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From the Clinical Research Centers |
Departments of Pediatrics and Internal Medicine and the Yale Children’s General Clinical Research Center, Yale University School of Medicine, New Haven, Connecticut 06520
Address all correspondence and requests for reprints to: S. Caprio, M.D., Department of Pediatrics, 333 Cedar Street, Yale University School of Medicine, New Haven, Connecticut 06520. E-mail: caprio{at}cdmas.med.yale.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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IGF-I is structurally related to insulin; however, unlike insulin, it circulates bound to specific proteins (IGFBPs) with variable affinities (10). Six IGFBPs have been structurally identified, but only IGFBP-1, -2, and -3 have been well characterized in humans (11). In contrast to the lack of diurnal variation in IGFBP-2 and -3, circulating IGFBP-1 levels vary widely throughout the day in an inverse relationship with changes in plasma insulin (12, 13, 14). Acute and chronic elevations in plasma insulin lower IGFBP-1 by suppressing its production by the liver, which may, in turn, serve to increase the bioavailability of free IGF-I (15).
Previous studies in obese prepubertal and pubertal children have demonstrated that hyperinsulinemia and insulin resistance are well established even in these early stages of obesity (16). Moreover, as GH and IGF-I levels normally peak during puberty, adolescence would appear to be an ideal developmental stage to examine the influence of obesity on IGF-I regulation. To address this question, we determined basal GH, total and free IGF-I, and IGFBPs in healthy obese adolescents and compared their results to those in lean adolescents and young adults. In addition, euglycemic hyperinsulinemic clamp studies were performed in all three groups to examine the effects of acute elevations in circulating insulin on IGFBP-1 and free IGF-I levels.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Three groups of subjects were studied, and their clinical and
biochemical characteristics are indicated in Table 1
. The two adolescent groups were age,
gender, and Tanner stage matched. Tanner stage of development in the
pubertal children ranged between II and IV. Specifically, 2 lean and 3
obese adolescents were in Tanner stage II, 10 lean and 8 obese
adolescents were in Tanner stage III, and 8 lean and 8 obese
adolescents were in Tanner stage IV. Plasma samples for measurement of
estradiol and testosterone were also taken as biochemical markers of
pubertal development. Estradiol levels in the lean adolescents females
ranged from 22–100 pg/mL; in the obese females, the range was 26–94
pg/mL. Total testosterone levels ranged in the lean boys from 2.3–7.5
pg/mL; in the obese boys, the range was from 1–4.6 pg/mL. As shown in
Table 1, weight (kilograms), body surface area (square meters), and
body mass index (BMI; kilograms per m2) were significantly
greater in the obese adolescents vs. those in the lean
adolescents. Although height tended to be lower in the obese subjects,
the difference was not statistically significant (P >
0.09). The obese adolescents were recruited from the Yale Pediatric
Weight Management Clinic; they all had a BMI, calculated as weight (in
kilograms) divided by height (in meters) squared, greater than the 95th
percentile specific for age and sex (based on percentile curves for
Caucasian girls and boys computed from the first National Health and
Nutrition Examination Survey, 1971–1974) (16). All subjects were in
good health and taking no medications, and none was attempting to
restrict calorie intake before the study. All subjects were normally
active, and none was participating in an organized physical training
program. The nature and purpose of the study were carefully explained
to both parents and to children before obtaining written voluntary
consent to participate. The study protocols were approved by the human
investigation committee of Yale University School of Medicine. Data for
insulin sensitivity obtained from some of the obese subjects were
included in previous publications (17, 18).
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All subjects were seen in the out-patient department of the Yale General Clinical Research Centers (child and adult units) in the morning after a 10- to 12-h overnight fast. Two basal blood samples (separated by 30 min) were obtained between 0800–0900 h for determination of plasma GH, total and free IGF-I, and IGFBPs. A detailed medical and nutritional history and physical examination were obtained for each subject. During the physical examination, Tanner stages of pubic hair, breast, and genital development were assessed, and height and weight were measured while the subjects were wearing only their under-garments.
Euglycemic/hyperinsulinemic clamp
We used a two-step euglycemic hyperinsulinemic clamp to assess
insulin sensitivity and the insulin dose-response curve for suppression
of IGFBP-1 in a randomly selected subset of subjects in all three
groups. Two iv catheters were inserted before the clamp studies: one in
an antecubital vein for administration of test substances and the other
in a vein of the hand or distal forearm of the contralateral arm for
blood sampling. The hand chosen for blood sampling was placed in a
heated box (
65 C) to facilitate blood sampling. Insulin was
administered as a prime continuous infusion at rates of 8 and 40
mU/m2·min body surface area. Each step lasted 120 min.
