1 Unité de
Diabétologie et Nutrition and
2 Ecole de Santé Publique, This time-course
study further explored the mechanisms whereby monoclonal antibodies
(MAbs) may enhance growth hormone (GH) effects. Hypophysectomized rats
were killed 0, 1, 3, 6, 12, 24, and 48 h after a single injection of
bovine (b) GH alone or complexed with an anti-bGH MAb.
Serum insulin-like growth factor I (IGF-I) concentrations were
increased more and for a longer period after MAb-GH complexes (peak at
24 h: 295 ± 24 ng/ml) than after bGH alone (peak at 12 h: 219 ± 37 ng/ml; P < 0.01), whereas liver IGF-I mRNA was similar at 12 h in both groups but remained higher at 24 h (by 65%, P < 0.001) and 48 h (by
64%, P < 0.001) in the presence of
the MAb. Induction of serum insulin-like growth factor-binding protein
(IGFBP)-3 and liver IGFBP-3 mRNA by bGH also was markedly amplified by
the MAb (3.6- and 2-fold at 24 h, respectively;
P < 0.01). GH receptors (GHR)
remained occupied for a longer period after MAb-GH injection (36 ± 16 and 35 ± 8% at 6 and 12 h, respectively) compared with bGH
alone (0 ± 28 and
growth hormone receptor; receptor occupancy; hypophysectomy; bovine
growth hormone
SOME MONOCLONAL ANTIBODIES (MAbs) raised against bovine
growth hormone (bGH) markedly potentiate GH-induced growth (20, 31).
The mechanisms of this potentiation remain unclear and may involve
prolongation of GH systemic half-life, conformational changes of GH
resulting in enhanced bioactivity, or alteration of the
hormone-receptor interaction leading to sustained receptor binding
and/or increased activation of intracellular postreceptor events.
Previous studies in our laboratory have shown that effects of MAbs are
mediated, at least in part, through an increased binding of GH to its
liver somatogenic receptors (GHR), although the in vitro binding of
125I-labeled bGH to liver
homogenates was decreased in the presence of high doses of MAb (20).
This increase was observed 18 h after a single injection of MAb-GH
complexes and might have resulted from a greater occupancy of GH
receptors, from a prolongation of GH binding to its receptors, or from
both mechanisms together.
On the other hand, the action of insulin-like growth factor (IGF-I) is
strongly regulated by multiple IGF-binding proteins (IGFBPs) (27). Most
of the IGF-I in serum circulates as a 150-kDa complex consisting of
IGF-I, IGFBP-3, and a non-IGF-I binding component termed acid-labile
subunit (2). Formation of this large complex alters IGF-I distribution
and clearance and modifies IGF-I bioactivity (15). The GH dependence of
IGFBP-3 was inferred from the decreased levels in hypopituitary
subjects and GH-deficient children and from the increased levels in
acromegalic patients (2, 4). Also, GH administration to
hypophysectomized rats was reported to increase plasma IGFBP-3 levels
(33). Therefore, modification of IGFBP levels in response to injection
of MAb-GH complexes may also occur and contribute to potentiation of GH growth-promoting actions.
To further explore the mechanisms of the MAb-induced potentiation of GH
effects, we investigated in the present study the kinetics of hepatic
GH binding and serum IGFBP levels in response to MAb-GH complexes in
hypophysectomized rats.
Animals. Female Wistar rats were
hypophysectomized at 4 wk of age (body weight: 70.0 ± 0.1 g) (IFFA
CREDO, Lyon, France) and delivered to our animal quarters 1 wk after
surgery. They were housed under controlled conditions of lighting
(lights on from 7:00 AM to 7:00 PM) and temperature (22 ± 2°C),
with free access to standard rat chow pellets and tap water containing
0.9% (wt/vol) NaCl. Experiments were initiated after a 7-day
adaptation period, and hypophysectomized animals gaining more than 5 g
during that week were discarded from the study.
