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Department of Medicine (I.M.C., M.L.H., S.S.P., M.O.T), Division of Endocrinology and Metabolism, University of Virginia Health Sciences Center; Division of Biostatistics (F.E.H.) and Epidemiology, Department of Health Evaluation Sciences, University of Virginia, Charlottesville, Virginia 22908; Department of Pediatrics (R.L.H.), Stanford University Medical Center, Stanford, California 94305; and Department of Medicine (K.G.M.M.A.), University of Newcastle Upon Tyne, Newcastle Upon Tyne, United Kingdom NEZ 4HH
Address all correspondence and requests for reprints to: Dr. Michael O. Thorner, Dept of Medicine, Box 466, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908.
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Abstract |
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Introduction |
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It is not known why GH secretion declines with increasing age. Possible mechanisms, alone or in combination, include reduced GHRH secretion or action, increased SRIH secretion or action, reduced somatotroph numbers or function, and increased sensitivity to the negative feedback effects of IGF-I. This study was designed to investigate the last possibility.
IGF-I mediates the growth promoting and metabolic actions of GH. Several findings suggest a negative feedback role for endogenous IGF-I in the control of GH secretion. First, exogenous IGF-I suppresses GH secretion in both animals and man when hypoglycemia, which stimulates GH secretion (17), is prevented (18, 19, 20). Second, there are a number of conditions, including starvation/malnutrition, Laron-type dwarfism, and type I diabetes mellitus, in which plasma IGF-I concentrations are decreased as a result of GH resistance at the receptor or postreceptor level (21, 22, 23, 24, 25, 26). The elevated serum GH concentration in these conditions is consistent with a reduction of IGF-I negative feedback, as is the fall in GH concentrations that follows IGF-I administration (19, 27, 28).
In previous studies, we demonstrated that 2 days of fasting increases GH secretion approximately 4-fold in both young and older subjects, but the absolute levels are 50% lower in both fed and fasted conditions in the elderly (29, 30). In young men, a low-dose, euglycemic iv infusion of recombinant human IGF-I (rhIGF-I) rapidly suppressed fasting-enhanced GH secretion by reducing the amplitude and number of secretory pulses measurable in an immunoradiometric assay (IRMA) (19). In the present study, a similar protocol was used to administer graded rhIGF-I infusions to both young and older adults to define the dose-response characteristics of this suppression and to determine whether the GH-suppressive effect of exogenous IGF-I changes with age. A finding of greater sensitivity to exogenous rhIGF-I in older people would suggest that increased endogenous IGF-I negative feedback effects might be a cause of the aging-associated decline in GH secretion.
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Subjects and Methods |
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The study was approved by the Human Investigation and General Clinical Research Center Advisory Committees of the University of Virginia and Genentech, Inc. All subjects gave written informed consent. Eleven young subjects were studied: 6 men and 5 women, with a mean (±SD) age of 25.2 ± 4.6 (range 20–35) and body mass index of 23.7 ± 1.8 kg/m2 (range 20–25.9). Eleven older subjects were studied: 6 men and 5 women with a mean age of 69.5 ± 5.8 (range 62–81) and a mean body mass index of 24.2 ± 2.5 kg/m2 (range 19.5–27). All subjects were nonsmokers, were taking no medications, had not undertaken transmeridian travel for more than 6 weeks, and had unremarkable clinical histories and normal physical examinations. All had normal biochemical indices of renal, hepatic, and hematologic function, glycated hemoglobin, thyroxine, thyroid-stimulating hormone, prolactin, LH, FSH, testosterone (men), and estradiol (women). All postmenopausal women had serum estradiol concentrations less than 51 pmol/L (14 pg/mL) with appropriately elevated levels of LH and FSH.
