1 Equipe Accueil 2402, Université Henri-Poincaré, 54500 Vandoeuvre les Nancy; 2 Institut National de la Santé et de la Recherche Médicale Unité 449, Faculté de Médecine Laënnec, 69000 Lyon, France; 3 Service de Diabétologie, Maladies Métaboliques et Nutrition and 4 Centre d'Investigation Cliniques Inserm-Centre Hospitalier Universitaire de Nancy, 54520 Dommartin les Toul, France
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ABSTRACT |
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We have shown that
membrane sphingomyelin (SM) is an independent predictor of the variance
of fasting plasma insulin (FPI) concentrations and the homeostasis
model assessment (HOMA) estimate of insulin resistance in obese women.
The peroxisome proliferator-activated receptor- (PPAR-
) is a key
component in adipocyte differentiation that may also contribute to the
sensitivity of cells to insulin. PPAR-
is activated by fatty acids,
and the membrane composition may have an impact on the activity of
PPAR-
and thus on the sensitivity of adipocytes to insulin. We
investigated these possible links by determining the phospholipid
contents of adipocyte membranes, the mRNA expression of PPAR-
, and
the FPI and HOMA estimate of insulin resistance in obese women. The
mRNA levels of tumor necrosis factor-
(TNF-
), which is suspected
to play a role in insulin resistance and which downregulates PPAR-
expression, were also quantified. FPI and HOMA were strongly positively
correlated with membrane SM (P < 0.005) and cholesterol
(P < 0.005). PPAR-
mRNA levels were negatively
correlated with FPI (P < 0.05) and HOMA (P
< 0.05) and positively correlated with high-density lipoprotein (HDL) cholesterol (P < 0.05), membrane SM (P < 0.05), and cholesterol contents (P < 0.05). TNF-
mRNA
levels were not correlated with membrane parameters. In stepwise
multiple regression analysis, the variations in PPAR-
mRNA levels
were mainly explained by HDL cholesterol (31.9%) and membrane SM
(17.7%). Our study shows that the expression of PPAR-
, a major
factor controlling adipocyte functions, the lipid composition of the
membrane, and insulin sensitivity are probably closely associated in
the adipose tissue of obese women.
obesity; adipose tissue; plasma membrane; transcription factor; insulin resistance
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INTRODUCTION |
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CHANGES IN THE PHOSPHOLIPID composition of the plasma membrane can dramatically alter cell functions by modifying, for example, the interactions of extracellular ligands with their receptors. Changes in membrane composition can also alter the intracellular signal transduction pathways, because the membrane contains a pool of phospholipids available for the production of second messengers. Therefore, genetically and/or nutritionally induced changes in membrane lipid composition could be involved in several human diseases (20). This is particularly relevant for the insulin resistance state, which is associated with a cluster of metabolic disturbances (42). It has been reported that there is a connection between the fatty acid composition of phospholipids in skeletal muscle and insulin sensitivity (1, 8, 10, 52). In other studies, insulin resistance has been associated with alterations in the physical properties of the membranes (12). In addition, we have shown that an altered composition of the major classes of phospholipids in erythrocyte membranes was associated with insulin resistance in obese patients. The membrane sphingomyelin content represents a major independent predictor of the variance of markers of insulin resistance in obese women (9).
Sphingomyelin also accumulates in the cell membranes in diseases with
peroxisomal disorders, and it has been suggested that peroxisome
proliferator-activated receptors (PPARs) are involved (46). The PPAR- subtype is abundantly expressed in
adipose tissue, where it plays a major role in adipocyte
differentiation and in maintaining the adipocyte phenotype
(47). PPAR-
may also play a role in the sensitivity of
cells to insulin (49). This concept relies mainly on the
discovery that PPAR-
is a high-affinity receptor for the
thiazolidinediones, a class of hypoglycemic agents that enhance insulin
action in target tissues (53). However, the physiological
regulation of PPAR-
gene expression in vivo or its potential
dysregulation in obesity and insulin resistance is not well
characterized (5). The natural ligands of PPAR-
include
PGJ2, a metabolite derived from arachidonic acid (17, 29). In addition, certain saturated and polyunsaturated fatty acids that are major components of cell membranes also activate PPAR-
(31). Therefore, the membrane composition may
have important consequences for the activity of PPAR-
and thus the
insulin sensitivity of adipocytes.