Hormones. Methionyl bGH (M1 L126)
kindly supplied by Monsanto (St. Louis, MO) was used for injecting the
rats and for the development of monoclonal antibodies. The sodium salt
of L-thyroxine (T4) and cortisol hemisuccinate
(Solucortef) were purchased from Aldrich (Milwaukee, WI) and Upjohn
(Kalamazoo, MI), respectively. Human IGF-I (International, Amersham,
Buckinghamshire, UK) served as tracer and standard in the IGF-I RIA.
The bGH used as tracer in the binding studies was a generous gift from
Dr. A. C. Paladini (Buenos Aires, Argentina), whereas bGH (B1;
AFP-5200) prepared by Dr. A. F. Parlow and obtained through the U.S.
Department of Agriculture Animal Hormone Program (National Institutes
of Health, Bethesda, MD) was used as unlabeled hormone for the
determination of nonspecific binding. Hormones were labeled with
125I, with the lactoperoxidase
method (16).
Monoclonal antibodies. As described
previously (20), anti-bGH MAbs were raised by classical methods
[culture and cloning of hybridoma cell lines (mouse spleen cells × BALB/C myeloma cells) secreting mouse monoclonal
IgG antibodies against bGH; generation of ascitic fluid by
intraperitoneal injection in mice; sodium sulfate purification]
and were selected according to their ability to bind
125I-labeled bGH in solution.
Their affinity was determined by Scatchard analysis of displacement
curves (16), obtained with increasing amounts of unlabeled GH to
compete with 125I-labeled bGH for
binding to MAbs. One MAb (6B1) that had the most potentiating effects
on GH activity was further characterized and selected for subsequent
studies (20). A monoclonal antibody directed against a viral protein of
bovine leukemia virus (gp 51) was used as a negative control. No
potentiating effect of GH action was observed with this MAb anti-gp 51 (data not shown).
Experimental procedure.
Hypophysectomized rats were pretreated during 7 days with a daily
subcutaneous injection of T4 (1 µg/100 g body wt) and cortisol (50 µg/100 g body wt), which caused body weight to increase slightly in most animals (+ 5.4 ± 1.9 g
over 1 wk; means ± SD). Hypophysectomized rats
(n = 56) were injected on
day 8 with bGH (100 µg/100 g body wt) alone or complexed with anti-bGH MAb
6B1 (40 µg/100 g; molar ratio of MAb to GH = 0.05). Hormone-antibody
complexes were allowed to form before injection by preincubation for 3 h at 4°C.
Animals (n = 4/group) were weighed and
killed by decapitation without anesthesia 0, 1, 3, 6, 12, and 24 h
after a single injection of bGH alone or MAb-GH complexes. Trunk blood
was collected in glass tubes, and the serum was frozen at
Extraction of serum IGF-I and RIA.
Serum concentrations of IGF-I were measured by RIA after
acid-extraction of serum samples with 0.5 mol HCl/l and silicic acid
columns (C18 Sep-Pak cartridges; Waters Associates,
Milford, MA), as described previously (17). With the use
of this technique, 77 ± 5% (means ± SD;
n = 4) of the IGF-I was recovered from
adult rat serum and >99% of the binding protein was removed (17,
23). The human IGF-I antiserum (UB 286) was a kind gift from Dr. L. E. Underwood (Chapel Hill, NC). The sensitivity of this assay was 25 pmol/l, and intra- and interassay coefficients of variation including
the extraction procedure were 6.0 and 6.2%, respectively.
Northern blot analysis. RNA was
isolated from liver tissue of each animal with the guanidine
thiocyanate-cesium chloride method (6). Total RNA was size-fractionated
on 1% agarose gels and transferred to nylon membranes (Hybond-N,
Amersham, Buckinghamshire, UK) by overnight vacuum blotting (VacuGene
XL, Pharmacia LKB, Sweden). After ultraviolet cross-linking
(Stratalinker, Stratagene, La Jolla, CA), the membranes were
sequentially hybridized to rat 32P-labeled complementary RNA
probes specific for GHR, IGF-I, and IGFBP-3 mRNA, with previously
reported prehybridization, hybridization, and washing conditions (30).
