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Original Studies |
Department of Pediatrics, Tor Vergata University (S.C., M.L.M.B., D.G., B.B.), 00133 Rome; Department of Pediatrics, Scientific Institute H San Raffaele, Milan University (R.B., G.C.), 20132 Milan; and Division of Nuclear Medicine, S. Eugenio Hospital (S.B.), 00144 Rome, Italy
Address all correspondence and requests for reprints to: Stefano Cianfarani, M.D., Laboratory of Pediatric Endocrinology, Room E-178, Tor Vergata University, via di Tor Vergata 135, 00133 Rome, Italy. E-mail: stefano.cianfarani{at}uniroma2.it
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Abstract |
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42–39 kDa) in 46
of 60 IDDM patients, whereas the approximately 18-kDa band was absent
in all 34 control sera. No relationship was found between the endocrine
variables and stature at diagnosis. In conclusion, our results indicate
that IDDM children at the onset of disease are not taller than healthy
peers and have increased IGFBP-3 proteolytic activity. Finally,
although the IGF system is normal in younger IDDM children, older
patients have reduced IGF levels. |
Introduction |
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Linear growth is under control of the insulin-like growth factor (IGF) system, which consists of IGF-I and -II, type 1 and type 2 IGF receptors, at least six high affinity IGF-binding proteins (IGFBP-1 to IGFBP-6), and specific proteases for IGFBPs (13). In IDDM children, IGF-I levels have been found invariably low or in the low normal range (14, 15, 16, 17, 18, 19, 20, 21). IGF-II levels were reported to be normal in a small series of diabetics (14, 22), whereas, more recently, IGF-II has been found reduced in IDDM children although to a lesser extent than IGF-I (16, 17). Serum IGFBP-3 concentrations were moderately reduced in diabetic adults (23), adolescents (24), and children with IDDM (16, 17, 25). To date, IGFBP proteolysis at the onset of IDDM has been investigated only by Bereket et al. (26), who reported that proteolytic activity against IGFBP-3 is significantly increased in IDDM children at diagnosis, yielding the approximately 29-kDa IGFBP-3 fragment as the major circulating form of IGFBP-3. They also observed that after the initiation of insulin therapy, the intact form of IGFBP-3 increased significantly, whereas the approximately 29-kDa fragment decreased dramatically, and they suggested a regulatory role of insulin for IGFBP-3 proteases (26). Insulin has been proposed to modulate IGFBP-3 proteolysis either directly or indirectly via metabolic changes caused by insulinopenia (27). In rats, acute metabolic acidosis has, in fact, been shown to enhance IGFBP-3 proteolysis (28), and, therefore, in humans, ketoacidosis might contribute to increased IGFBP-3 protease activity in untreated diabetic children (27).
The major bias of most previous studies of growth in IDDM children before diagnosis is that height was compared with growth reference data without taking into account the effect of a secular trend. In addition, to date, no study has been carried out to relate the IGF system status to growth status in children with newly diagnosed IDDM. Finally, the only data available for IGF system and IGFBP-3 proteolysis at the onset of the disease come from a small group of children, and the effect of age was not investigated.
In the present study we assessed the growth of 60 children with IDDM at diagnosis, comparing their stature with target height, normal growth standards, and height of 102 sex- and age-matched controls. Furthermore, we investigated IGF system status and IGFBP-3 proteolysis, relating them to patients’ ages and heights at disease onset.
