| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Department of Endocrinology (M) and Center for Eating Disorders, Odense University Hospital (R.K.S., J.H., M.H.-N., C.H.), DK-5000 Odense; and Medical Department M (Endocrinology and Diabetes), Medical Research Laboratory and Institute of Experimental Clinical Research, University Hospital of Aarhus (A.F., J.F., S.F.), DK-8000 Aarhus, Denmark
Address all correspondence and requests for reprints to: René Klinkby Støving, M.D., Department of Endocrinology (M), Odense University Hospital, DK-5000 Odense C, Denmark.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The genesis and the consequences of the altered IGF-IGFBP axis in AN are far from understood. The present study was performed to address the questions of whether the abnormalities are similar to those of other catabolic GH resistance conditions and especially to clarify whether the levels of free IGF-I and -II are changed and whether AN is associated with increased IGFBP-3 proteolytic activity.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
We studied 24 women with AN and 10 healthy age-matched women as
controls. The clinical and biochemical characteristics of the subjects
are summarized in Table 1
. The patients
met the DSM-IV criteria for AN (16), and the diagnosis of AN was made
by the division of psychiatry of the same hospital. The duration of
disease (defined as significant weight loss according to the DSM-IV
criteria) for all the patients was 1 yr or longer (range, 1–22 yr; for
19 of 24 of the patients, it was between 1–10 yr). None of the
patients received any drug therapy for at least 6 months before
entering the study. The patients were all normohydrated, and they had
low serum levels of gonadotropins, estradiol (E2)
and total T3. The controls were not receiving any
medication, and they all passed a physical examination and a routine
laboratory screening, including gonadotropins,
E2, T3, fasting blood glucose, and
insulin. All of the control subjects had regular menstrual cycles, and
the blood sampling took place during the follicular stage (days 2–8).
Twelve of the patients gained weight, as illustrated in Table 1, and
were restudied after 3 months. Before the second blood sampling, the
patients were out-patients, and their weights were relatively
stationary (the weight changes in the previous 2 weeks were <5%).
Twelve patients were not restudied; they either failed to gain weight,
were medicated, or had finished their treatment in our department. All
blood samples were obtained at 0800 h in the supine position after
an overnight fast. For determination of GH, two samples were taken at
0800 and 0900 h, respectively. The study was approved by the local
ethical committee, and all the participants signed informed
consent.
|
All measurements were performed in duplicate within the same assay. With the exception of free IGF-I and free IGF-II, all intra- and interassay coefficients of variation (CVs) were less than 5% and 10%, respectively. GH levels were determined by an immunofluorometric assay (Delfia, Wallac Oy, Turku, Finland). GHBP was determined by an immunofunctional time-resolved fluoroimmunoassay, as described previously (17). Serum total IGF-I and -II were determined after acid-ethanol extraction using noncompetitive time-resolved monoclonal immunofluorometric assays as previously described (18). Serum free IGF-I and -II were determined using ultrafiltration by centrifugation as previously described (19). In brief, Amicon YMT 30 membranes and MPS-1 supporting devices were used (Amicon Division, W. R. Grace, Beverly, MA). Before centrifugation, serum samples were diluted (1:11) in Krebs-Ringer bicarbonate buffer (pH 7.4) containing 50 g/L human serum albumin (Behring AG, Marburg, Germany). From each dilution, triplicate samples of 600 µL were applied to the membranes, incubated (30 min at 37 C), and centrifuged (1500 rpm at 37 C; model Rotixa/RP, Hettich Zentrifugen, Tuttlingen, Germany). The lower detection limits of free IGF-I and -II in the ultrafiltrates were 50 and 150 ng/L, respectively. The intraassay CVs, including ultrafiltration and immunoassay, averaged 19% for free IGF-I and 13% for free IGF-II.
Serum IGFBP-2 and IGFBP-3 were measured by RIA and immunoradiometric assay, respectively (Diagnostic System Laboratories, Inc., Webster, TX). Serum IGFBP-1 was determined by an enzyme-linked immunosorbent assay (Medix Biochemica, Kauniainen, Finland).
Serum E2 was determined by RIA (Orion Diagnostica, Espoo, Finland), serum LH and FSH were determined by immunofluorometric assay (Delfia, Wallac Oy), total T3 was determined by RIA (Amerlex-M, Ortho Clinical Diagnostics, Amersham, Aylesbury, UK), insulin was determined by a double antibody RIA (Kabi Pharmacia Diagnotics AB, Uppsala, Sweden), and P-glucose was determined by glucose dehydrogenase (D-6100, Merck, Darmstadt, Germany).