During the study, three arterialized samples were collected at baseline
and during the last 30 min of each step of the clamp for determination
of insulin, IGFBP-1, IGFBP-2, and free IGF-I. The euglycemic
hyperinsulinemic clamp study was performed in 10 lean adolescents (6
males and 4 females), 9 obese adolescents (6 males and 3 females) and 6
lean adults (3 males and 3 females). In the lean adolescent group, 9
children were in Tanner stage III, and 1 was in Tanner stage IV. In the
obese group, 6 subjects were in Tanner stage III, and 3 in Tanner stage
IV.
Determinations
Plasma glucose levels were measured by the glucose oxidase method with a Beckman glucose analyzer (Beckman Instruments, Brea, CA). Plasma GH and insulin were measured by a double antibody RIA. Plasma total IGF-I was measured by acid-ethanol precipitation (Nichols Institute, San Juan Capistrano, CA). Plasma free IGF-I, IGFBP-1, and IGFBP-3 were measured by a two-site immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX), as described by Takada et al. (19). Plasma IGFBP-2 was determined by double antibody RIA.
Recently, there has been a great interest in the measurement of "free" IGF-I, which, theoretically, is the biologically active fraction. Various methods have been used to measure the free (or freely dissociated) IGF fraction. We used a two-site immunoradiometric assay kit (Diagnostic Systems Laboratories) that is highly sensitive and is used as a direct assay to measure the dissociable fraction of IGF-I, which is considered the free IGF-I fraction. As described in detail by Juul et al. (20), this immunoradiometric assay is a noncompetitive assay in which the analyte is sandwiched between two antibodies. The free IGF-I and IGFBP-1, -2, and -3 measurements were performed in our laboratory. The intraassay coefficients of variation were 10.6% for IGFBP-1, 10.5% for IGFBP-2, and 6.1% for IGFBP-3; the interassay coefficients of variation were 8% for free IGF-I, 9.1% for IGFBP-1, 7.5% for IGFBP-2, and 16% for IGFBP-3.
Statistical analysis
All values are presented as the mean ± SEM. Multiple group comparisons were performed by using repeated measures ANOVA to compare the responses of different groups over time (Sistat+ version 5, SPSS, Chicago, IL). Dunnett’s procedure for multiple comparisons was used post-hoc to localize effects. Differences were considered significant at the 0.05 level.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and 2)
Basal total IGF-I was significantly higher in lean adolescents
(535 ± 37 ng/mL) than in obese adolescents (354 ± 29 ng/mL,
respectively; P < 0.01), whereas basal insulin was
elevated in obese adolescents (108 ± 12 pmol/L) compared to
levels in both groups of lean subjects (adolescents, 60 ± 14
pmol/L; adults, 30 ± 14 pmol/L; P < 0.001).
Elevated basal insulin in the obese group was associated with lower
IGFBP-1 (Fig. 2; 14 ± 3 ng/mL) vs. levels in both
groups of lean subjects (adolescents, 40 ± 5; adults, 57 ±
7; P < 0.001) and increased free IGF-I concentrations
(3.0 ± 0.5 ng/mL) vs. those in lean adults (1 ±
0.3; P < 0.02). Free IGF-I also tended to be higher in
obese vs. nonobese adolescents (2.0 ± 0.3 ng/mL;
P < 0.07), which may explain why basal GH (1.8 ±
0.2 ng/mL) and IGFBP-3 values were lowest in the obese group
(P < 0.02 vs. lean adolescents). IGFBP-2
levels were significantly lower in both obese and lean adolescents than
in lean adults (Fig. 2; P < 0.05). Univariate analysis
showed that in all three groups basal insulin was inversely related to
basal IGFBP-1 levels (Fig. 3; r =
-0.49; P < 0.01) and that IGFBP-1 was inversely
correlated with free IGF-I (r = -0.32; P <
0.05).
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To determine the dose-response effects of insulin on the
availability of IGFBP-1 and free IGF-I, changes in plasma
concentrations were measured under steady state conditions during low
and relatively high physiological dose insulin infusions. As shown in
Table 2, fasting and steady state plasma
insulin concentrations were higher in obese adolescents than in both
lean groups. Obese adolescents were very insulin resistant, as
indicated by the M values that were significantly reduced. Plasma
IGFBP-1 levels remained virtually unchanged in the obese adolescents
despite greater peripheral insulin levels. In contrast, in lean
adolescents, suppression of plasma IGFBP-1 occurred only during the
higher insulin dose infusion, as opposed to the insulin-induced
dose-dependent suppression of plasma IGFBP-1 concentrations observed in
lean adults. The unresponsiveness of plasma IGFBP-1 to the 8 and 40
mU/m2·min insulin infusion of the obese adolescents is
clearly illustrated in Fig. 4.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the postabsorptive state, obese adolescents had 1) reduced circulating GH and total IGF-I levels; 2) markedly suppressed IGFBP-1, IGFBP-2, and IGFBP-3 concentrations; and 3) slightly higher circulating levels of free IGF-I compared to lean adolescent subjects. Moreover, free IGF-I and insulin concentrations were higher and IGFBP-1 levels were lower in obese adolescents compared to lean adults. The differences in basal GH, IGF-I, and IGFBP levels observed in our obese vs. lean subjects can be interpreted as expected compensatory adaptations to the insulin resistance and basal hyperinsulinemia that characterize the obese state. It is intriguing to speculate that in obese adolescents, increased portal insulin concentrations overnight are likely to suppress hepatic production and secretion of IGFBP-1, which, in turn, is likely to account for the increase in circulating free IGF-I concentrations. GH levels fall via negative feedback of free IGF-I on GH secretion, resulting in reductions in total IGF-I and IGFBP-3 and a new steady state of normal or only modestly increased free IGF-I levels compared to those in lean adolescents.