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Subjects and Methods |
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Sixty patients (25 females and 35 males; mean age ± SD, 6.4 ± 3.9 yr) with newly diagnosed IDDM were recruited at the Departments of Pediatrics, Scientific Institute H San Raffaele (Milan, Italy), Milan University (Milan, Italy), and Tor Vergata University (Rome, Italy). Forty-seven subjects were prepubertal, and 13 were pubertal. Of the 13 pubertal patients, 4 were Tanner stage II, 5 were Tanner stage III, 1 was Tanner stage IV, and 3 were Tanner stage V (29). Height was compared with the growth reference standards of Tanner and Whitehouse (29) and was expressed as the z-score for chronological age and sex according to the following formula: z-score = (x - average x)/SD, where x is the actual height, average x is the mean height at that age and for that sex, and SD is the SD from the mean. Anthropometric measurements and fasting venous blood samples were taken within 24 h of diagnosis and the initiation of insulin therapy. The diagnosis of IDDM was established by a fasting plasma glucose concentration greater than 140 mg/dL (7.7 mmol/L) or a random plasma glucose greater than 200 mg/dL (11 mmol/L) with ketosis and classical symptoms of diabetes (30). The heights of the parents of each diabetic child were measured, and target height (TH) was calculated with the following formulas: boy TH (cm) = father’s height + (mother’s height + 13)/2; girl TH (cm) = mother’s height + (father’s height - 13)/2. Patients’ statures were also corrected for their target heights according to the formula: corrected height (z-score) = actual height (z-score) - TH (z-score). One hundred and two sex-, age-, and pubertal stage-matched healthy schoolchildren (mean age ± SD, 5.2 ± 2.7 yr) were chosen as the control group for height. An additional group of age-matched 34 children (mean age ± SD, 10.2 ± 1 yr; pubertal stage I or II) was chosen as the control group to compare the endocrine variables of 26 IDDM patients with age above 6 yr and pubertal stage I or II (mean age ± SD, 10.2 ± 2.8 yr). All control children were selected from a population of 1040 schoolchildren attending primary and secondary schools in Rome and included in a regional survey on the prevalence of endemic goiter (31). They underwent height and weight measurements, clinical evaluation, assessment of urinary iodine excretion, and thyroid ultrasound scan. Informed consent was obtained from the parents of all patients.
Assays
Serum IGF-I was measured by immunoradiometric assay (IRMA; Nichols Institute Diagnostics, San Juan Capistrano, CA). The intraassay coefficient of variation (CV) was 3.3–4.6%, the interassay CV was 9.3–15.8%, and the sensitivity limit was 6 ng/mL. Serum IGF-II was measured by IRMA (Diagnostics Systems Laboratories, Inc., Webster, TX). The intraassay CV was 3.4–6.5%, the interassay CV was 4.5–6.3%, and the sensitivity limit was 12 ng/mL. Serum IGFBP-3 was measured by IRMA (Diagnostics Systems Laboratories, Inc.). The intraassay CV was 1.8–3.9%, the interassay CV was 0.5–1.9%, and the sensitivity limit was 0.5 ng/mL. Serum C peptide was measured by RIA (Byk-Sangtec Diagnostica, Dietzenbach, Germany). The intraassay CV was 1.9–5.0%, the interassay CV was 3.2–10.%, and the sensitivity limit was 0.05 ng/mL. Serum cortisol was measured by RIA (Byk-Sangtec Diagnostica). The intraassay CV was 2.1–4.0%, the interassay CV was 3.2–9.0%, and the sensitivity limit was 0.5 µg/dL. Hemoglobin A1c (HbA1c) was measured by high pressure liquid chromatography.
Western immunoblot analysis
To visualize circulating proteolytic fragments of IGFBP-3, Western immunoblotting analysis was performed as previously described (32). Briefly, after addition of nonreducing SDS sample buffer, serum samples (3 µL) were processed by SDS-PAGE (12% gel). Separated proteins were electroblotted onto nitrocellulose filters in a Hoeffer semidry Tansphor unit (San Francisco, CA). Filters were blocked with 1% BSA and sequentially incubated with sheep anti-IGFBP-3 antibody (provided by Dr. J. M. P. Holly, University of Bristol, Bristol, UK) overnight at 4 C and with goat antisheep IgG conjugated with horseradish peroxidase (Amersham International, Aylesbury, UK) for 2 h at room temperature. Filters were exposed to enhanced chemiluminescence reagents (Amersham International) for 1 min at 20 C and were exposed to Hyperfilm enhanced chemiluminescence for 1 min to 1 h at 20 C. Densitometric analysis of bands was performed using a GS 700 imaging densitometer (Bio-Rad Laboratories, Inc., Richmond, CA). The relative amount of each IGFBP-3 circulating fragment was estimated by calculating the absorbance of the fragment band over the sum of the intact IGFBP-3 and IGFBP-3 fragments in the same lane.