In addition to being an important constituent of cell membranes,
sphingomyelin is a mediator in some of the actions of tumor necrosis
factor- (TNF-
; see Ref. 27), a peptide that downregulates PPAR-
expression (54), inhibits insulin action
(22, 33), and increases lipolysis (15) when
added to adipose cell lines and human adipocytes. Moreover, TNF-
is
produced by adipose tissue in proportion to the mass of fat (21,
26) and, at least in rodents, it has been demonstrated that
adipose-derived TNF-
is a key factor in obesity-related insulin
resistance (23).
Thus all of these data suggest that the expression and activity of
important factors controlling adipocyte functions, the composition of
the membrane, and insulin sensitivity are probably closely associated
in adipose tissue. The present work was done to investigate these
possible links in the subcutaneous adipose tissue of obese individuals.
The distribution of cholesterol and major phospholipids in adipocyte
plasma membranes and the mRNA expression of PPAR- and TNF-
were
determined in fat biopsies taken from obese women with very different
body mass indexes, fasting plasma insulin concentrations, and
homeostasis model assessment (HOMA) estimates of insulin resistance.
PPAR-
and TNF-
mRNA levels were quantified by RT-competitive PCR (cPCR).
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SUBJECTS AND METHODS |
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Selection of patients. We studied 20 obese Caucasian patients [age: 41.2 ± 2.6 yr; body mass index (BMI): 33.6 ± 0.7 kg/m2] whose body weights had been stable for several months. None of the subjects had been involved in a weight reduction program over the previous 3 mo. The glucose tolerance of the patients, based on the oral glucose tolerance test, was normal according to World Health Organization criteria. None of the subjects was treated with any drug that could influence plasma lipid concentrations or glucose tolerance. Written informed consent was obtained from all patients, and the study was approved by the Ethics Committee of the Nancy University Hospital.
Biochemical determinations. Blood samples were taken from an antecubital vein in the morning after a 12-h overnight fast. Fasting plasma glucose was measured by the glucose oxidase method with a Beckman BGA II Glucose Analyzer (Beckman Instruments, Fullerton, CA). Plasma triglycerides and cholesterol were measured enzymatically (Boehringer, Mannheim, Germany). High-density lipoprotein cholesterol (HDL cholesterol) was determined in the plasma after precipitation of very low density and low-density lipoproteins (LDL) with phosphotungstic acid. LDL cholesterol was estimated using Friedewald's formula. Plasma insulin was measured by RIA (CIS Biointernational, ORIS group, Gif-sur-Yvette, France). The sensitivity of the RIA was 1 mU/l, and the intra- and interassay coefficients of variation were 8.2 and 8.8%, respectively. The cross-reactivity with proinsulin was 14%. Fasting plasma insulin and HOMA, which is correlated with the euglycemic-hyperinsulinemic clamp (36), were chosen as insulin resistance markers. The HOMA estimate of insulin resistance was assessed by the formula [fasting insulin (mU/l) × fasting glucose (mmol/l)]22.5.
Adipose tissue biopsies.
Subcutaneous fat tissue was obtained by incisional biopsy performed
under local anesthesia from the lower abdominal wall. Five of 20 fat
biopsies were not large enough (<5 g) for adipocyte plasma membrane
preparations. Adipose tissue was frozen in liquid nitrogen immediately
and was stored at 80°C.
RNA preparations. Total RNA from adipose tissue (~200 mg of frozen tissue) was obtained using the RNeasy total RNA kit (Qiagen, Courtaboeuf, France). The absorption ratio at 260 to 280 nm was between 1.7 and 2.0 for all preparations. RNA integrity was verified by agarose gel electrophoresis.
Quantification of the target mRNAs.