Western ligand blot (WLB) of serum IGFBPs
Two microliters of serum were subjected to WLB to attain an
additional confirmation of the immunoreactive IGFBP-1, IGFBP-2, and
IGFBP-3 levels and to determine changes in IGFBP-4.
SDS-PAGE and ligand blot analysis were performed according to the
method of Hossenlopp et al. (20), as previously
described (21). Serum was subjected to SDS-PAGE (10% polyacrylamide)
under nonreducing conditions. The specificity of the IGFBP bands was
ensured by competitive coincubation with unlabeled IGF-I purchased from
Bachem (Bubendorf, Switzerland). All samples from each subject were
analyzed in the same gel. A representative WLB autoradiograph is shown
in Fig. 1.
|
The IGFBP-3 protease assays were performed as previously
described using human recombinant [125I]IGFBP-3 obtained
from Diagnostic System Laboratories, Inc. (22).
[125I]IGFBP-3 (
30,000 cpm) was incubated for 18 h
at 37 C with 2 µL serum from controls/patients and subjected to
SDS-PAGE as described above. On each gel, internal control sera from
normal controls and term pregnant subjects were included.
Electrophoresed gels were fixed in a solution of 7% acetic acid,
dried, and autoradiographed. The amount of proteolysis for each sample
was given as the percentage of proteolytic cleavage products for each
lane (in vitro proteolysis). A representative IGFBP-3
protease autoradiogram is shown in Fig. 2.
|
Autoradiograms of WLBs and IGFBP-3 protease assay were quantified by densitometry using a Shimadzu CS-9001 PC dual wavelength flying spot scanner (Shimadzu Europe, Duisburg, Germany). The relative density of the bands was measured as arbitrary absorbance units (AU) per mm2.
Dual energy x-ray absorptiometry
The percentage of body fat was measured by dual energy x-ray absorptiometry, using a Hologic QDR-2000 densitometer (Waltham, MA). All scans were performed in single beam mode.
Statistics
Results are expressed as group mean values (±SEM), and comparisons between groups were performed using the Mann-Whitney test (except in the paired weight gain study, where the Wilcoxon test was used). Bivariate correlations were estimated using Spearman coefficients. P < 0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Serum levels of basal GH, GHBP, IGFs, and IGFBPs are summarized in
Table 1
. The GHBP level was significantly decreased in the anorectic
patients. In the AN group (n = 24), GHBP was a significantly
correlated to body mass index (BMI; r = 0.65; P <
0.01) and percent body fat (r = 0.66; P <
10-4). In the whole group (n = 34), GHBP levels were
correlated with total IGF-I levels (r = 0.66; P <
0.01), but not with free IGF-I levels (r = 0.26; P = 0.22).
Total IGF-I levels were significantly lower in the AN group than in the
control group, whereas there was no difference in total IGF-II levels.
However, the free fractions of IGF-I as well as IGF-II were markedly
decreased in the anorectic patients. When comparing patients with AN
and controls, free IGF-I (69%) and free IGF-II (54%) were relatively
more reduced than total IGF-I (32%) and total IGF-II (12%). The
free/total IGF-I ratios were 2.4 ± 0.4 x 10-3
for the anorectics and 5.5 ± 0.7 x 10-3 for
the controls (P < 0.001). The free/total IGF-II ratios
were 0.7 ± 0.1 x 10-3 for the anorectics and
1.4 ± 0.2 x 10-3 for the controls
(P < 0.001). In the AN patients, total IGF-I levels
were significantly correlated to BMI (r = 0.56; P
< 0.01), whereas free IGF-I levels were not (r = 0.36;
P = 0.08).
The mean levels of serum RIA-determined IGFBP-3 were slightly lower in AN than in controls (P < 0.05), whereas WLB-determined IGFBP-3 only tended to be lower in AN (1857 ± 112 vs. 2160 ± 113 AU/mm2 in the controls; P = 0.12). There was a significant correlation between IGFBP-3 concentrations measured by RIA and those measured by WLB (r = 0.77; P < 10-4). There were no significant differences between the groups with respect to [125I]IGFBP-3 proteolysis (21.7 ± 0.7 vs. 20.1 ± 1.2% in the controls). The mean IGFBP-1 level was increased in the anorectic patients, and this was confirmed by the WLB technique (172 ± 42 vs. 92 ± 9 AU/mm2 in the controls; P < 0.05). The correlation between the RIA and the WLB measurements of IGFBP-1 was significant (r = 0.64; P < 0.01). The RIA-determined IGFBP-2 levels were not significantly changed in AN (P = 0.26), whereas for WLB measurements, the IGFBP-2 levels were significantly higher in the anorectic group (410 ± 50 vs. 214 ± 37 AU/mm2; P < 0.05). The Spearman coefficient for the relation between the IGFBP-2 levels measured by RIA and those measured by WLB was r = 0.94; P < 10-4. IGFBP-4 was only determined semiquantitatively by WLB, and we did not find a significant difference between the anorectics and the controls.