Statistics
Results are reported as the mean ± SD. Differences between means were assessed using unpaired two-tailed t test and one-way ANOVA. Significance was assigned for P < 0.05. After ascertaining that all variables were normally distributed, the relationships among parameters were evaluated by Pearson correlation. All of the relationships between variables were controlled for the effect of pubertal stage. A computer program was used for all statistical calculations (SOLO 3.0, BMPD Statistical Software, Los Angeles, CA).
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Results |
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The mean height of diabetics was higher than growth reference
values (mean height ± SD, 0.64 ± 1.4 z-score)
and their target height (mean target height ± SD,
0.1 ± 0.84 z-score; P < 0.005). However, no
significant difference in height was found between IDDM children
and con- trols (mean height ± SD,
0.64 ± 0.95 z-score). Even subdividing subjects according to age,
no significant difference in height was seen between diabetics and
age-matched controls (Table 1
). Younger
diabetics (age, <6 yr) were relatively taller, although not
significantly, than older ones (age, >6 yr; n = 26; mean
height ± SD, 0.79 ± 1.2
vs. 0.43 ± 1.6 z-score).
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Endocrine variables
Overall, IDDM children showed reduced serum levels of IGF-I
(mean ± SD, -0.65 ± 1.9 z-score), and normal
levels of IGF-II (mean ± SD, -0.05 ± 1.2
z-score) and IGFBP-3 (mean ± SD, -0.06 ± 1.2
z-score; Table 2). However, whereas
patients younger than 6 yr showed normal levels of IGF-I, IGF-II, and
IGFBP-3, IDDM children older than 6 yr showed significantly reduced
values of IGF-I, IGF-II, and IGFBP-3 compared with either the
age-matched control group or younger patients (P <
0.01; Fig. 1). IGF-II correlated
inversely with HbA1c (r = -0.45;
P < 0.01). Finally, IGF-II and IGFBP-3 were inversely
related to the age at diagnosis (IGF-II: r = -0.48;
P < 0.001; IGFBP-3: r = -0.32; P
= 0.01). No relationship was found between the endocrine variables and
stature.
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IGFBP-3 immunoblot analysis revealed the presence of an
approximately 18-kDa fragment of IGFBP-3 in addition to the major
approximately 29-kDa fragment and the intact form (42–39 kDa) in
serum of 46 of 60 IDDM patients at diagnosis, whereas in none of the 34
controls was the approximately 18-kDa band found (Fig. 2). Densitometric analysis of IGFBP-3
bands did not show any relationship between the relative amount of the
fragments and the anthropometric variables. The intensity of the
approximately 18-kDa band correlated inversely with IGF-II (r =
-0.33; P < 0.05). Neither the approximately 18- or
30-kDa fragment relative amounts correlated with acidosis (evaluated by
determining the values of pH and bicarbonates) and residual ß-cell
function (expressed by serum C peptide levels).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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According to previous reports (14, 15, 16, 17, 18, 19, 20, 21), we have found significantly reduced concentrations of IGF-I and, in contrast with previous data (16, 17), almost normal levels of IGF-II and IGFBP-3 in the whole group of patients. However, differently from the previous studies that investigated small numbers of patients with age close to or in the adolescence, the recruitment of a large group of IDDM children at diagnosis enabled us to subdivide the subjects according to age. Further data analysis revealed that although younger patients (age, <6 yr) had normal levels of IGF-I, IGF-II, and IGFBP-3, older subjects showed significantly reduced concentrations of the three peptides compared with either reference values or the control group. Insulin is a well known regulator of IGF-I expression (33, 34, 35, 36); chronic hypoinsulinism may directly affect the production of IGF-I. The finding that IGF-II and IGFBP-3 levels are less affected than IGF-I may also be explained by the fact that circulating levels of IGF-II and IGFBP-3 are dependent on genetic, rather than environmental, components to a greater extent than IGF-I (37).