Experimental protocol
Each subject was studied on two occasions, separated by at least 4 weeks, and took ferrous sulphate between study days. The young women were studied between days 1 and 6 of the menstrual cycle (31) and had a negative ß HCG pregnancy test immediately before each study. For each study, the subjects fasted for 40.5 h, during which time they ingested only water, potassium chloride (20 meq/day), and multivitamins (one tablet/day). Compliance with the fast was monitored by twice-daily measurement of weight and urine ketones. On each morning of the fast and at the end of the study, blood was obtained for serum chemistries and hepatic enzymes to monitor for adverse effects of the fast and rhIGF-I infusion. The studies were performed on day 2 of the fast (31–40.5 h of fasting), and subjects remained supine and awake during the studies. In a single-blind, randomized order, the subjects received infusions of rhIGF-I on one admission and normal saline on the other.
rhIGF-I admission. By 0600 h on day 2 of the fast, a cannula was placed anterograde in an antecubital vein for continuous infusion of test substances, and retrograde in a wrist vein of the other arm for blood sampling. The hand from which blood samples were collected was kept in a heated box at 70 C to ensure arterialization of venous blood (32). From 0700–1630 h, arterialized venous blood samples were obtained at 5-min intervals for measurement of GH and at 0700, 0800, 0840, and 0850 h and every 10 min from 0900–1630 h for measurement of glucose. Blood samples for total and free IGF-I were drawn every 30 min from 0700–1630 h; samples for measurement of serum insulin, free fatty acids (FFAs), and ß-hydroxybutyrate were drawn at 0700, 0900, 1130, 1400, and 1630 h. All serum samples were frozen at -20 C until analyzed.
The following iv infusions were given at a rate of 5.6 µL/kg·min: 0700–0900 h saline; 0900–1130 h rhIGF-I at 1 µg/kg·h; 1130–1400 h rhIGF-I at 3 µg/kg·h; and 1400–1630 h rhIGF-I at 10 µg/kg·h.
The rhIGF-I (Genentech, Inc., South San Francisco, CA) was prepared in normal saline from a stock 5 mg/mL solution. An infusion of 20% dextrose was started if plasma glucose fell more than 15% below the pre-rhIGF-I baseline (mean of 0840 and 0850 h plasma glucose values). The rate of dextrose infusion was adjusted every 10 min to keep plasma glucose within 10% of this (15% below baseline) value, on the basis of the plasma glucose measurements and a negative feedback algorithm (32). The actual glucose concentration of the 20% dextrose solution was measured for each experiment. A glucose analyzer (Beckman Analytical System Group, Columbia, MD) was used to measure plasma glucose concentrations during the clamp, and dextrose infusions were delivered by microprocessor pumps (Harvard Apparatus Model 22, South Natick, MA) controlled by a computer program (running on an IBM-compatible computer) written and kindly supplied by Dr. David Krusch (University of Rochester, Rochester, NY). Blood sampling ended at 1630 h, and the subjects were fed.
Saline (control) admission. The protocol was the same as on the rhIGF-I admission, except that saline was infused instead of rhIGF-I. Dextrose (20%) was administered as on the rhIGF-I admission, as needed to prevent plasma glucose from decreasing by more than 15% from baseline.
Assays
GH. Serum GH concentrations were measured in duplicate with the Nichols LumaTag hGH chemiluminescence assay (Nichols Institute, San Juan Capistrano, CA); the protocol was modified as previously described (33). The sensitivity of the assay is 0.002 µg/L. The interassay coefficients of variation were 7.2% at 1.7 µg/L, and 7.2% at 4.2 µg/L. The intraassay coefficients of variation were 4.9% at 0.2 µg/L, 6.7% at 2 µg/L, and 6.4% at 4.9 µg/L.
IGF-I. Total plasma IGF-I was measured by RIA after acid ethanol extraction, using a commercially available kit (Nichols Institute). Interassay coefficients of variation were 5.2% at 122 µg/L and 8.4% at 185 µg/L. Intraassay coefficients of variation were 2.4% at 119 µg/L and 3% at 207 µg/L. The sensitivity of this assay is 32 µg/L. Serum-free IGF-I was measured by a noncompetitive IRMA (Diagnostic Systems Laboratories, Inc., Webster, TX). This is a two-site IRMA which is done in two stages. The readily available or free IGF-I in the sample is first bound by an anti-IGF-I capture antibody coated on the side of the tube. The epitope on this capture antibody has preference for unbound IGF-I. The tube is then emptied and washed, removing any IGF-I in the sample that is not bound by the capture antibody. The amount of free IGF-I bound to the tube is determined using an I125-labeled anti-IGF-I antibody that binds to an epitope on the IGF-I different from the capture antibody, and the binding of the second antibody to IGF-I is independent of binding to the first. The free IGF-I standards are calibrated to the World Health Organization international reference reagent for IGF-I (code 87/5181). Interassay coefficients of variation were 8.4% at 0.26 µg/L, 3.6% at 5.52 µg/L, and 10.8% at 13.87 µg/L. Intraassay coefficients of variation were 10.4% at 0.29 µg/L, 5.2% at 6.26 µg/L, and 3.3% at 14.2 µg/L. The assay sensitivity is 0.03 µg/L.