The specific IGF-I antisense probe was generated from the 194-bp
Ava
II-Hinf I cDNA rat IGF-I exon 3 fragment inserted into the Bluescript plasmid vector; the
GHR/GH-binding protein (GHBP) antisense probe was generated from the
951-bp Bgl II rat GHR cDNA fragment
subcloned into the
pT7T3
vector; and the IGFBP-3 riboprobe was generated from the 452-bp
Hind
III-BamH I rat IGFBP-3 cDNA fragment
inserted into the Bluescript vector, with
T7 or
T3 RNA polymerase (Life
Technologies, Merelbeke, Belgium). An 18S
32P-labeled oligonucleotide was
used as a control probe. Relative expression of mRNA in each liver was
quantified by scanning autoradiograms with a LKB Ultroscan XL Laser
Densitometer (Pharmacia, Sweden). Results were normalized by assigning
a value of 1 arbitrary densitometric unit (ADU) to hepatic mRNA from
hypophysectomized rats killed at time
0.
Binding studies. Somatogenic binding
sites were measured in each rat individually on 2,500 g pellets of liver homogenates previously treated with 4 mol/l
MgCl2 to remove endogenous ligand to provide a measure of the total GH-binding sites (18, 19). Parallel
studies were performed on water-treated homogenates to measure the free
binding sites. Fractional occupancy of the receptors was calculated by
the equation: 100 × (total binding sites Western ligand blot analysis. To
assess IGFBPs in serum, ligand blotting was performed as previously
described (14). Sodium dodecyl sulfate-polyacrylamide gels (12.5%)
were cast with a Hoefer Mighty Small II slab gel system (Hoefer, San
Francisco, CA). Electrophoresed proteins were transferred to
nitrocellulose membranes that were incubated for 24 h with 250,000 counts · min Statistical analyses. Data were
analyzed with the BMDP New System professional (University of
California Press, Berkeley, 1995) statistical software. All parameters
were compared between GH and MAb-GH groups at the different time points
with a two-way analysis of variance with Fisher's
F tests when variances were equal, or
with Brown-Forsythe F tests when the
assumption of equal variances was not satisfied (5). Serum IGF-I, liver
IGFBP-3 mRNA, serum IGFBPs, and GH-receptor occupancy values were log transformed before analysis because of a proportional relationship between means and SD in raw data (11). Results are reported as means ± SE. The P values were corrected
for multiple comparisons with a Bonferroni criterion (11). All tests
were two-sided, and a P value <0.05
was considered significant.
Injection of MAb-GH complexes in hypophysectomized animals caused both
a potentiation and a prolongation of the body weight gain response to
bGH. A significant weight gain was already observed 12 h after
injection of MAb-GH complexes (2.6 ± 0.7 g), whereas no change in
body weight was recorded after bGH alone at this time ( To further explore the mechanisms of MAb growth-promoting effects, we
studied the time-dependent effects of the MAb on the serum IGF-I
response to GH. After a single injection of bGH, serum IGF-I
concentrations peaked at 12 h (219 ± 37 ng/ml) and rapidly returned
to preinjection values by 24 h (Fig. 1).
When bGH was injected complexed to the anti-bGH MAb, a higher IGF-I
peak was reached after 24 h (295 ± 24 ng/ml;
P < 0.01), and after 48 h, serum
IGF-I levels were still significantly elevated compared with values
observed after bGH alone. When bGH was complexed with a monoclonal
antibody directed against an unrelated viral protein (MAb anti-gp 51),
no enhancing effect was observed on body weight gain or serum IGF-I
(data not shown). Further studies were therefore not pursued with this
antibody.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
15 ± 11%), whereas total liver GH-binding sites and GHR mRNA levels were not affected by the
MAb. We conclude that MAbs against GH amplify and prolong the serum
IGF-I response to GH, which may result from both a prolongation of
liver IGF-I synthesis and an enhanced induction of IGFBP-3. These two
effects may in turn be the consequences of sustained GH binding to its
liver receptors in the presence of MAb.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20°C for determination of IGF-I concentrations. Livers were
quickly removed, and a piece was flash-frozen in liquid nitrogen for
determination of liver IGF-I mRNA. The remaining tissue was homogenized
in 10% (wt/vol) ice-cold sucrose buffer (0.3 mol/l), as described
previously (16). The homogenates were stored at
20°C until
assayed for total and free bGH-binding sites.
free binding
sites)/total binding sites. Specific binding (B) was the difference
between the radioactivity bound in the absence and in the presence of
unlabeled hormone and expressed as a percentage of the total
radioactivity (T): B/T × 100. MgCl2 treatment caused a protein
loss of 19.0 ± 3.7% (means ± SD), and the binding values were
corrected for this protein loss (19).