The concentrations of PPAR- and TNF-
mRNA were measured by
RT-cPCR which, after a specific RT reaction, exploits the
coamplification of the target cDNA with known amounts of a specific DNA
competitor molecule added in the same PCR tube (4). The
reverse transcription reactions were performed on 0.2 µg of total
tissue RNA in the presence of one of the specific antisense primers.
The experimental conditions, the primer sequences, and the validation
of the RT-cPCR mRNA assays of PPAR-
and TNF-
have been described
in detail previously (3, 6).
Adipocyte plasma membrane isolation.
At first, a total membrane fraction was prepared as previously
described (7), with minor modifications. Ten milliliters of a HEPES-sucrose buffer [buffer 1: 20 mmol/l HEPES, 255 mmol/l sucrose, and 1 mmol/l EDTA, pH 7.4, containing protease
inhibitors (5 µg/ml leupeptin, 5 µg/ml
pepstatin, and 5 µg/ml aprotinin)] were added to adipose
tissue (5-7 g), and the tissue was homogenized at 4°C using an
Ultra-Turrax (TP18) homogenizer. The homogenate was first centrifuged
at 500 g for 10 min at 4°C. The supernatant was separated
from the pellet, and fat cake and then was centrifuged at 150,000 g for 2 h at 4°C. The pellet (total membrane
fraction) was then treated as previously described (34).
It was suspended in buffer 1 and layered carefully on a
sucrose cushion (41% in buffer 1) that was then centrifuged
for 1 h at 95,000 g. The plasma membrane fraction that
settled at the interface was collected, diluted in buffer 1,
and washed at 4°C by centrifugation at 200,000 g for 20 min. The resulting pellet was suspended in Tris-EDTA buffer
(buffer 2: 1 mmol/l Tris, 1 mmol/l EDTA, and 10 mmol/l NaCl), and the protein content was estimated by the Lowry method. The
membrane suspensions obtained with this method have been characterized by use of marker enzyme activities. The plasma membrane fractions were
enriched four- to sevenfold in 5'-nucleotidase activity compared with
homogenized tissue. The plasma membrane fractions were stored at
80°C until further analysis.
Membrane lipid determination. Lipids were extracted from adipocyte membranes with methanol and chloroform (16) containing 50 mg/l 2,6-di-tert-butyl-p-cresol to prevent lipid peroxidation. The organic phase was evaporated to dryness under a stream of nitrogen at room temperature. The lipid residue was dissolved in chloroform, and its components were separated by HPLC (51). Lysophosphatidylcholine (10 µg/100 µg proteins) was added as internal standard before extraction. Phospholipids were detected with an evaporative light scattering detector. Phospholipid data were expressed as a weight concentration calculated from calibration curves established for each phospholipid. Membrane cholesterol was determined by the method of Zlatkis and Zak (55).
Statistical analysis.
Data are presented as means ± SE or medians with 25th and 75th
percentiles. Variables were assessed for normality by the skewness and
kurtosis test. To improve the skewness and kurtosis of the distributions, waist-to-hip ratio (WHR), fasting blood glucose, phosphatidylcholine, and phosphatidylserine were logarithmically transformed for statistical analysis and then were backtransformed to
their natural units for presentation in Tables 1-5. Medians and
25th and 75th percentiles are presented when the data follow a log
normal distribution. Univariate statistical analysis was performed by
linear regression analysis to identify correlations between variables.
Stepwise multiple regression analysis was carried out to determine the
independent predictors of PPAR- mRNA levels. All calculations were
performed using Statview software (Abacus Concepts, Berkeley, CA).
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RESULTS |
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The relevant clinical and metabolic characteristics of the 20 patients are shown in Table 1. Their BMIs ranged from 28 to 39.5 kg/m2. One patient had a BMI <30 kg/m2. None of the patients had major lipid abnormalities. Their fasting plasma insulin ranged from 5.2 to 40.1 mU/l. The HOMA values ranged from 1.1 to 11.8.
The mean values of cholesterol and the five classes of phospholipids of adipocyte membranes are shown in Table 2. Cholesterol ranged from 110 to 570 µg/mg proteins. Phosphatidylcholine (range: 225-1,217 µg/mg proteins) and phosphatidylethanolamine (range: 110-877 µg/mg proteins) were the more abundant phospholipids, whereas the concentrations of sphingomyelin (range: 33-167 µg/mg proteins), phosphatidylinositol (range: 21-103 µg/mg proteins), and phosphatidylserine (range: 45-276 µg/mg proteins) were less important.