Other assays. Previously described methods were used to measure plasma insulin, unesterified free fatty acid (FFA), and ß-hydroxybutyrate (BOH) (19). The sensitivity of the insulin assay was 6 pmol/L. Samples with undetectable amounts of the above analytes were assigned the value of the sensitivity of the assay for statistical analysis. Other studies were performed in the Clinical Laboratories or the General Clinical Research Center Core Laboratory at the University of Virginia Health Sciences Center using standard methods.
Statistical analyses
Results are expressed as mean ± SE, unless otherwise stated. Because each subject served as his/her own control, comparisons between the results from rhIGF-I and control treatment were made with a paired t test, or Wilcoxon signed rank test if the results were not normally distributed, as determined by the Wilk-Shapiro test. When comparisons were made between young and older subjects, an unpaired t test was used, or Wilcoxon rank sum test for results not normally distributed. All hypothesis testing was two-tailed. Statistical significance was assumed at P < 0.05.
Determination of timing of GH suppression by rhIGF-I. Because GH was measured at 5-min intervals for 9.5 h, paired comparisons at each time point were not appropriate. To objectively assess the inhibitory effects of IGF-I, over time, against a background of spontaneous GH secretion that includes both basal and varying amplitude bursts of secretion, we sought to normalize each subject’s GH concentration time trend during the rhIGF-I infusion for the subject’s GH concentration time trend during the control (saline) infusion. Both absolute change and percentage change have been used to analyze such data. To determine the best method to adjust for control conditions, we plotted for each subject both the difference in GH concentrations (IGF-I day - control day) and the ratio of GH levels (IGF-I day/control day) against time over the 9.5-hour period (34). We observed that differences were more independent of the control conditions than ratios and, therefore, we chose differences as the method to analyze for rhIGF-I effects over time.
The time-course of the suppression of serum GH concentrations by rhIGF-I infusion was determined for each age group, and a comparison of this time-course was made between the age groups. To look for an rhIGF-I treatment effect, we subtracted each subject’s serum GH concentration, at each time point during the saline infusion, from its GH concentration at the same time point during the rhIGF-I infusion (rhIGF-I day - saline day). We then derived a GH concentration-time regression curve with simultaneous (taking into account multiple timepoints) 95% confidence bands for each age group, as described in detail in the Appendix. To estimate the difference in the time-course of GH suppression for young vs. older subjects, we determined the difference between the regression curves for young and older subjects at each time point (young - older subjects) and simultaneous 95% confidence bands were again calculated (Appendix).
Relationship between GH suppression and changes in serum free and total IGF-I concentrations. The difference between serum GH concentrations on the rhIGF-I day and the saline day (rhIGF-I day - saline day) was compared to: 1) the change in free IGF-I concentrations; and 2) the change in total IGF-I concentrations (rhIGF-I day - saline day) for each age group. Regression curves and simultaneous 95% confidence bands were calculated as described in the Appendix. To test for differences between age groups, the regression lines were subtracted (young - older subjects) at each time point and simultaneous 95% confidence bands were calculated.
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Results |
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Within each age group, mean glucose concentrations did not differ,
at any time point, between control and rhIGF-I infusion days. Glucose
concentrations did decline slightly, as expected, during fasting but
were prevented from falling more than 15% below baseline by infusion
of 20% dextrose as necessary (Table 1). These infusions were needed
more often during rhIGF-I than control infusions, in both young (9/11
vs. 3/11) and older subjects (10/11 vs. 4/11),
consistent with the known glucose-lowering action of IGF-I (17). Mean
glucose infusion rates, over the three treatment periods, were not
significantly different between young and older subjects for either
control or rhIGF-I infusions. The mean glucose infusion rate during the
entire treatment period (0900–1630 h) was significantly greater on the
rhIGF-I day than on the control day in the older subjects and
nonsignificantly greater in the young subjects. Significantly more
glucose was infused during the 10 µg/kg·h rhIGF-I treatment period
in the older subjects but not during any other treatment period in
either age group. On the rhIGF-I treatment day, 4 of 11 young and 5 of
11 older subjects had glucose infused before the end of the 3
µg/kg·h treatment period at 1400 h.