1 · ml
1
of 125I-labeled IGF-II (250 µCi/µg) and were washed and subjected to autoradiography. The autoradiograms were scanned with an
Ultroscan XL (LKB) apparatus. For each sample, density signals were
determined for the 45,000-39,000 relative molecular weight
(Mr) (glycosylated variants of
IGFBP-3), 34,000-29,000 Mr
(IGFBP-1 and -2, and glycosylated IGFBP-4, -5, and -6), and 24,000 Mr (nonglycosylated IGFBP-4) bands. The 34,000-29,000 Mr
IGFBP cluster was analyzed together because it could not be reliably
separated for quantification by scanning densitometry, and it was
assumed to contain IGFBP-1 and -2 predominantly.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0.5 ± 0.6 g; P < 0.01). After 24 and 48 h,
the body weight gain of the MAb-GH injected rats was 1.5- and 6-fold
higher than that of the rats injected with bGH alone (body wt gain: 6.6 ± 0.7 vs. 4.3 ± 0.5 g; P < 0.05 and 5.9 ± 0.6 vs. 0.9 ± 1.0 g;
P < 0.001, respectively).
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Fig. 1.
Serum insulin-like growth factor I (IGF-I) concentrations of female
hypophysectomized rats at different time intervals after a single sc
injection of bovine growth hormone (bGH, 100 µg; ), either alone
or complexed with monoclonal antibody (MAb) 6B1 (40 µg;
). Results
are shown as the means ± SE of 4 rats/group.
*** P < 0.001 vs. group
receiving bGH alone.
We next studied the effect of MAb on the time course of GH-induced
IGF-I gene expression. Liver IGF-I mRNA levels were markedly induced by
GH injection, with a maximal increase by ninefold at 6 and 12 h (Fig.
2). Similar peak levels were reached after
injection of MAb-GH complexes, but thereafter IGF-I mRNA abundance
remained significantly higher than after bGH alone (by 65% at 24 h,
P < 0.001 and by 64% at 48 h,
P < 0.05). This sustained
increase of IGF-I gene expression was similarly observed for the four
IGF-I mRNA size classes of transcripts (7.5-, 4.5-, 1.7-, 1.2- 0.7-kb transcripts).
|
Because the IGFBPs may modulate IGF-I clearance and actions, we also
investigated the effects of the MAb on serum IGFBPs. GH injection to
hypophysectomized rats increased serum IGFBP-1 to -4 (Fig.
3). Serum IGFBP-3 levels were increased
fivefold (P < 0.001), and serum
IGFBP-4 levels were increased twofold
(P < 0.01) 12 h after bGH injection,
whereas IGFBP-1 and -2 levels rose twofold at 24 and 48 h
(P < 0.05). The MAb did not show any significant potentiating effect on the response of IGFBP-1, -2, and -4 to GH. In contrast, the elevation of serum IGFBP-3 levels in response
to GH was markedly amplified and prolonged, being 3.6-fold higher
(P < 0.01) 24 h after the MAb-GH
injection compared with bGH injection alone.
|
GH administration after 12 h caused a twofold increase in IGFBP-3 mRNA
levels (P < 0.05) whether injected
alone or complexed to the MAb (Fig. 4).
This response was sustained in the presence of MAb, as IGFBP-3 mRNA
levels remained twofold higher (P < 0.01) 24 h after MAb-GH injection compared with levels observed after injection of bGH alone.
|
To explain this MAb-related prolongation of IGF-I and IGFBP-3
synthesis, we evaluated the effects of the MAb at the hepatic GH-receptor level. We determined its effects on liver GHR and GHBP mRNA
levels and on liver GH binding at different time points. After GH
injection, a slight increase in liver GHR and GHBP mRNAs levels was
observed 12 h after injection of bGH
(P < 0.01 and P < 0.05, respectively, compared
with time
0) (Fig.