We previously reported that protein glycation secondary to high concentrations of glucose has no protective effect against IGFBP protease activity (38). To date, only Bereket et al. (26) have investigated the IGFBP-3 protease activity in sera of hyperglycemic IDDM children at the onset of disease, reporting increased proteolysis of IGFBP-3. In the present study IGFBP-3 immunoblot analysis showed both the typical approximately 42/39-kDa doublet, representing the intact form of the molecule, and the major approximately 29-kDa fragment, which in many cases was the predominant form, thus indicating, according to the previous report, augmented proteolytic activity (26). In addition, we demonstrated the presence of an additional IGFBP-3 circulating form with a molecular mass of about 18 kDa. This fragment was previously identified in urine and serum from healthy children (39), GH-deficient patients (39), healthy neonates (32), and infants with intrauterine growth retardation (32). Recently, in adolescents and young adults with IDDM for at least 5 yr a urinary 18-kDa N-terminal IGFBP-3 fragment has been characterized and found to correlate with urinary albumin excretion (40). In the same group of patients, an approximately 18-kDa fragment was also detectable in serum by Western immunoblot analysis, and it is likely that it corresponds to the IGFBP-3 fragment we have identified in our IDDM children, suggesting that whatever its pathophysiological significance might be, it is already present in most IDDM children at the onset of disease. We previously proposed that the approximately 18-kDa fragment may be produced either by a protease acting on a cleavage site different from that yielding the major approximately 29-kDa fragment or by increasing concentrations of a single protease that may lead to progressively smaller fragments of IGFBP-3 (32). The finding that diabetics with the approximately 18-kDa fragment showed increased IGFBP-3 proteolysis and reduced body mass index is consistent with previous reports suggesting that nutritional status is a major regulator of IGFBP-3 proteolysis (41, 42).
Insulin has been proposed to be one of the major regulators of IGFBP-3 proteolysis, and according to this hypothesis, insulin therapy was reported to dramatically reduce IGFBP-3 proteolysis in IDDM children after the initiation of treatment (26). Moreover, IGFBP-3 proteolysis was increased in patients with insulin resistance (43, 44). In contrast with these previous findings, no correlation between the intensity of the approximately 18- and/or 29-kDa band and C peptide levels was found in our patients. However, it has to be pointed out that we did not assess proteolytic activity directly, but only by determining the relative amounts of IGFBP-3 fragments. Contrarily to what was previously proposed (27, 28), we did not find any relationship between the relative amounts of IGFBP-3 fragments and the severity of acidosis. In our patients, however, blood samples were collected within 24 h from admission, and in most of them the correction of metabolic acidosis had already been undertaken.
As the IGF system plays a major role in growth regulation, we investigated the relationships between height and the IGF system-related variables at diagnosis, but no relationship was found. Stature at diagnosis is the result of growth in the years preceding the onset of disease, whereas IGFs, IGFBPs, and IGFBP proteases recorded at diagnosis reflect the actual metabolic and nutritional status of patients rather than the IGF-dependent growth-promoting system in the years before the onset of disease, and the inverse correlation found between IGF-II and HbA1c is in line with this interpretation.
In conclusion, our results suggest that IDDM children at the onset of disease are not taller than their healthy peers and show, for the first time, that the IGF system is normal in younger diabetics, but it deteriorates with age. Finally, we have demonstrated the presence of a small fragment of IGFBP-3 that might represent the product of a novel IGFBP-3 protease. The challenge is now to understand the physiological significance of the increased IGFBP-3 proteolysis and whether it can be exploited to achieve a better control of growth and metabolism in IDDM children.
Received March 1, 2000.
Revised July 17, 2000.
Accepted August 8, 2000.
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References |
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