1 Laboratory of Immunogenetics, Department of Genetics, Biology and Biochemistry, University of Torino Medical School, and 3 Experimental Medicine Center, 10126 Torino; 2 Unit of Endocrinology, Scientific Institute "Casa Sollievo della Sofferenza," San Giovanni Rotondo, 71013 Foggia; 4 Division of Obstetrics and Gynecology, Department of Medical Science, University A. Avogadro of Eastern Piedmont, 28100 Novara; and 5 Department of Clinical Science, University of Rome "La Sapienza," 00161 Rome, Italy
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ABSTRACT |
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We evaluated whether insulin signaling modulates plasma cell glycoprotein (PC-1) plasma membrane recruitment, posttranslational processing, and gene expression in human cultured cell lines. Insulin induced a fourfold increase (P < 0.01) of membrane PC-1 expression by rapid and sensitive mechanism(s). This effect was reduced (P < 0.05-0.01) by inhibition of phosphatidylinositol 3-kinase (200 nmol/l wortmannin) and S6 kinase (50 nmol/l rapamycin) activities and intracellular trafficking (50 µmol/l monensin) and was not accompanied by PC-1 gene expression changes. Moreover, at Western blot, insulin elicited the appearance, in both plasma membrane and cytosol, of a PC-1-related 146-kDa band (in addition to bands of 163, 117, 106, and 97 kDa observed also in absence of insulin) that was sensitive to endoglycosidase H. Finally, inhibition of PC-1 translocation to plasma membrane, by wortmannin pretreatment, increases insulin-stimulated receptor autophosphorylation. Our data indicate that insulin stimulates PC-1 posttranslational processing and translocation to the plasma membrane, which in turn impairs insulin receptor signaling. Bidirectional cross talk between insulin and PC-1, therefore, takes place, which may be part of the hormone self-desensitization mechanism.
insulin receptor; insulin resistance; growth factor and ectoenzyme; insulin desensitization; plasma cell glycoprotein-1
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INTRODUCTION |
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INSULIN RESISTANCE is
a central feature of type 2 diabetes, obesity, and cardiovascular
disease (1, 15, 20), all major causes of morbidity and
mortality in developed countries. The cellular mechanisms of insulin
resistance are mostly unknown (29). Among the inhibitors
of insulin action is plasma cell glycoprotein (PC-1), a type II
membrane glycoprotein expressed by apparently unrelated tissues and
cells. Within the immune system, PC-1 is confined to plasma cells and
plasmocytomas, where it was first described (4, 13).
However, it is also expressed by skeletal muscle, fat, liver, pancreas,
testis, osteoblasts, hepatocytes, skin fibroblast, and continuous
lines, including breast and liver cancer cells (4, 13).
Although it has not yet been clustered, PC-1 is a member of the
ectoenzyme family, now scoring >3% of the cell surface proteins
(12). PC-1 exhibits phosphodiesterase I/pyrophosphatase
activities and is a member of a multigene family, which currently
includes two other members (i.e., PD-1 and PD-1
) (33). At the present time, there is no definitive
information on the biological functions of the ectophosphodiesterases.
Ectophosphodiesterase activity may vary from tissue to tissue and may
include recycling of nucleotides from extracellular fluid.
Ectophosphodiesterases may also be involved in modulation of the
concentrations of pharmacologically active substances (e.g., adenosine)
that act on cellular receptors and thereby influence cellular response
at the local level, or in the control of local concentrations of
pyrophosphate and, consequently, in the regulation of bone
calcification (13).
