85 and the catalytic activity of PI3K were
enhanced by 2.6 and 2.4 times, respectively, in the microsomes isolated
from refed rats as compared with fasted rats (Fig.
2, A and B). This
increase was the consequence of a translocation process and not of a
global increase in the amount of p85, because the amount of
immunoreactive p85 in the homogenates from total livers was similar in
refed rats and in fasted rats (Fig. 2D). From microsomal
protein recovery determinations and comparisons of the amounts of
immunoreactive p85 in microsomes and homogenates, we could estimate
that p85 bound to microsomes represented about 10-15% of total liver
p85 in fasted rats and about 25-30% in refed rats. In 3T3-L1
adipocyte cells, it has been strongly suggested that the specificity of
insulin to stimulate glucose transport is dependent on its ability to
target PI3K to low density microsomes, from which glucose transporters
translocate (18). In contrast, platelet-derived growth factor, which
does not stimulate glucose transport, mainly activates PI3K in the plasma membrane fraction (18). It has been suggested that IRS1 could be
the key transduction factor in this specificity of insulin to target
PI3K to intracellular membranes (19). In agreement with such a specific
PI3K-targeting pathway taking place in the liver during the
postprandial period, the amount of immunoreactive IRS1 was increased by
2.6 in microsomes purified from refed rats as compared with fasted rats
(Fig. 2C). IRS1 and p85 bound to microsomes were present as
a complex (Fig. 2E), strongly suggesting that the presence
of PI3K in microsomes was the result of an insulin-signaling process.
Unit 449,
ABSTRACT
Top
Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
80 °C for
subsequent determination of protein and Western blotting analyses. In
some experiments, microsomes were also isolated according to the
classical procedure described in our previous papers (4, 7).
-32P]ATP), 15 µg of
phosphatidylinositol-4,5-bisphosphate (Sigma) and 30 µg of
phosphatidylserine (Sigma), pH 7.3. After a 15-min incubation at
37 °C under agitation, the reaction was stopped by the addition of
400 µl of chloroform/methanol (1:1, vol:vol). Phospholipids were
extracted and deacylated, and
[32P]glycerophosphoinositol-3,4,5-trisphosphate was
quantified by HPLC technique as described previously (23, 24).
5'-Nucleotidase was determined according to a previously published
procedure (21).
RESULTS AND DISCUSSION
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Fig. 1.
Effect of refeeding on the liver microsomal
Glc6Pase activity. Liver microsomes were extracted from rats
fasted for 48 h (F) or fasted for 48 h and refed
for 360 min (R) according to the rapid procedure, and
Glc6Pase was immediately assayed at 1 and 20 mM Glc6P. The
results are expressed as the mean ± S.E.(n = 12).
*, significantly different from the fasted value, p < 0.001. In the upper panel, immunodetection of Glc6Pase
protein in liver microsomes (15 µg analyzed in each lane)
prepared from fasted and refed rats according to the rapid procedure
(F, R) and from fasted rats according to the
classical procedure (M). The experiment shown is
representative of six yielding comparative results.
View larger version (49K):
[in a new window]
Fig. 2.
Quantification of PI3K and IRS1 in liver
microsomes from fasted and refed rats. Microsomes were isolated
from fasted (F) and refed rats (R) as in Fig. 1.
The left hand upper panel shows the immunodetection of p85
(15 µg of protein/lane) from a representative experiment,
and panel A shows the results of the densitometric analysis
of 6 tracks in each condition (a.u. = arbitrary
densitometric unit). The results are expressed as the mean ±S.E. The
quantification of the catalytic PI3K activity was performed as
described in the text. The results are given as the mean ± S.E.
(n = 6) in panel B. The right hand
upper panel shows the immunodetection of IRS1 in a representative
experiment, and panel C shows a densitometric analysis as in
panel A. *, significantly different from the fasted value,
p < 0.01. Panel D shows the immunodetection
of p85 in the whole liver homogenates from refed as compared with
fasted rats (15 µg of protein/lane). In panel
E, microsomes from refed rats were solubilized in the presence of
0.5% (mass/vol) cholate for 20 min at 4 °C. Insoluble material
(I) was removed by centrifugation for 1 h at
100,000 × g. p85 was immunoprecipitated from the
solubilized material using p85 antibody (1:200) and protein A-Sepharose
according to previously described procedures (28). The
p85-immunoprecipitated pellet (P) was analyzed in terms of
immunoreactive IRS1 content in parallel with I and with the supernatant
of p85 immunoprecipitation (S) as in panel C. To
be the most meaningful, the detection was performed with overloading 50 µg of protein in tracks I and S, whereas less than 3 µg of total
protein were present in the track P.