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)On the control day, the total IGF-I concentrations of younger subjects were more than double those of older subjects (219 ± 15 vs. 103 ± 19 µg/L, P < 0.01; mean of 0700, 0900, 1130, 1400, and 1630 h values). Free IGF-I levels also were greater in young subjects, but the difference was not significant (2.1 ± 0.3 vs. 1.3 ± 0.3 µg/L, P = 0.08). In both young and older subjects, about 1% of the IGF-I was free (0.94 ± 0.09% vs. 1.3 ± 0.2%, P = 0.08). The percentage of IGF-I that was free was significantly greater in women than men in the younger subjects (1.1 ± 0.09 vs. 0.78 ± 0.07%, P = 0.02), and there was a trend in the same direction for the older subjects (1.7 ± 0.2 vs. 1.0 ± 0.2%, P = 0.08).
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Serum GH concentrations (see
Figs. 2–6)
Baseline serum GH concentrations were greater in young than older
subjects (3.3 ± 0.7 vs. 1.9 ± 0.5 µg/L, mean
of 0700–0900 h values on control and IGF-I days; P =
0.02). There was no significant gender difference in either age group.
Infusion of rhIGF-I suppressed serum GH concentrations in both age
groups and in both men and women. The GH concentrations of a
representative young and a representative older subject are shown in
Fig. 2 and mean GH concentrations for each age group
during saline, and the 3 rhIGF-I treatment periods are shown in Fig. 3. In both young and older subjects, serum GH
concentrations were significantly suppressed during infusion of 3 and
10 µg/kg·h rhIGF-I, but not 1 µg/kg·h (Fig. 4, top and middle panels). In the older subjects, there was a
transient decrease during the 1 µg/kg·h dose that was not
sustained. As shown in Fig. 4, lower panel, there was no
significant difference in suppression of GH concentrations between
young and older subjects during the 1 or 3 µg/kg·h doses of
rhIGF-I. There were periods of time during the infusion of 10
µg/kg·h rhIGF-I when the young subjects transiently had greater
suppression of serum GH than the older subjects, although this was not
the case at the end of the 10 µg/kg·h infusion. Note, in Fig. 3, that during the last hour of the rhIGF-I infusion, mean serum GH
concentrations did not differ between young (0.78 ± 0.24 µg/L)
and older (0.61 ± 0.16 µg/L) subjects, despite the fact that
the older subjects started at a lower baseline GH concentration before
rhIGF-I infusion.
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, middle panel), small increases in free
IGF-I concentrations of up to 2–3 µg/L were associated with
significant suppression of GH concentrations, but further increases
produced little or no additional GH suppression. In contrast, the young
subjects displayed progressive suppression of GH levels with increasing
free IGF-I levels (Fig. 5, top panel), although rhIGF-I
infusion produced smaller overall rises in free IGF-I levels in the
young than older subjects. Reflecting these differences, the more that
free IGF-I levels increased as a result of rhIGF-I infusion, the
greater the difference between the suppression of circulating GH
concentrations in young and older subjects. The suppression was
significantly greater in the young subjects when free IGF-I
concentrations were more than 3–4 µg/L above baseline (Fig. 5, lower panel). Although less dramatic, the young subjects
also had greater suppression of GH, relative to changes in total IGF-I
levels; GH suppression was significantly greater in the young than
older subjects at total IGF-I levels more than about 150 µg/L above
baseline (Fig. 6). Insulin, free fatty acid, and BOH concentrations
Baseline insulin concentrations were nonsignificantly higher in older than young subjects (23.7 ± 1.4 vs. 17.3 ± 1 pmol/L, mean of 0700 and 0900 h values, P = 0.3) and declined during the saline (control) infusions as the fast continued. Infusion of rhIGF-I had an equivalent minor suppressive effect on plasma insulin concentrations in both age groups. Mean insulin levels decreased 33% in the young and 35% in the older subjects during the IGF-I infusion, compared with decreases of 11% and 18%, respectively, on the control day. Insulin levels (pmol/L) were significantly lower on the IGF-I infusion day than on the control day at the end of both the 3 µg/kg·h (9.9 ± 2.3 vs. 17 ± 2.4, P = 0.005) and 10 µg/kg·h (10.8 ± 3.2 vs. 18.1 ± 2.9, P = 0.007) infusion periods in the young, and at the end of the 3 µg/kg·h (14.9 ± 2.3 vs. 19.9 ± 3.3, P = 0.02) but not the 10 µg/kg·h (16.3 ± 4 vs. 20.2 ± 3.6, P = 0.6) infusion period in the older subjects.