PC-1 is reported to inhibit the tyrosine kinase activity of the insulin receptor, and it is well documented that its overexpression in human tissues and cells is paralleled by whole body insulin resistance (7-10, 17, 24, 27) and impaired insulin receptor function (7-10, 16, 17), glucose transport (28, 32), and glycogen synthesis (8, 27); these data, however, have not been replicated in animal models of insulin resistance (21), thus suggesting that the negative role on insulin action may be restricted to human PC-1 (i.e., it may be species specific). This relationship clearly indicates functional interactions between the insulin-induced effects and human PC-1. In fact, a physical association between insulin receptor and PC-1 has recently been demonstrated (5, 16). Therefore, a putative role attributable to PC-1 in the pathogenesis of insulin resistance is likely related to the allosteric regulation of the insulin receptor at the plasma membrane level (5,16). Whether the functional interaction between insulin action and PC-1 is bidirectional and whether insulin stimulation of intact cells modulates PC-1 plasma membrane expression are still unknown.
To explore these issues, we evaluated whether insulin signaling modulates PC-1 plasma membrane recruitment, posttranslational processing, and gene expression in human cultured cells.
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MATERIALS AND METHODS |
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Materials. Cell culture reagents and fetal calf serum (FCS) were purchased from Seromed (Berlin, Germany). Human insulin, IGF-I, monensin, wortmannin, rapamycin, and H-7 were from Sigma Chemical (St. Louis, MO). Chemicals were from BDH. Acylamide/bis 40%, temed, and ammonium persulfate were purchased from Bio-Rad Laboratories (Hercules, CA). Enhanced chemiluminescence (ECL) Western blotting detection reagents and Hybond ECL nitrocellulose membrane were obtained from Amersham Pharmacia Biotech. Superscript II M-MLV RNase H-RT and deoxyribonucleoside 5'-triphosphate dNTP (dATP, dGTP, dCTP, and dTTP) were purchased from Life Technologies Laboratories (Paisley, UK). RNasin, Taq polymerase, and CellTiter 96 Aqueous One Solution Cell Proliferation Assay were purchased from Promega (Madison, WI).
Cell culture. HepG2, a human hepatocarcinoma insulin-responsive cell line, and MCF-7, a well-differentiated insulin- and estrogen-responsive human breast cancer cell line, were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in RPMI-1640 medium with 5% heat-inactivated FCS, 2 mmol/l L-glutamine, and antibiotics at 37°C and 5% CO2. All experiments were performed on 80-90% confluent cell monolayers grown in 75-cm2 flasks or in 10-cm petri dishes. Before each experiment, the cells were cultured in serum-free medium as detailed below.
Monoclonal antibodies and controls. 3E8 and 4H4 were murine monoclonal antibodies (MAb) specific for human PC-1, the former used for staining techniques and the latter for Western blot analysis. Both MAb were kindly provided by Dr. James W. Goding (Prahran, Victoria, Australia) (3, 25). A locally produced and purified anti-HIV-1 gp120 was selected as an isotype-matched negative control.
The MAb specific for phosphotyrosine-containing proteins, p-Tyr (PY20), was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Analysis of PC-1 expression by flow cytometry. After 18 h in serum-free medium supplemented with 0.1% BSA, cells were washed with 30 mmol/l PBS and incubated for the indicated times in RPMI-1640 medium + 0.1% BSA in the absence or the presence of human insulin or IGF-I and/or different chemicals, including protein-trafficking and insulin-signaling inhibitors. Cells were then immediately washed twice in cold PBS, trypsinized, resuspended in PBS + 0.2% BSA and 0.1% NaN3, and incubated with anti-PC-1 MAb for 1 h at 4°C. Cells were then washed twice and incubated with FITC-conjugated goat anti-mouse Ig (30 min at 4°C) and fixed in 2.5% paraformaldehyde (10 min at room temperature). The analysis was performed on a FACSort (Becton Dickinson, Milan, Italy) with the Lysis II software. The threshold on the forward scatter signal was adjusted so that debris did not trigger the cytometer. Excitation was from an argon laser at 488 nm. Background antibody binding was estimated by isotype-matched negative control MAb (11).
Preparation of cytosolic and plasma membrane cell fractions.