The mean values of PPAR- and TNF-
mRNA levels are shown in Table
3. For PPAR-
, only the total
(
1 +
2) mRNA amount of the nuclear
receptor was analyzed. PPAR-
mRNA concentrations ranged from 7.3 to
68.5 amol/µg total RNA. TNF-
mRNA was very low (0.52 ± 0.07 amol/µg total RNA).
Table 4 shows the Pearson correlation
coefficients between the insulin resistance markers (fasting plasma
insulin levels and HOMA values), the clinical and metabolic
characteristics of the subjects, and adipocyte membrane parameters.
Fasting plasma insulin correlated positively with BMI
(P < 0.05), WHR (P < 0.01), fasting
blood glucose (P < 0.05), membrane sphingomyelin
(P < 0.005), phosphatidylcholine (P < 0.05), and cholesterol (P < 0.005) contents. The most
important correlations were with sphingomyelin (r = 0.698, P <0.005) and cholesterol (r = 0.723, P < 0.005) membrane contents (Fig.
1, A and B). The
correlations with the HOMA estimates of insulin resistance were similar
to those observed with fasting plasma insulin, except for a positive
correlation with phosphatidylethanolamine (P < 0.05)
that does not exist with fasting plasma insulin. Because membrane
sphingomyelin and cholesterol contents were strongly correlated with
the insulin resistance markers, these two membrane components were also
analyzed with regard to the anthropometric variables that characterized
obesity in this study. There was no correlation with BMI or WHR.
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Next, we analyzed the possible relationships with the amounts of the
target mRNAs (Table 4). PPAR- mRNA levels were negatively correlated
with the fasting plasma insulin concentrations (r =
0.470, P < 0.05), and HOMA (r =
0,482,
P < 0.05) and positively correlated with HDL
cholesterol (r = 0.467, P < 0.05).
Figure 2, A and B,
shows the correlations between PPAR-
mRNA levels and fasting plasma
insulin and HDL cholesterol concentrations. There was also a negative
correlation between PPAR-
mRNA levels and the amounts of
sphingomyelin (r =
0.553, P < 0.05)
and cholesterol (r =
0.512, P < 0.05) in the membrane (Fig. 3,
A and B). The concentrations of TNF-
mRNA were
not correlated with any other measured parameter, except with WHR
(r = 0.535, P < 0.05).
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Stepwise multiple regression analyses were performed to evaluate the
relative contribution of biological parameters and membrane lipid
concentrations to the relationships with PPAR- mRNA. In models with
BMI or WHR, plasma lipid parameters, membrane lipid concentrations,
fasting plasma insulin, or HOMA values, the variations in PPAR-
mRNA
concentrations were mainly explained by HDL cholesterol (31.9%) and
membrane sphingomyelin (17.7%; Table
5).
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DISCUSSION |
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We have investigated the relationship between the cholesterol and
phospholipid composition of the adipocyte membranes, insulin resistance
markers, and the expression of the adipose-specific nuclear receptor
PPAR- in subcutaneous adipose tissue in obese women.
The main phospholipids of the adipocyte plasma membrane are phosphatidylcholine and phosphatidylethanolamine, with sphingomyelin, phosphatidylserine, and phosphatidylinositol being less abundant. This phospholipid profile is compatible with data for rat adipocytes (19). The mean values of total phospholipids-to-protein (1.10 ± 0.10), phosphatidylcholine-to-protein (0.45 ± 0.06), and cholesterol-to-protein (0.26 ± 0.04) mass ratios are close to those reported for the adipocyte plasma membranes of massively obese patients (24).