5), but addition of the MAb did not modify
this slight induction of GHR and GHBP gene expression. On the other
hand, total liver GH receptors were not significantly affected by
injection of bGH alone or complexed with the MAb (respectively 4.0 ± 0.4 vs. 4.0 ± 0.9 B/T×100/mg protein, at 12 h).
|
After a single injection of bGH or MAb-GH complexes, free liver
GH-binding sites decreased rapidly to a similar extent in both groups
(94% after bGH and
95% after MAb-GH at 1 h). In the
group treated with bGH alone, free binding sites returned to
preinjection values by 12 h, whereas a slower recovery of the free
binding sites occurred after MAb-GH injection (4.6 ± 0.5 after bGH
vs. 2.4 ± 0.3 B/T×100/mg protein after MAb-GH, at 12 h;
P < 0.05). At 24 and 48 h, the
number of free binding sites was again similar in both groups. From the
determination of total and free binding sites, occupancy of liver GH
receptors could be calculated (Fig. 6).
This occupancy was nearly complete 1 h after bGH was injected either
alone or complexed to the MAb. Thereafter, GH-receptor occupancy
decreased rapidly in GH-injected hypophysectomized animals but remained
significantly elevated up to 12 h after the injection of MAb-GH
complexes, suggesting a sustained binding of GH to its liver receptors
when injected complexed to the monoclonal antibody.
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DISCUSSION |
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The present time-course study demonstrates that a MAb against bGH both amplifies and prolongs the serum IGF-I response to GH in hypophysectomized rats. This effect seems to result in part from a prolongation of liver IGF-I synthesis and also from an enhanced induction of IGFBP-3 in serum, leading to an extended half-life of IGF-I in the circulation. Increased liver IGF-I and IGFBP-3 synthesis may in turn be the consequences of sustained binding of GH to its liver receptors in the presence of MAb, as evidenced by our time-course study of GH-receptor occupancy.
We show here that the potentiation of IGF-I response by the MAb is due to amplified and sustained IGF-I levels in the circulation rather than early greater induction of the growth factor. In fact, only the late phase of the GH response is affected by the MAb. Indeed, serum IGF-I levels raised similarly during the first 12 h regardless of the administration of the MAb. Later, the MAb complexed to GH delayed and amplified the peak of serum IGF-I concentrations. MAbs also seem to slow down the return of serum IGF-I to preinjection values. This accumulation of IGF-I in serum might result either from a reduction of the peptide clearance rate from the circulation or from a prolonged synthesis in the presence of the MAb. Our data suggest that both mechanisms might be operative.
No change in body weight was observed 12 h after GH injection, whereas, at the same time, MAb-GH injection already caused a significant increase in body weight. This early effect of MAb on weight gain contrasts with its delayed effects on IGF-I, therefore suggesting that part of the GH effects on body weight is independent of IGF-I stimulation. MAbs could indeed enhance the direct effects of GH on sodium and water retention by the kidney, which are commonly recorded during the initial phase of GH treatment (24). In addition, MAbs are able to potentiate other anabolic, galactopoietic, and diabetogenic effects of GH unrelated to IGF-I production (1, 25).
GH administration increased serum IGFBP-3 levels, and this induction was markedly potentiated by coadministration of the MAb, as higher serum IGFBP-3 levels were observed at 12, 24, and 48 h. This might account in part for the prolongation of high IGF-I concentrations in serum as the half-life of IGF-I is extended when associated with the IGFBP-3 in a ternary complex (32). Evidence for potentiation of IGF-I action by IGFBP-3 is also provided by several studies (7, 9, 10, 13). In particular, it has been shown that injection of human IGFBP-3 together with IGF-I to GH-deficient rats causes significant increase in body weight gain and epiphyseal width compared with IGF-I alone (7). These effects may be related to prevention of type I IGF-receptor downregulation (8) or to the ability of IGFBP-3 to associate with cellular membrane, lowering its affinity for IGF-I and favoring receptor availability of IGF-I (22). Finally, binding protein-complexed IGF-I may constitute a slow-release form that may also temporarily protect IGF-I from degradation until it is released from the complexes by proteolytic processing of the IGFBP, which reduces its affinity for the ligand (34). The marked potentiating effect of MAb-GH coadministration on IGFBP-3 levels may therefore be one of the pivotal mechanisms by which monoclonal antibodies amplify GH growth-promoting actions.