MCF-7 cells were grown in RPMI-1640 full medium, starved for 20 h,
and then stimulated with 10 nmol/l insulin, as just described. Cells
were then collected, washed with ice-cold PBS, and lysed in 250 mmol/l
mannitol, 0.5 mmol/l EGTA, 5 mmol/l HEPES (pH 7.4), 1 mmol/l
Na3-orthovanadate, 1 mmol/l PMSF, and 10 µg/ml aprotinin for 5-10 min on ice. Nuclei were removed by centrifugation at 500 g for 5 min. The supernatant was then centrifuged at 15,000 g for 10 min, which separated the intracellular light
membranes in the supernatant from a heavy plasma membrane in the pellet (22, 23). The resulting pellet, further referred to as the crude plasma membrane fraction, was resuspended in 50 mmol/l HEPES (pH
7.5) with 1% Triton X-100, 10% glycerol, 1.5 mol/l MgCl2, 150 mmol/l NaCl, 1 mmol/l EGTA, 100 mmol/l NaF, 10 mmol/l
NA4P2O7, 1 mmol/l
Na3-orthovanadate, 1 mmol/l PMSF, and 10 µg/ml aprotinin for 30 min on ice and then centrifuged at 4°C for 10 min at 14,000 g to remove the insoluble fraction. Crude cytosolic (i.e.,
supernatant of the 15,000-g spin) and soluble plasma
membrane extracts were then frozen and stored at 80°C. Protein
concentration of each extract was determined by the Bradford assay
(Bio-Rad).
Western blot analysis. MCF-7 cells were grown in RPMI-1640 full medium, starved for 20 h, and then stimulated with 10 nmol/l insulin, lysed, and collected as described in Preparation of cytosolic and plasma membrane cell fractions. Soluble proteins (100 µg) were then denatured with 5× Laemmli buffer for 5 min at 95°C, separated by 7.5% SDS-PAGE, and transferred to nitrocellulose by semidry Transblot (Bio-Rad). Membranes were then blocked with NET-G buffer (150 mmol/l NaCl, 5 mmol/l EDTA, 50 mmol/l Tris, 0.05% Triton X-100, and 0.25% gelatin) and probed with 4H4 anti-PC-1 MAb. Membranes were washed and incubated with peroxidase-conjugated anti-mouse Ig for 1 h at room temperature. Visualization of immune complexes was obtained by ECL.
Measurement of insulin receptor tyrosine kinase activity. MCF-7 cells were grown in RPMI-1640 full medium, starved for 20 h, and then incubated in the absence or the presence of 200 nmol/l wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, for 20 min. After two washes, cells were stimulated with 10 nmol/l insulin for 60 min at 37°C. Cells were then lysed and subjected to Western blot analysis, as previously described, using a p-Tyr (PY20) MAb as probe.
Cell proliferation assay. Cells were grown on 96-well plates in RPMI-1640 full medium, starved for 20 h, and then preincubated for 20 min in the absence or the presence of 200 nmol/l wortmannin. Insulin (10 nmol/l) was then added, and MTS assay [3-(4,5-olimethylthiozoe-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrosoline] was performed after 3 days (14). Briefly, 20 µl of MTS tetrazolium compound (CellTiter 96 Aqueous One Solution Cell Proliferation Assay) were added to each well. After a 4-h incubation, the absorbance was recorded at 490 nm with a 96-plate reader.
RNA extraction and reverse transcription.
RNA was extracted from cultured cells by TRIzol LS Reagent (GIBCO Life
Technologies). cDNA first strand was synthesized from 0.5-µg total
RNA samples as follows. RNA (preheated at 80°C for 5 min) was reverse
transcribed in 10 µl of reaction buffer (in mM: 50 Tris · HCl, pH 8.3, 75 KCl, 15 MgCl2,
10 DTT, and 500 dNTP) containing 10 pmol of random primers and 100 units of Superscript II M-MLV RNase H-RT, in the presence of 15 units
of RNasin at 42°C for 50 min. After cDNA synthesis reaction, the
samples were diluted fivefold with sterile water and stored at 70°C
(7).