Both FFA and BOH concentrations rose gradually throughout both saline and rhIGF-I infusions, as expected in subjects fasting for 31–40.5 h. There were no age-related differences in the concentrations of these analytes, and graded infusion of rhIGF-I had no effect in either age group.
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Discussion |
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A greater absolute suppression of serum GH occurred in the young than the older subjects during most of the 3 and 10 µg/kg·h rhIGF-I infusion periods. The greater suppression of serum GH in young subjects occurred despite the fact that serum total and free IGF-I concentrations increased to a greater degree in older subjects during rhIGF-I infusion. As a result, given increases in total and free IGF-I concentrations were associated with significantly greater absolute suppression of serum GH concentrations in the young than older subjects. We offer two possible explanations of these results.
First, aging may result in a reduction in the sensitivity of GH secretion to IGF-I negative feedback. Such a reduction could result from a decreased number of IGF-I receptors, decreased delivery of IGF-I to IGF-I receptors, or decreased IGF-I action at the postreceptor level in the pituitary and/or hypothalamus. IGF-I binding to its pituitary receptors is affected by prevailing nutritional and hormonal conditions; fasting and estrogen therapy both increase this binding in rats (35, 36). We are unaware, however, of any reports of the effects of aging on IGF-I binding or receptor concentrations in any species.
Second, there may be a limit to the suppressive effects of IGF-I on GH secretion. Increases in serum IGF-I levels above a certain point may not produce additional suppression of GH secretion and serum GH concentrations. Thus, although free IGF-I levels were significantly higher in the older than young subjects at the end of the rhIGF-I infusion, no further suppression of GH concentrations occurred. Perhaps more likely, there may be a baseline below which it is difficult or impossible for rhIGF-I to suppress GH secretion and serum GH concentrations. In support of this, there were no significant differences between the mean serum GH concentrations in the young and older subjects during the last hour of the 10 µg/kg·h dose, despite the fact that serum GH concentrations were higher in the young subjects during the control (saline) infusion. The greater absolute decline of serum GH concentrations in young subjects therefore might reflect their higher baseline GH levels rather than a greater sensitivity to IGF-I negative feedback in this age group. In either case, our data demonstrate clearly that older subjects are not more sensitive to IGF-I negative feedback than young subjects and exclude this hypothetical mechanism for the decline in GH secretion associated with aging. Therefore, it seems more likely that decreased GHRH or increased SRIH secretion accounts for the lower GH concentrations observed in older subjects.
We do not know why the increases in serum total and free IGF-I concentrations during the rhIGF-I infusion were greater in older than young subjects. The effects of aging on IGF-I volume of distribution (Vd) and clearance have not been reported. Vd for hydrophilic compounds, such as IGF-I, correlates better with lean body weight than with total body weight (37). On average, lean body weight declines as a percentage of total body weight with advancing age (37). Older adults may therefore have a smaller Vd for IGF-I than weight-matched young adults. Administering rhIGF-I on a per-kilogram-total-body-weight basis, as we did in this study, therefore, in effect, might deliver a higher dose of rhIGF-I to the older subjects.