Obese subjects that had a higher sphingomyelin and cholesterol content in their adipocyte membrane are less sensitive to insulin, as assessed by HOMA. These results confirm our recent observations that insulin resistance is associated with an increased sphingomyelin content in erythrocyte membranes (9). Twenty percent of the women in the obese group studied were menopausal, and none was on hormone replacement therapy. The hormonal status may influence the phospholipid composition of the adipocyte plasma membrane. It has been documented that several hormones may influence phospholipid synthesis (35, 39), but there was no correlation between age and membrane sphingomyelin content in the obese group studied, indicating that the observed differences are not likely related to the hormonal status of the patients.
As a nuclear receptor, PPAR- participates in the regulation of genes
involved in the differentiation of adipocytes and also in lipid
metabolism (47). Its activation by the thiazolidinediones leads to the improvement of insulin action in vivo and in vitro (45). PPAR-
is therefore considered as playing a
pivotal role in insulin sensitization and glucose metabolism
(5). We find large individual variations in the amounts of
PPAR-
mRNA in subcutaneous adipose tissue. This is in keeping with
our previous data (3, 43). The variability in the amount
of PPAR-
mRNA is not linked to the BMI of the subjects, as
previously reported (43). The concentration of PPAR-
mRNA is positively correlated with the plasma concentration of HDL
cholesterol and negatively with some markers of insulin resistance,
membrane cholesterol, and sphingomyelin contents. Interestingly, in
another recent study, we found a similar association between HDL
cholesterol and the amounts of mRNA for PPAR-
subcutaneous adipose
tissue (6). Nearly similar correlations in the
intraperitoneal adipose tissue were also recently reported (30). The authors reported that PPAR-
mRNA levels were
negatively correlated with fasting plasma insulin in obese nondiabetic
subjects and positively with HDL cholesterol in postobese subjects. A
high PPAR-
expression and activity may therefore be associated with a high sensitivity to insulin in the adipose tissue of obese subjects. Its is, however, important to note that this relationship between insulin resistance and adipose tissue PPAR-
expression has not been
found when lean controls, obese nondiabetic subjects, and type 2 diabetics patients have been investigated together (43). It seems, therefore, that PPAR-
gene expression may be associated with insulin sensitivity in obese subjects characterized by large differences in fasting plasma insulin, sphingomyelin, and cholesterol contents of their adipocyte membranes.
There may be several links between PPAR- gene expression, plasma
metabolic parameters, adipocyte membrane composition, and impaired
insulin action. In our study, stepwise multiple regression analysis
indicated that plasma HDL cholesterol concentration could explain
~30% of the variance in PPAR-
mRNA levels. This relationship is
supported by recent data showing that adding purified HDL particles to
the culture medium stimulates human preadipocyte differentiation in
vitro (50). It is possible that the induction of the
expression of PPAR-
, a key molecular factor of this process, could
be involved in the adipogenic effect of HDL. However, the mechanisms
whereby plasma HDL cholesterol concentrations are related to the
amounts of PPAR-
mRNA in adipose tissue are not yet clear. Perhaps
the changes in the bidirectional transfer of cholesterol between cells and the HDL particles can participate in the regulation of the expression of the PPAR-
gene. In fact, HDL delivers cholesterol in
an esterified form to adipocytes and removes free cholesterol from the
cells. Adipocytes are enriched in the transcription factor ADD1/SREBP1c
(adipocyte determination differentiation-dependent factor/sterol
regulatory element-binding protein), which is activated by proteolytic
cleavage after cholesterol depletion of the cell (44).
Interestingly, ADD1/SREBP1c has recently been shown to regulate
PPAR-
gene expression in cultured adipocytes, and response elements
have been found in the promoter sequence of the human PPAR-
gene
(14). Moreover, the expression of the activated form of
ADD1/SREBP1c in cultured adipocytes leads to the production of ligands
for PPAR-
(28). Therefore, the intracellular amounts of
free cholesterol may be a link between plasma HDL cholesterol concentrations and PPAR-
gene expression in adipocytes.