Competitive PCR. A small MseI-MseI fragment (nt 267-321) was removed from the 5-min portion of PC-1 cDNA nt 152-588, according to the published sequence (GenBank accession no. M57736), and the deleted cDNA was cloned in pCR-Blunt Vector. The recombinant plasmid pPC55, whose identity was assessed by both PCR and restriction mapping, was used in competition experiments. Two microliters of each cDNA were mixed with equal volumes of the competitor plasmid pPC55 by serial 1:2 dilutions starting from 4,000 to 1,000 copies of competitor. The primers adopted anneal to nt 234-255 and 502-523 of PC-1 cDNA. PCR generates a product that is 289 base pairs long and a competitor product, which is 55 base pairs shorter. The target mixtures represented by a fixed amount of cDNA and decreasing amounts of competitor were then amplified in 12.5 µl of PCR buffer (1.5 mM MgCl2, 10 mM Tris · HCl, pH 9, 50 mM KCl, and 0.1% Triton X-100) containing each primer at the concentration of 1 µM, the four triphosphate deoxynucleotides each at 200 µM concentration, and 0.5 unit of Taq polymerase. PCR was carried out for 37 cycles, for 40 s at 94°C, 40 s at 62°C, and 30 s at 72°C. An initial denaturation of 2 min and a final extension of 5 min were also included. PCR products were finally resolved by electrophoresis on 12% gels, visualized by silver staining, and quantified by laser scanning densitometry (7).
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RESULTS |
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Insulin treatment and PC-1 cell membrane expression in MCF-7 and
HepG2 cell lines.
The experiments were aimed at linking the expression of PC-1 with
exposure of MCF-7 and HepG2 cell lines to different amounts of insulin
and at different incubation times. The amounts of the staining 3E8
anti-PC-1 MAb adopted were preselected to operate in a range of maximum
sensitivity of the test. Isotype-matched control antibodies confirmed
the specificity of the staining observed. The results obtained indicate
that the exposure of MCF-7 and HepG2 to insulin is followed by an
upmodulation of PC-1 plasma membrane expression, which is time and dose
dependent (Fig. 1). The effect of insulin
treatment becomes detectable at 0.01 nmol/l and is maximal (i.e.,
4-fold increase) at 0.1-1 nmol/l (Fig. 1, A and C). The effect exerted by 10 nmol/l insulin appears 15 min
after treatment, peaks after 60 min, and remains detectable up to
18 h (Fig. 1, B and D). The effect was
entirely reversible within 18 h after cessation of insulin
treatment (data not shown). IGF-I had no effects on PC-1 expression,
either in MCF-7 or HepG2 cell lines (data not shown).
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Insulin effect on PC-1 gene expression. These findings raise the question of how PC-1 expression is increased. To address this question, we comparatively evaluated the PC-1 gene expression in MCF-7 cells cultured in the presence or in the absence of insulin. The results obtained indicate that treatment with 10 nmol/l insulin for 60 min has no effects on PC-1 gene expression in MCF-7 cells, as evaluated by competitive RT-PCR in insulin-treated (1,030 ± 321 copies of cDNA/40 ng RNA, n = 3) vs. control cells (1,300 ± 265 copies of cDNA/40 ng RNA, n = 3), respectively. Similar results were also obtained after 24 h of insulin treatment (800 ± 354 vs. 900 ± 283 copies of cDNA/40 ng RNA, n = 3).
Insulin treatment induces surface recruitment of PC-1 mobilized
from cytosolic stores.
Indirectly supporting the view that PC-1 is mobilized from cytosolic
compartments is the observation that the effects of 10 nmol/l insulin
in MCF-7 cells are completely abolished at 4°C (data not shown) and
significantly reduced by pretreatment of the cells for 15 min with 50 µmol/l monensin, an inhibitor of intracellular protein traffic (Fig.
3).
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Insulin treatment induces structural changes of the PC-1 molecule.