Effect of weight gain
The results are reported in Table 1. After a weight gain from BMI
14.1 ± 0.6 to 16.1 ± 0.5 kg/m2
(P < 0.05) over 3 months, the serum levels of GHBP did
not change significantly (P = 0.24). The free and total
IGF levels increased significantly. The increases in the free factions
were more pronounced than those in the total IGFs (increase in mean
serum IGF-I during weight gain, 41% in total IGF-I vs.
119% in free IGF-I; increase in mean serum IGF-II during weight gain,
21% in total IGF-II vs. 46% in free IGF-II). All IGFBPs
(both immunoassay and WLB measurements) changed in the control
direction; however, during this minor weight gain, the changes in
IGFBP-3 and IGFBP-4 were not significant.
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
In contrast to our results, Argente et al. (6) recently
reported normal levels of free IGF-I in anorectic patients, and thereby
suggested that although they have low total IGF-I levels, AN patients
may not be deficient in biologically active IGF-I. In general,
discrepant findings in endocrine studies of AN might be related to
differences in study populations, as early stage patients probably
differ from those with long term histories (23). The study population
of Argente et al. (6) was comparable with that of ours with
respect to diagnostic criteria and duration of disease (all patients
had 
1-yr duration of disease). However, it is not clear whether the
patients reported by Argente et al. (6) were treated with
psychotropic drugs, whereas none of our patients were medicated.
Although there is no evidence that antipsychotic or antidepressants
should change the equilibrium between free and bound IGFs, this cannot
be excluded. Most likely, the discrepancy between the two studies is
due to methodological differences, as the analysis of Argente et
al. (6) was based on a nonequilibrium direct immunoradiometric
determination of free IGF-I in serum using antibodies directed against
the unbound peptide under conditions far different from those in
vivo. It has been shown previously that the equilibrium between
free and bound IGF is strongly dependent on temperature, pH, ionic
milieu, and dilution (19). Therefore, we used a validated equilibrium
assay for the determination of free IGFs (19). Using this method, we
observed a highly significant reduction in free IGF-I and free IGF-II
levels in AN. Moreover, the free/total ratios for IGF-I and IGF-II were
significantly lower, suggesting that the relative bioavailable IGF
fractions are also reduced in AN patients. After a minor weight gain,
the relative increases in the free fractions were more pronounced than
those in the total, indicating that ultrafiltrated free IGF levels are
more sensitive as nutritional markers than are total IGF levels. One
mechanism by which the IGF actions are modified by nutritional factors
may be through alterations in IGFBPs. In the present study, the effects
of refeeding on IGFBP-2, IGFBP-3, and IGFBP-4 were modest compared with
those of IGFBP-1, suggesting that the mechanism of the nutritional
effect on IGFBP-1 is different from that on the other IGFBPs.
Presumably, IGFBP-1 is not mainly a carrier of IGFs, but, rather, acts
as a modulator of IGF metabolic activities. It has been shown in animal
experiments that IGFBP-1 is an inhibitor of IGF-I activity (24). In
this role, IGFBP-1 can be viewed as an insulin and IGF-I
counterregulator, which protects AN patients from hypoglycemia.
Proteolytic cleavage of IGFBP-3 may be an important regulation of IGF
action, although the consequences of IGFBP-3 proteolysis on IGF
bioavailability are still a matter of dispute. It has previously been
shown that IGFBP-3 fragments have a markedly reduced affinity for IGF-I
(25, 26). In line with this, induction of IGFBP-3 proteolysis may be an
adaptive mechanism by which the availability of IGF-I is increased or
maintained at a normal level in various physiological and
pathophysiological conditions. From a methodological point of view, the
induction of IGFBP-3 proteolysis may cause problems when IGFBP-3 is
measured by conventional assays, as IGFBP-3 fragments in most
immunoassays may be indistinguishable from intact IGFBP-3, but when
measured by the WLB technique only intact IGFBP-3 is determined.
Accordingly, conditions characterized by the presence of IGFBP-3
proteolytic activity will show a discrepancy between IGFBP-3 levels
measured by the two methods (15). In the present study, good accordance
was found between immunoassayable and WLB-measured IGFBP-3, giving no
evidence for the presence of IGFBP-3 proteolytic activity in patients
with AN. Furthermore, we observed no increased IGFBP-3 proteolytic
activity in AN patients using a direct, in vitro IGFBP-3
proteolytic assay.