Human adipocytes synthesize little cholesterol de novo: intracellular
and membrane cholesterol is derived primarily from lipoproteins (2). The binding capacity of HDL to the membrane receptor
(48) of adipocyte is affected by membrane lipid
composition, particularly by the type of fatty acids in membrane
sphingomyelin (56). In addition, the phospholipid contents
of HDL particles and the cell membranes play a major role in the
distribution of free cholesterol between the cell membrane and HDL
(25). We have found in this study that PPAR- mRNA
levels are negatively correlated with the cholesterol and sphingomyelin
content in the adipocyte membranes. Therefore, changes in intracellular
free cholesterol could be favored by changes in the distribution of
phospholipid classes, especially sphingomyelin and also cholesterol
contents in the adipocyte membrane.
Differences in sphingomyelin concentration appear to explain ~17% of
the variability of PPAR- mRNA in the stepwise multiple regression
analysis. An increased sphingomyelin content in the adipocyte membrane
might also participate in PPAR-
gene expression through an indirect
effect on the transcriptional activity of ADD1/SREBP1c. The
accumulation of sphingomyelin in adipocyte plasma membranes may be due
to a decreased sphingomyelinase activity, as previously reported for
other cell types (13). Recently, sphingomyelinase has been
reported to induce the activation of ADD1/SREBP1 through a
sterol-independent mechanism (32). As discussed above, the
activation of ADD1/SREBP1c could then produce ligands for PPAR-
and
increase PPAR-
expression. A high sphingomyelin content in the
adipocyte membrane may therefore be related to a decreased PPAR-
gene expression and activity through this mechanism.
TNF- was another candidate for the link between insulin resistance
and lipid composition of the membrane. In fact, the intracellular action of TNF-
involves, at least in part, plasma membrane
sphingomyelin to generate ceramide (27). In addition,
TNF-
in cultured adipose cells leads to a decreased expression of
PPAR-
in cultured adipose cells (54). Some data have
also suggested that increased expression of TNF-
in adipose tissue
might be associated with insulin resistance during obesity (23,
26). In agreement with recent works (6, 38), we
found extremely low concentrations of adipose TNF-
mRNA, even in fat
samples from extremely obese women. There was no correlation between
subcutaneous adipose tissue TNF-
mRNA levels and the phospholipid
composition of the adipocyte membrane, insulin resistance markers, or
the BMI in the studied obese women. This extremely low level of
expression of TNF-
was consistent with the lack of significant in
vivo production of the cytokine by human adipose tissue in obese
subjects (37). These results do not, however, exclude the
possibility that TNF-
plays a role in insulin resistance in humans.
In fact, elevated concentrations of circulating TNF-
have been found
in insulin-resistant obese patients with and without type 2 diabetes
mellitus (11, 40, 41). However, it is likely that plasma
TNF-
does not originate from adipose tissue in these situations.
In conclusion, our findings in the adipocytes of insulin-resistant
obese patients pointed to a potentially important role for
sphingomyelin and cholesterol in the membrane. Because strong interactions exist between the cholesterol and sphingomyelin contents of the adipocyte membranes, those that are enriched in cholesterol are
also enriched in sphingomyelin (18). It is likely that
cholesterol levels in the membrane reflect the intracellular
cholesterol concentration, which may in turn affect the regulation of
the expression of several important genes, including the gene for the
nuclear receptor PPAR-. Differences in membrane cholesterol content
between obese individuals may then affect both the structural membrane
properties and the function of the adipocytes, two effects that may
contribute to modifications of insulin action. It is now important to
discover what determines the difference in cholesterol content of the
adipocyte membrane between obese individuals. The altered metabolism of lipoproteins and the interaction of HDL with cell membranes are presently being investigated.
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ACKNOWLEDGEMENTS |
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We thank Drs. Saury and Dinh Doan for help in performing the biopsies. The English text was corrected by Dr. Owen Parkes.
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FOOTNOTES |
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This work was supported in part by a grant from the Association de Langue Française pour l'Etude du Diabète et des Maladies Métaboliques-Institut Servier du Diabète, 1997 and the Fondation pour la Recherche Médicale.
Address for reprint requests and other correspondence: M. Donner, EA 2402, Bâtiment INSERM, CHU de Brabois 54511, Vandoeuvre les Nancy Cédex, France (E-mail: donner{at}u14.nancy.inserm.fr).
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.
Received 21 October 1999; accepted in final form 2 May 2000.
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