The discrepancy between the IGF-I concentration increases in older and young subjects was more marked for free than total IGF-I. The increase in free IGF-I concentrations was 2-fold greater in older than in young subjects, compared to 1.25-fold greater for total IGF-I. It is possible that this difference results from higher numbers of unoccupied IGF binding protein (IGFBP) binding sites in the younger subjects, thus less of the infused rhIGF-I remains unbound to be measured as free IGF-I. The majority of IGF-I in the circulation is carried in a 150 Kd complex consisting of IGF-I, an 85 Kd acid-labile subunit and IGFBP-3 (38). With advancing age, circulating concentrations of IGFBP-3 fall (39) and those of IGFBP-1 increase (40); the effect of age on plasma levels of the other IGFBPs has not been clearly determined, but may be minimal (41).
Measurement of free or unbound IGF-I has been technically difficult, and as a result the effect of a given intervention or variable on free IGF-I concentrations is often inferred from the levels of total IGF-I and one or more of the IGFBPs (42). The free IGF-I results in this paper must be interpreted with some caution. The free IGF-I assay we have used may provide a measure of IGF-I free of the major binding protein, IGFBP-3, rather than of all 6 IGFBPs. Juul et al. have reported that free IGF-I concentrations in pubertal boys measured by this IRMA are approximately double those measured by an ultrafiltration technique (43). Nevertheless, they found a strong positive correlation between the results of the two assays and it is not known which of these two assays more closely measures bioavailable IGF-I (43). Our finding that increases in serum free IGF-I concentrations resulting from rhIGF-I infusion were associated with greater suppression of GH concentrations in young than older subjects is consistent with our finding that increases in total IGF-I concentrations were also associated with greater GH suppression in the young subjects.
The GH-suppressive effect of rhIGF-I appears to be independent of its insulin-like actions. In this study, GH release was suppressed by rhIGF-I doses that had minimal, if any, effect on blood glucose concentrations. By the end of the 3 µg/kg·h treatment period, rhIGF-I administration had caused a significant suppression of mean GH concentrations to approximately half of control day values, however less than half of the subjects required glucose infusion to maintain blood glucose concentrations within 15% of baseline, and mean glucose infusion rates were not significantly different from those on the control day in either age group. Furthermore, the rhIGF-I infusion did not affect circulating levels of FFAs or BOH at any time point in the study, whereas they are suppressed by insulin and higher doses of rhIGF-I (19, 44). Increases in plasma FFA concentrations suppress GH secretion (45), and higher circulating levels have been proposed as a cause of the reduced GH secretion associated with obesity (46). Our finding that fasting FFA concentrations were not different in young and older adults, is consistent with other reports (47), and suggests that differences in FFA concentrations are not a cause of the aging-associated decline in GH secretion, although it does not exclude the possibility that the elderly are more sensitive to the GH-suppressive effects of FFAs.
Our study was designed to produce carefully controlled conditions and to maximize the chance of detecting any differences in the effects of rhIGF-I on GH release between young and older adults. rhIGF-I was given intravenously to avoid possible differences in absorption after subcutaneous injection. Subjects were fasted to remove the confounding effects of food intake on GH release; protein meals and some amino acids are stimulatory (48), carbohydrates and fats suppressive (33, 49) and the effect of mixed meals has not been clearly defined. Fasting also increases GH and suppresses IGF-I concentrations (22, 29), changes that are rapidly reversed with re-feeding (50). How the effects observed with intravenous rhIGF-I administration in fasting subjects correlate with those of endogenous IGF-I under a variety of other nutritional states, is not known. By the end of the 3 µg/kg·h rhIGF-I infusion period, significant suppression of GH concentrations had occurred in both age groups. Mean total IGF-I concentrations at that time point were higher than baseline, but well within age-adjusted nonfasting normal ranges, and also within the lower fasting normal ranges for each age group derived from the results of this study (mean ± 2 SD at the same time point on the control day).
Whether the observed suppression of GH release occurred at physiological concentrations of free IGF-I is not clear. Free IGF-I has a half-life of about 10 min, compared to longer than 10 h for IGF-I bound to IGFBP-3 as part of the 150 Kd complex (51). Free IGF-I concentration profiles based on frequent blood sampling have not been reported, and the degree to which free IGF-I concentrations fluctuate throughout the day is not known. The mean free IGF-I concentration at the end of the 3 µg/kg·h IGF-I infusion in younger subjects was 1.7 times that at the same time on the control day, and within the normal range for the control day (see above). The mean free IGF-I in the older subjects was 3.7 times that on the control day, and above the control day normal range, although 4 of the 11 subjects had values within that range.