Because microsomes isolated from the whole liver still contain a little proportion of contaminating plasma membranes (usually 10% of microsomal protein), we wished to ascertain that PI3K was associated to endoplasmic reticulum membranes and not only to the residual plasma membranes. Therefore, we further fractionated microsomal membranes in sucrose gradients. As shown in Fig. 3, p85 was present in all subfractions, either enriched in plasma membranes (fractions 2, 3, and 4 with high 5'-nucleotidase activity as a plasma membrane marker enzyme) or in endoplasmic reticulum (fractions 6 to 8, with high Glc6Pase activity). Because equal amounts of proteins from each fraction were loaded on the gel and because fractions 6 to 8 contained high amounts of proteins as compared with the other fractions of the gradient (about 65% total microsomal protein), the distribution of p85 as studied in Fig. 3, which suggests that the amount of p85 could be rather low in these fractions, is misleading. In fact, we could calculate from densitometric measurements and protein concentrations that about 70% total microsomal p85 was associated to these Glc6Pase-enriched fractions in the liver from refed rats. Although the total amount of immunoreactive p85 was lower in microsomes from fasted rats (see Fig. 2A), the repartition of p85 along the gradient was comparable with that in refed rats (not shown). This could be expected, assuming that IRS1 is the targeting factor of PI3K to intracellular membranes, because the ratio of immunoreactive IRS1 to immunoreactive p85 was similar in isolated microsomes from both fasted and refed rats (Fig. 2, A and C). Noteworthy, Glc6Pase activity was significantly lower in fractions 6 to 8 (endoplasmic reticulum) from refed rats as compared with the corresponding fractions from fasted rats (Fig. 3). This suggested that the inhibition was stable after microsomes had been rapidly extracted from the liver homogenates. In contrast, it was not lower in fractions 2 to 4 (plasma membranes) in which a substantial proportion of Glc6Pase activity might be accounted for by the presence of nonspecific phosphatase activities such as alkaline phosphatase.
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It is now admitted that phosphatidylinositol-3,4-bisphosphate and, more likely, phosphatidylinositol-3,4,5-trisphosphate (PI3,4,5P3) are the key lipid metabolites of the PI3K-signaling pathway, because they are almost absent from resting cells, whereas their intracellular concentration substantially increases upon stimulation (29). Previously, we performed reconstitution experiments of microsomes purified from fasted rats using our classical procedure in which Glc6Pase is fully active, with pure phosphatidylinositol-3,4-bisphosphate and PI3,4,5P3. We have shown that Glc6Pase activity is inhibited in vitro in the presence of minute amounts of both D3-phosphoinositides (PI3,4,5P3 being the most efficient inhibitor) and not in the presence of various other phospholipids (16). To document in vivo that the products of translocated PI3K are the inducing factor in the Glc6Pase inhibition, we quantified PI3,4,5P3 in microsomes from fasted and refed rats by two approaches. The one involved the metabolic labeling of the ATP pool by 32P in rats before refeeding experiments and the analysis of [32P]glycerophosphoinositol-3,4,5-trisphosphate by HPLC (see "Materials and Methods"). The other involved the separation of phosphoinositides by thin layer chromatography and quantification of PI3,4,5P3 by densitometry. Unfortunately, neither approach allowed us evidence of a higher total PI3,4,5P3 content in microsomes from refed rats as compared with fasted rats (data not shown). Although this is not a positive result, we would like to point out that it does not exclude that PI3,4,5P3 is the inhibitor of Glc6Pase in refed rats. Indeed 1) the PI3,4,5P3 fraction bound to (and inhibiting) Glc6Pase should likely represent only a minor fraction from the total microsomal PI3,4,5P3 content; 2) the bulk microsomal PI3,4,5P3 content might have returned to its basal fasting level because of the action of counter-regulatory phosphoinositide phosphatases during the isolation procedure; 3) those PI3,4,5P3 molecules inhibiting Glc6Pase could be protected from the action of phosphoinositide phosphatases because of tight binding to the enzyme. We have previously suggested that the binding of the lipidic part of the PI3,4,5P3 molecule to some hydrophobic region of the enzyme is able to stabilize the enzyme-inhibitor interaction (16). One may thus hypothesize that this particular PI3,4,5P3 pool bound to Glc6Pase does return to basal level with a lengthened time course as compared with the bulk microsomal PI3,4,5P3 content. This might explain why the Glc6Pase inhibition did not decrease in parallel with the total inhibitor concentration as the isolation of microsomes was ongoing. Alternatively, the reversibility of the PI3,4,5P3-mediated Glc6Pase inhibition might involve the action of a regulatory molecule that is not present in rapidly isolated microsomes. That the Glc6Pase inhibition seemed stable in the endoplasmic reticulum after fractionation in sucrose gradients (Fig. 3) is in agreement with the latter proposal.