In accordance with previous reports (4, 5, 6), we found markedly decreased levels of serum GHBP in AN, correlating with total IGF-I levels. This is consistent with the findings of low GHBP levels in other forms of malnutrition (27, 28). Estrogens have been found to increase GHBP levels in young (29) as well as postmenopausal women (30). In AN, hypoestrogenemia is a well known feature. However, we found no significant correlation between GHBP levels and levels of E2, suggesting that E2 is not an independent determinant of GHBP levels. In a study population consisting of patients with AN, normal weight subjects, and obese subjects, Postel-Vinay et al. (27) found a strong correlation between BMI and GHBP levels. Our data confirm this finding; moreover, GHBP levels were significantly correlated to body fat percentages in the AN patients, which is in accordance with the observations that abdominal fat correlates positively to GHBP levels in healthy subjects (31) as well as in GH-deficient adults (32). It has not been clarified whether circulating GHBP derives solely from GH receptors in the liver or GH receptors in other tissues, e.g. fat tissue. In AN patients, diminished production of circulating GHBP from adipose tissue in addition to reduced density of GH receptors in the liver may explain the low levels of GHBP.
In critical illness, GH resistance has been considered a stereotype stress reaction, along with hypercatabolism (14) and autocannibalism (33), which may delay recovery. The present data suggest that the adaptation of the IGF-IGFBP axis in AN is different from that in other catabolic GH resistance syndromes, as the low free IGF-I level was not associated with increased IGFBP-3 proteolysis. We speculate that this difference could be due to the fact that the emaciation in AN is usually a chronic condition developed slowly over several months, in contrast to the more rapid changes in most critically ill patients. Further studies are warranted to clarify this matter.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received August 3, 1998.
Revised November 17, 1998.
Accepted November 23, 1998.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
2-Adrenoceptor sensitivity in anorexia nervosa: GH
response to clonidine or GHRH stimulation. Biol Psychiatry. 25:256–264.[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  A. Giustina, G. Mazziotti, and E. Canalis Growth Hormone, Insulin-Like Growth Factors, and the Skeleton Endocr. Rev., August 1, 2008; 29(5): 535 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Stoving, J.-W. Chen, D. Glintborg, K. Brixen, A. Flyvbjerg, K. Horder, and J. Frystyk Bioactive Insulin-Like Growth Factor (IGF) I and IGF-Binding Protein-1 in Anorexia Nervosa J. Clin. Endocrinol. Metab., June 1, 2007; 92(6): 2323 - 2329. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Priego, M. Granado, A. I. Martin, A. Lopez-Calderon, and M. A. Villanua Dexamethasone administration attenuates the inhibitory effect of lipopolysaccharide on IGF-I and IGF-binding protein-3 in adult rats J. Endocrinol., June 1, 2005; 185(3): 467 - 476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Heer, C. Mika, I. Grzella, N. Heussen, and B. Herpertz-Dahlmann Bone turnover during inpatient nutritional therapy and outpatient follow-up in patients with anorexia nervosa compared with that in healthy control subjects Am. J. Clinical Nutrition, September 1, 2004; 80(3): 774 - 781. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Modan-Moses, A. Yaroslavsky, I. Novikov, S. Segev, A. Toledano, E. Miterany, and D. Stein Stunting of Growth as a Major Feature of Anorexia Nervosa in Male Adolescents Pediatrics, February 1, 2003; 111(2): 270 - 276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Heer, C. Mika, I. Grzella, C. Drummer, and B. Herpertz-Dahlmann Changes in Bone Turnover in Patients with Anorexia Nervosa during Eleven Weeks of Inpatient Dietary Treatment Clin. Chem., May 1, 2002; 48(5): 754 - 760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Gianotti, A. I. Pincelli, M. Scacchi, M. Rolla, D. Bellitti, E. Arvat, F. Lanfranco, A. Torsello, E. Ghigo, F. Cavagnini, et al. Effects of Recombinant Human Insulin-Like Growth Factor I Administration on Spontaneous and Growth Hormone (GH)-Releasing Hormone-Stimulated GH Secretion in Anorexia Nervosa J. Clin. Endocrinol. Metab., August 1, 2000; 85(8): 2805 - 2809. [Abstract] [Full Text]
|
|
|
|
|
|
R. K. Støving, J. D. Veldhuis, A. Flyvbjerg, J. Vinten, J. Hangaard, O. G. Koldkjær, J. Kristiansen, and C. Hagen Jointly Amplified Basal and Pulsatile Growth Hormone (GH) Secretion and Increased Process Irregularity in Women with Anorexia Nervosa: Indirect Evidence for Disruption of Feedback Regulation within the GH-Insulin-Like Growth Factor I Axis J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 2056 - 2063. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