These increases in free IGF-I concentrations with intravenous rhIGF-I infusion most likely reflect saturation of available IGFBP binding sites. In humans, IGF-I administered alone has little if any effect on IGFBP-3 concentrations (52). Administration of IGF-I to fasting or calorically-deprived subjects does result in increased serum IGFBP-1 and IGFBP-2 concentrations (52, 53). In the case of IGFBP-1, this increase is consistent with the suppressive effect that IGF-I administration has on plasma insulin concentrations (19), as IGFBP-I concentrations are inversely related to insulin concentrations (38). The increase in free IGF-I concentrations observed in this and other studies after IGF-I administration probably reflects the fact that IGFBPs 1 and 2 account for too small a proportion of total IGF-I binding, and the synthesis and release of new IGFBPs occurs too slowly, to prevent saturation of binding sites. Bearing in mind that suppression of GH secretion by rhIGF-I precedes decreases in circulating GH concentrations (19), and also that the suppressive effect of rhIGF-I on GH concentrations in the present study started before the end of the 3 µg/kg·h rhIGF-I infusion period, a likely interpretation of our findings is that the inhibition of GH release produced by exogenous rhIGF-I occurred at physiological blood levels of total IGF-I in both age groups, at physiological levels of free IGF-I in the younger subjects, and at physiological or slightly supraphysiological levels of free IGF-I in the older subjects. Thus, although continuous intravenous infusion of rhIGF-I is certainly not a physiologic intervention, we believe these findings are more likely to reflect the sensitivity of the hypothalamic-pituitary axis to endogenous IGF-I feedback than those of previous studies in which higher circulating concentrations of IGF-I were achieved.
In conclusion, intravenous infusion of exogenous rhIGF-I, in doses as low as 3 µg/kg·h, suppresses GH release in fasted young and older adults, and this GH-suppressive effect of rhIGF-I most likely is exerted independently of its glucose- lowering action. Advancing age is associated with an apparent reduction in sensitivity to the GH-suppressive effects of exogenous IGF-I. Because the circulating IGF-I concentrations produced by the rhIGF-I infusion approximated those under physiological conditions, these results suggest that increased sensitivity to the negative feedback effects of endogenous IGF-I is not a cause of the aging-associated decline in GH secretion.
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Acknowledgments |
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Footnotes |
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2 Current Address: Department of Medicine, Royal Adelaide Hospital,
North Terrace, Adelaide, South Australia, 5000.
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We derived confidence bands for the time trends in the younger and older subjects, and for the difference in the two time trends, using a nonparametic approach called the bootstrap A2 . The bootstrap does not assume normality or any other distribution for the residuals about the fitted regression curve. The bootstrap is a simulation technique in which a large number, here 500, of samples with replacement from the original sample are used to study the statistical variation in any quantity of interest. The particular form of the bootstrap we used considers time and subjects to both be fixed effects. This is done by simulating the distributions of the residuals using the coefficients of the spline function from the original sample, rather than simulating the actual concentrations. The sampling was done by sampling with replacement from subjects rather than from all of the data points, to take into account the fact that measurements made within a subject are correlated A3 . No assumption is made about the form of the correlations over time within subjects for this analysis.
It is common to compute pointwise confidence limits of the mean concentration at each of a series of time points, whether using a parametric approach or using the bootstrap. This method does not adjust for multiple comparisons caused by the attempt to make inferences about the time-concentration curve at multiple time points. Simultaneous confidence regions on the other hand allow one to make inferences about the whole regression curve, thereby making it safer to interpret nuances about the population mean curves. The method of Tibshirani A4 was used to easily obtain simultaneous confidence sets for the set of coefficients of the spline time profile function as well as the average intercept parameter (over subjects). Here one computes regression coefficients for the original and the 500 bootstrapped samples. For each set of regression coefficients, -2 times the log likelihood criterion is computed assuming a Gaussian distribution, taking care that the log likelihood is evaluated with respect to the original response vector and design matrix. The confidence set of the regression coefficients is the set of all coefficients that are associated with -2 log likelihood values that are less than or equal to say the 0.95 quantile of the vector of 501 -2 log likelihoods. For the coefficients satisfying this condition, predicted curves are computed at the time grid, and minima and maxima of these curves are computed separately at each time point to derive the final simultaneous confidence band.