The apparent molecular mass of the PC-1 molecule was analyzed
by Western blot on total lysates derived from MCF-7 cells. The PC-1
molecule as expressed by untreated MCF-7 includes four bands of 163, 117, 106, and 97 kDa. Exposure of the cells to 10 nmol/l insulin is
followed by the appearance of an additional band of 146 kDa,
accompanied by a simultaneous decreased intensity of the 117-, 106-, and 97-kDa bands. The effect mediated by insulin treatment is time
dependent, becoming apparent after 15 min, peaking around 2 h, and
remaining detectable after 18 h (Fig.
4). After insulin treatment, the 146-kDa
band was detectable in both cytosol and crude plasma membrane fractions
(Fig. 5). This new pool of PC-1 likely
differs in glycosylation from the conventional PC-1. Indeed, treatment
of cell lysates with 5 mU/ml endoglycosidase H (endo H) for 16 h
at 37°C is followed by the complete disappearance of the 146-kDa band
(Fig. 6). The finding that insulin
induces structural changes in PC-1 localized on both cytosolic and
crude plasma membrane fractions is compatible with the hypothesis that part of PC-1 mounted on the cell surface after insulin stimulation comes from internal stores.
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Inhibition of PC-1 expression at the plasma membrane increases
insulin stimulation of receptor autophosphorylation but not cell
proliferation.
Wortmannin pretreatment (200 nmol/l) for 20 min, which inhibits
stimulation of PC-1 plasma membrane expression (Fig. 2), increases insulin-induced receptor autophosphorylation (Fig.
7). These data suggest, therefore, that
upmodulation of PC-1 plasma membrane expression decreases the ability
of insulin to stimulate receptor autophosphorylation.
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DISCUSSION |
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This work offers evidence for the view that some receptors having key roles in cell life (in this case, the insulin receptor) do not simply operate through interaction with the ligand. Indeed, the signals implemented by the ligand-receptor interaction in many instances induce all-or-nothing events, with no means of modulating the intensity and length of the signal through the help of neighboring molecules or receptors.
The present results indicate that there is a functional interplay between the exposure of a human cell to insulin stimulation and PC-1, a surface molecule initially studied as a plasma cell marker and successively identified as an ectoenzyme with pleiotropic functions (4). Indeed, insulin stimulation is followed by increased plasma membrane PC-1 expression, at least in the breast and liver cancer cell lines analyzed. These effects are dose and time dependent and highly sensitive, as indicated by the low insulin concentrations needed to implement the event (i.e., 0.01 nmol/l), whereas 0.1-1 nmol/l are necessary to obtain the maximal effects. The specificity of the effects observed is witnessed by the finding that IGF-I stimulation does not resemble any aspect of the insulin-induced PC-1 modulation.
More difficult to explain is the mechanism(s) by which PC-1 is
modulated, and this is the focus of in-depth investigations that are
currently ongoing. So far, our data suggest the need for at least
intact PI 3- and S6-kinase activities (i.e., both key
insulin-signaling steps) for the modulation of membrane PC-1, further
proof of the specificity of the phenomenon observed. Also possible is
the involvement of atypical PKC- and -
, which are believed to be
inhibited by wortmannin (26). In contrast, no role of
typical PKC isoforms is indicated by our data. The signals involved do
not progress through the activation of the PC-1 gene, which remains in
its resting state during insulin-mediated signaling. No role is also
inferred by the rapidity of the modulation, which takes place in
minutes. These findings suggest two possible mechanisms, one linked to
the mobilization of PC-1 and the second to modifications undergone at
the cell membrane level. The results obtained seem to support the
hypothesis that both mechanisms are simultaneously operative.