Although we only measured basal GH levels just before performing the clamp studies, low GH concentrations have been consistently reported in obese children (22, 23) and adults (4) during 24-h sampling and in response to provocative stimuli. In contrast, a number of studies have failed to observe reduced concentrations of total IGF-I in obese adults (24, 25). As total IGF-I levels normally fall with aging (26), the ability to distinguish mildly suppressed IGF-I values in obese vs. lean adults is limited, especially in patient samples that have a wide range in age. Nutritional status is another factor that may confound interpretation of IGF-I levels, and particular care was taken to ensure that none of our patients was attempting to restrict food intake before the study. It is conceivable that the increase in total IGF-I observed in nonobese adolescents might be due to the relative preponderance of more females in the lean group (10 vs. 8 girls in the obese group). It should be noted, however, that in our study we have not found any significant gender effect on total IGF-I levels in either obese or nonobese adolescents. Although obesity is known to lower basal IGFBP-1 concentrations in inverse correlation to increasing plasma insulin levels (27), the effect of obesity on IGFBP-2 has not been previously determined. We found low basal IGFBP-2 levels in the obese and lean adolescents compared to those in lean adults, and no acute suppression was observed during the clamp study. The reason for the low IGFBP-2 levels in both obese and lean adolescents is unclear and may not be totally due to the elevated insulin concentrations, as the levels were similar even in the face of higher insulin levels in the obese compared with the lean adolescents. Although insulin may have some role in regulating plasma IGFBP-2, our data suggest that the effect may not be acute, as plasma IGFBP levels remained virtually unchanged during the insulin infusions. This is in marked contrast with the acute suppressive effect of insulin on IGFBP-1 levels.
Although there were substantial alterations in the IGF-I/IGFBP axis in obese subjects in the postabsorptive state, additional abnormalities were observed in plasma IGFBP-1 responses to acute elevations in plasma insulin, as would be observed postprandially. In obese subjects, acutely raising insulin to even high physiological levels during the 40 mU/m2·min clamp had virtually no effect on already low IGFBP-1 levels, whereas lean adults rapidly lowered IGFBP-1 levels even in response to the low dose insulin infusion. Intermediate responses were observed in lean adolescents, consistent with the physiological insulin resistance that accompanies normal puberty (28, 29, 30). Contrary to our expectations, the marked suppression of IGFBP-1 seen in lean adults during the clamp study did not lead to an increase in free IGF-I levels. The duration of the clamp procedure (4 h) may have been insufficient to observe a rise in circulating free IGF-I concentrations, an observation that raises questions regarding the physiological importance of acute compared to chronic suppression of IGFBP-1 in human subjects. Timing of the study may have also played a role. Suppressed levels of IGFBP-1 during the night may have a greater impact on free IGF-I levels when IGF-I production is increased in response to nocturnal peaks in GH. Conversely, GH secretion tends to be suppressed under euglycemic-hyperinsulinemic clamp conditions (31).
In summary, the metabolic syndrome induced by increased body fat appears to have profound effects on the complex interplay among GH, IGF-I, and IGFBPs during adolescence. Nevertheless, the net effect was to increase the ratio of free to total IGF-I in obese subjects, which may help explain why the pubertal growth spurt is not altered in obese adolescents even in the face of lower GH levels. Indeed, it is intriguing to speculate that such decreases in circulating GH may serve a beneficial role, as the antiinsulin effect of the rise in GH levels that normally occur during puberty has been implicated as the cause of the insulin resistance that was observed in the lean adolescents in this study (28). We have previously demonstrated that the insulin resistance in obese preadolescents does not differ from that in obese adolescents (16). In contrast, in teenagers with poorly controlled diabetes, in whom GH and IGFBP-1 are increased and free IGF-I decreased (31, 32), the adverse effects of puberty and diabetes on insulin sensitivity are additive (28).
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Acknowledgments |
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Footnotes |
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Received October 6, 1997.
Revised January 28, 1998.
Accepted February 10, 1998.
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