We also show in the present report that the MAb is able to prolong the GH-induced IGFBP-3 mRNA synthesis up to 24 h after injection. The parallel time course between serum IGFBP-3 peptide and hepatic IGFBP-3 mRNA concentrations in response to MAb-GH injection suggests that the MAb potentiates the GH-induced IGFBP-3 levels at the level of gene expression.
In a previous study, we reported that GH-receptor occupancy was increased sixfold 18 h after injection of MAb-GH complexes (20). We now demonstrate that this increase is due to a prolongation rather than an amplification of receptor occupancy and that GHR gene expression and total liver GH-binding sites are not modified in the presence of the MAb. Two separate mechanisms may explain the prolongation of GH binding to its liver receptors: prolonged half-life of the hormone in serum or retarded internalization of the GH-GHR complex in the presence of the MAb.
Prolongation of the systemic half-life of exogenous insulin may occur in diabetic patients with insulin autoantibodies and may enhance its biological activity (12). Likewise, the MAb anti-bGH might induce prolonged GH levels in circulation, and this would account for prolonged GH-receptor occupancy (3). Serum GH concentrations and receptor occupancy have indeed been shown to correlate well in hypophysectomized rats (18). However, it is unlikely the sole explanation for MAb-mediated enhancement of GH action. Indeed, continuously raised GH levels rather produce attenuated body growth gain and lower serum IGF-I in hypophysectomized rats when compared with intermittent peaks of hormone levels (18). Furthermore, we found that a continuous infusion of GH for 3 days was less efficient to induce body weight gain and serum IGF-I than daily injections of MAb-GH complexes for the same period (unpublished data).
It has been reported that after MAb-GH complexes have been injected in intact rats, their uptake by the liver is delayed, compared with GH alone (28). Moreover, MAb-GH complexes are recovered to a large extent within sinusoidal cells, whereas the hormone alone is mostly found in hepatocytes (28). These observations support the view that prolonged receptor occupancy might be due to modification in the intracellular processing of the GH-GHR complex in the presence of the MAb. Increased affinity of the receptor for GH could also be implicated through allosteric conformational change of the hormone induced by the MAb (21). Indeed in vitro studies show that MAbs that potentiate GH-induced proliferation of cultured Nb2 cells also enhance the affinity of GH for its receptors (26). This possibility cannot be ruled out in our in vivo experiment as Scatchard analysis was not performed.
Little is known about the quantitative relationship between liver GH binding and biological activity. Our data provide support for a tight and sustained coupling between both phenomenons. Prolonged receptor occupancy seems to lead to sustained IGF-I synthesis, in agreement with in vitro data showing that incubation of cultured hepatocytes with GH progressively increased IGF-I mRNA levels up to 24 h (29). Furthermore, in hypophysectomized rats, continuous infusion of GH induces a dose-related receptor occupancy that correlates with serum IGF-I levels (18).
In summary, our kinetics study provides new insights into the mechanisms by which monoclonal antibodies may potentiate growth hormone action. The MAbs affect mainly the late phase of the somatogenic response to GH. They prolong the binding of GH to its liver receptors, leading thereby to both a prolonged liver IGF-I synthesis and an increase of IGFBP-3 production. These two mechanisms act concurrently to maintain elevated IGF-I levels in serum and to potentiate GH growth-promoting actions.
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ACKNOWLEDGEMENTS |
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We wish to thank P. Malvaux and M. Maes for continuous support. This work was supported by a grant (2.4555.92) from the National Fund for Scientific Research (Belgium).
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Maiter, Unité de Diabétologie et Nutrition, UCL DIAB 5474, Av. Hippocrate 10, B-1200 Brussels, Belgium (E-mail: maiter{at}diab.ucl.ac.be).
Received 13 November 1998; accepted in final form 22 April 1999.
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