To obtain another type of confirmation of the role of PI3K in the Glc6Pase inhibition in vivo, we carried out refeeding experiments with rats treated by intraperitoneal injections of wortmannin, a specific inhibitor of PI3K activity (15). A similar approach was successfully used to inhibit skeletal muscle P70 S6 kinase by rapamycin in mice (30). Four injections (one every 2 h from 1 h before refeeding) were given at doses of 100 µg/kg by intraperitoneal injection. This represented a total dose close to the maximally tolerated daily dose in mice (31). However, the liver microsomal PI3K activity was not substantially inhibited under these in vivo conditions (by about 30% only, data not shown), and Glc6Pase activity was inhibited in wortmannin-treated refed rats as in control saline-injected refed rats (61 ± 4 versus 63 ± 4 nmol/min/mg of protein at 1 mM Glc6P and 237 ± 16 versus 228 ± 2 nmol/min/mg of protein at 20 mM, respectively, compared with the results of Fig. 1). Another attempt was made with LY294002, another specific inhibitor of PI3K (4 injections as above at 25 mg/kg/injection), which was also unsuccessful in relieving Glc6Pase activity from inhibition (not shown). Unfortunately, we could not obtain this additional confirmation for a role of PI3K in the control of Glc6Pase activity. Again, we would like to emphasize that this result is not counter to the basic proposition that PI3K is responsible for Glc6Pase inhibition. It is likely that a 30% inhibition only of PI3K activity might not be sufficient to alter the inhibition of Glc6Pase significantly. In addition, it cannot be excluded that the PI3K isotype involved in the control of Glc6Pase is insensitive to the inhibitors used in this work. PI3K species insensitive to one or both of these inhibitors have indeed been described (32-34).
A remaining important question is to know why Glc6Pase inhibition could only be evidenced in the most physiological situation of hyperinsulinemia, e.g. rats refed after a period of fasting and not in rats perfused with insulin (9), whereas compelling indirect evidence has been provided that insulin inhibits hepatic glucose production by acting on Glc6Pase (10, 12, 13). This could be explained by a differential stability of Glc6Pase inhibition, which could be accounted for by the presence of a co-factor during refeeding experiments and its absence during insulin perfusion experiments. This might explain why isolated hepatocytes have often revealed a weakly suitable model to study the short term metabolic effects of insulin in the liver. We have previously discussed the possibility that some liver metabolite(s) dependent on nutrient availability could be required together with insulin for a full inhibition of Glc6Pase to take place in vivo (9, 10). Noteworthy, recent results from us (35)2 and others (36) have emphasized the crucial role that hyperglycemia could play in the inhibition of the hepatic Glc6Pase flux in vivo. Efforts are ongoing to identify the putative factor(s) required for the inhibition mechanism of Glc6Pase under the action of insulin to be clearly demonstrated in a simplified experimental model. Such a demonstration in isolated hepatocytes, for example, would allow us to question the role of PI3K by means of PI3K inhibitors.
In conclusion, we report the definitive demonstration in isolated
microsomes that a mechanism of inhibition of Glc6Pase activity takes
place in the liver of rats during the postprandial period. In addition,
our results strongly suggest that this inhibition mechanism could be
dependent on a translocation process of PI3K onto the liver endoplasmic
reticulum membranes, a likely consequence of the activation of the
insulin-signaling pathway, and mediated by the main lipid product of
PI3K, e.g. PI3,4,5P3. Together with the recent
reports that the targeting of PI3K to intracellular membranes could be
a key mechanism in the activation of glucose transport by insulin in
isolated adipocytes (15, 17-19) and in the inhibition of
apolipoprotein B secretion by insulin in isolated hepatocytes (37), the
data presented here further document in vivo that
translocation processes of PI3K might constitute a general process
in insulin signaling in both the liver and peripheral tissues. In the
liver, they bring new insights into the mechanisms of control of
hepatic glucose production at the level of Glc6Pase and the involvement
of PI3K in these processes. They also provide a new rationale regarding
the impairment of insulin in suppressing hepatic glucose production in
insulin-resistant animals, because the IRS1-PI3K activation pathway is
strongly altered in the liver of such animals (14, 38, 39).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. J. C. Bordet for precious help in performing some of the experiments described in this paper and Dr. M. Croset for helpful advice and editing during the preparation of the manuscript.
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FOOTNOTES |
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* 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.
¶ To whom correspondence should be addressed: Faculty of Medicine R. T. H. Laennec, rue G. Paradin, 69372 Lyon Cedex 08, France. Tel.: (33) 4 78 77 87 88; Fax: (33) 4 78 77 87 62; E-mail: mithieux{at}laennec.univ-lyon1.fr.
The abbreviations used are: Glc6Pase, glucose-6-phosphatase; IRS1, insulin receptor substrate 1; PI3K, phosphatidylinositol 3-kinase; PI3, 4,5P3, phosphatidylinositol-3,4,5-trisphosphate; HPLC, high performance liquid chromatography.
2 L. Guignot and G. Mithieux, submitted for publication.
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REFERENCES |
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