Calculations were done using S-PLUS Version 3.3 (MathSoft, Inc.) in conjunction with the rm.boot function written by Frank Harrell, in the Hmisc library of public domain S functions on the statlib archive (Web address http://lib.stat.cmu.edu).
Harrell FE, Lee KL, Pollock BG. 1988 Regression models in clinical studies: Determining relationships
between predictors and response. J Natl Cancer Inst. 80:1198–1202.[Abstract][Medline]
Efron B, Tibshirani R. 1993 An introduction to the
bootstrap. New York: Chapman and Hall.
Feng Z, McLerran D, Grizzle J. 1996 A comparison
of statistical methods for clustered data analysis with Gaussian error. Stat Med. 15:1793–1806.[Abstract][Medline]
Tibshirani R, Knight K. 1996 Model search and
inference by bootstrap "bumping." Technical report, Department of
Statistics, University of Toronto,
http://www.utstat.toronto.edu/
tibs. Presented at the Joint
Statistical Meetings, Chicago, IL, 1996.
Received March 7, 1997.
Revised May 15, 1997.
Accepted June 2, 1997.
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  J. D. Veldhuis, D. M. Keenan, J. N. Bailey, A. Adeniji, J. M. Miles, R. Paulo, M. Cosma, and C. Soares-Welch Estradiol Supplementation in Postmenopausal Women Attenuates Suppression of Pulsatile Growth Hormone Secretion by Recombinant Human Insulin-like Growth Factor Type I J. Clin. Endocrinol. Metab., November 1, 2008; 93(11): 4471 - 4478. [Abstract] [Full Text] [PDF]
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 M. G. S. Frutos, L. Cacicedo, C. F. Mendez, D. Vicent, M. Gonzalez, and F. Sanchez-Franco Pituitary Alterations Involved in the Decline of Growth Hormone Gene Expression in the Pituitary of Aging Rats J. Gerontol. A Biol. Sci. Med. Sci., June 1, 2007; 62(6): 585 - 597. [Abstract] [Full Text] [PDF]
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 J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, A. D. Rogol, J. C. Lovejoy, M. Sheffield-Moore, N. Mauras, and C. Y. Bowers Endocrine Control of Body Composition in Infancy, Childhood, and Puberty Endocr. Rev., February 1, 2005; 26(1): 114 - 146. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, J. Y. Weltman, A. L. Weltman, A. Iranmanesh, E. E. Muller, and C. Y. Bowers Age and Secretagogue Type Jointly Determine Dynamic Growth Hormone Responses to Exogenous Insulin-Like Growth Factor-Negative Feedback in Healthy Men J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5542 - 5548. [Abstract] [Full Text] [PDF]
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K. Yuen, J. Frystyk, M. Umpleby, L. Fryklund, and D. Dunger Changes in Free Rather Than Total Insulin-Like Growth Factor-I Enhance Insulin Sensitivity and Suppress Endogenous Peak Growth Hormone (GH) Release following Short-Term Low-Dose GH Administration in Young Healthy Adults J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 3956 - 3964. [Abstract] [Full Text] [PDF]
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 R. Nass, S. S. Pezzoli, I. M. Chapman, J. Patrie, R. L. Hintz, M. L. Hartman, and M. O. Thorner IGF-I does not affect the net increase in GH release in response to arginine Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E702 - E710. [Abstract] [Full Text] [PDF]
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S. M. Anderson, L. Wideman, J. T. Patrie, A. Weltman, C. Y. Bowers, and J. D. Veldhuis E2 Supplementation Selectively Relieves GH's Autonegative Feedback on GH-Releasing Peptide-2-Stimulated GH Secretion J. Clin. Endocrinol. Metab., December 1, 2001; 86(12): 5904 - 5911. [Abstract] [Full Text] [PDF]
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)
young subjects on control day; (•) young subjects on rhIGF-I day;
(
) older subjects on control day; and (
) older subjects on
rhIGF-I day. Statistical comparisons were performed at 0700 h,
0900 h and at the end of each treatment period; *,
P < 0.05 (IGF-I vs. control); **,
P < 0.01 (IGF-I vs. control); #,
P < 0.05 (older vs. young
subjects); 