The use of monensin, an inhibitor of the cytosolic protein traffic, is able to block the upmodulation of PC-1, further evidence against a de novo synthesis of the molecule and, indeed, evidence of mobilization from internal stores. Also, the need for intact PI 3-kinase activity, which is known to modulate the effect of insulin on the intracellular trafficking of other proteins (31), is compatible with PC-1 mobilization under insulin stimulation. It is reported that a portion of the PC-1 molecular pool is localized in the context of the endoplasmic reticulum (2). Our findings indicate that insulin signaling induces upmodulation of PC-1 through recruitment of a pool of molecules almost identical to surface PC-1, even if structurally different. Western blot analysis with specific MAb of total and subcellular fraction lysates allowed the identification of two discrete pools of PC-1. The first shows bona fide correspondence to cell surface PC-1 and is characterized as a tetrameric complex. The second pool, which is recruited upon insulin and which is detectable also at the plasma membrane level, is characterized by an extra chain in the range of 146 kDa, in addition to the tetrameric complex. This chain is glycosylated and sensitive to treatment with Endo H. A reasonable scenario is that the surface PC-1 pool may be flanked upon request by an Endo H-sensitive pool of the same molecule, which is able to perform the extra functions required.
This view is probably too schematic or, at least, biased on the basis
of limitation stemming from the use of MAb specific for discrete
epitopes for the analysis of the PC-1 expression. The other possible
mechanism, here only highlighted, is that insulin binding to its
receptor might induce cell surface ruffling, which leads to the
unmasking of hidden PC-1 determinants or which permits readier exposure
to MAb binding (18). This view is supported by recent
findings indicating that ectoenzymes tend to form clusters of
functionally linked molecules that are localized in specialized areas
of the membrane (34). Whatever mechanisms ultimately prove to be involved, the recruitment to plasma membrane would facilitate the
lateral interactions between PC-1 and the -subunit of the insulin
receptor, a prerequisite for the inhibition of insulin signaling
(5, 16). A strong relationship between PC-1 at the plasma
membrane and inhibition of insulin action is suggested by our present
data showing that, when PC-1 recruitment at the plasma membrane is
inhibited by wortmannin, insulin-stimulated receptor
autophosphorylation is, in fact, increased. The increased receptor
phosphorylation we observed did not, in turn, induce increased insulin
action (i.e., stimulation of cell growth), probably because the
wortmannin upstream stimulatory effect on insulin receptor function is
counterbalanced by its downstream inhibitory effect on PI 3-kinase activity.
PC-1 overexpression impairs insulin receptor tyrosine kinase activity and induces insulin resistance in several tissues and cells (7-10, 17, 24, 27, 28, 32). Conversely, PC-1 expression is reduced in cells with severe forms of insulin resistance due to receptor or postreceptor downstream genetic defects (30). Taken together, these data indicate that PC-1, rather than simply being an inhibitor of insulin action, is likely to be a fine tuner of the modulation of the insulin receptor. Such activity would occur through lateral associations taking place between the insulin receptor and PC-1 (5, 16). If this were the case, one would then expect insulin to modulate PC-1 plasma membrane localization and exposure as part of the hormone self-desensitization mechanism. Indeed, the conclusions of the present work indicate that, by rapid, sensitive, and specific mechanism(s), insulin increases membrane PC-1 expression, which, in turn, induces a negative modulation of insulin receptor autophosphorylation. This evidence gives further support to the idea that PC-1 is a physiological modulator of insulin sensitivity and that qualitative (5, 6, 19) and quantitative (7-10, 17, 24, 27, 28, 32) variations of the molecule may have a significant role in conditions in which altered insulin sensitivity is pathogenic.
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ACKNOWLEDGEMENTS |
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This research was supported by the Italian Ministry of Health Ricerca Corrente 2001 (to V. Trischitta), by Associazione Italiano per la Ricerca sul Cancro (Milano, Italy), by Special Projects "Biotecnologie" Consiglio Nazionale delle Ricerca and Cofinanziamento (Ministero dell' Università e della Ricerca Scientifica e Tecnologica) (to F. Malavasi), and by Fondo per gli Investimenti dello Ricerca di Base 2002.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. Menzaghi, Unit of Endocrinology, Scientific Institute "Casa Sollievo della Sofferenza," San Giovanni Rotondo, 71013 Foggia, Italy (E-mail: endocrinologia{at}operapadrepio.it).
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.
First published November 19, 2002;10.1152/ajpendo.00503.2001
Received 7 November 2001; accepted in final form 17 November 2002.
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