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Regulation of GH Secretion in Acromegaly: Reproducibility of Daily GH Profiles and Attenuated Negative Feedback by IGF-I

Division of Endocrinology and Metabolism, Department of Internal Medicine, Department of Veterans Affairs Medical Center, and University of Michigan Medical Center and Department of Biostatistics, School of Public Health (W.P., M.B.B.), University of Michigan, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Craig A. Jaffe, M.D., Division of Endocrinology and Metabolism, University of Michigan Medical Center, 3920 Taubman Center, 1500 Medical Center Drive, Ann Arbor, Michigan 48109-0354. E-mail: cjaffe{at}umich.edu

Abstract

GH hypersecretion is a hallmark of acromegaly. It is unknown whether the secretory activity of somatotroph adenoma is autonomous or is still governed by central or peripheral mechanisms. In this study we investigated whether GH secretion in acromegaly 1) has a reproducible circadian pattern and 2) is inhibited by exogenous IGF-I. Eleven patients with newly diagnosed acromegaly were studied in 2 protocols. In protocol 1, peripheral blood was sampled every 10 min for 48 h in 6 patients for the determination of concordance between 24-h GH profiles. There was no significant day to day variability in mean 24-h output. There was, however, a significant time effect, and the 24-h GH secretion pattern was maintained between days. In protocol 2, 5 patients were sampled for GH every 10 min twice, once during infusion of normal saline and once during iv infusion of recombinant human IGF-I (10 µg/kg·h). The recombinant human IGF-I infusion increased plasma IGF-I to approximately 230% of the baseline concentration. This resulted in GH suppression (4220 ± 1950 vs. 3223 ± 1472 µg/liter·min; P = 0.001), but did not alter GH secretion pattern. There were highly significant cross-correlations for 10 of the 11 of the subjects in the two protocols when the lag was 0 min. By harmonic analysis, nocturnal augmentation of GH was maintained, and maximum daily GH occurred at approximately 2300 h. These data demonstrate that the pattern of GH secretion in acromegaly is not random, but is highly preserved with 24-h periodicity. In addition, negative feedback regulation by IGF-I is preserved, although the degree of negative feedback is grossly attenuated. Thus, secretory activity of somatotroph adenomas is not autonomous or haphazard, but is still subject to both feedback and feedforward regulatory mechanisms.

WHETHER GH SECRETION in acromegaly is autonomous or is still controlled by the stimulatory and inhibitory feedback mechanisms regulating GH secretion in normal humans is unknown. The observations that GHRH, which is necessary for GH secretion in normal subjects (1, 2), does not stimulate GH release in all acromegalic patients (3), that periadenomatous tissue does not exhibit somatotroph hyperplasia (4), and that many somatotroph adenoma have G_s-activating mutations (5) suggest that these tumors function independently from normal hypothalamic regulation. Yet, nocturnal augmentation of GH secretion, which is the hallmark of central GH regulation, is still maintained in many patients (6, 7). This implies that GH release from somatotroph adenoma is somehow coordinated and supports the hypothesis that the central regulatory mechanisms of GH secretion are at least partially intact.

The integrity of negative feedback by IGF-I on GH in acromegaly is uncertain. In other examples of endocrine neoplasia, shifts in dose-inhibition curves might contribute to trophic hormone hypersecretion. For example, in Cushing’s disease pharmacological, but not physiological, doses of glucocorticoids shut off ACTH hypersecretion from pituitary adenoma (8). Similarly, parathyroid adenomas continue to secrete PTH at a time when serum calcium concentrations are pathologically elevated, but PTH secretion is suppressed at higher serum calcium concentrations (9, 10). Persistent GH hypersecretion in the face of grossly elevated IGF-I implies impaired negative feedback by IGF-I. This hypothesis is supported by in vitro data suggesting that some GH-secreting tumor cell lines may be poorly responsive to direct suppression by IGF-I (11). However, GH secretion in acromegaly is augmented by maneuvers that directly affect circulating IGF-I (12, 13, 14).

Complementary paradigms of raising or lowering the target hormone concentrations have been traditionally used to investigate the physiological mechanisms governing trophic hormone secretion. For example, inhibition of cortisol by metyrapone or by the administration of dexamethasone has been used to study the integrity of the hypothalamic-pituitary-adrenal axis. Fasting-induced decrease in plasma IGF-I potently stimulated GH secretion in normal subjects (14), implying that IGF-I negative feedback is important in the normal physiological control of GH.

The recent availability of recombinant human IGF-I (rhIGF-I) allows direct investigation into the role of IGF-I in neuroendocrine GH control and eliminates the confounding variables that arise with IGF-I-lowering manipulations. We previously demonstrated that infusion of rhIGF-I potently suppressed both spontaneous GH secretion and the GH response to GHRH in normal men (15) and women (16). We subsequently used rhIGF-I to study negative feedback regulation of GH secretion in acromegaly. In addition, we investigated the day to day reproducibility of the GH secretion pattern in this disease.

Materials and Methods

The study was approved by the University of Michigan institutional review board and the General Clinical Research Center advisory committee. All subjects signed informed consent documents before their participation. Eleven patients with newly diagnosed acromegaly were studied in 1 of 2 protocols. None had received any prior treatment for the disease. Clinical details of the individual patients in protocol 1 (patients 1–6) and protocol 2 (patients 7–11) are given in Table 1. All had clinical evidence for acromegaly, elevated baseline plasma GH and IGF-I concentrations, and pituitary tumors demonstrated by magnetic resonance imaging scanning. None of the patients had acromegaly in association with another endocrine tumor syndrome. Subsequently, each subject underwent surgery, and the diagnosis of somatotroph adenoma was established histologically in all. The tumors were immunostained for pituitary hormones in 9 of 11 patients (Table 1). All of the patients were clinically and biochemically euthyroid, as determined by a normal serum free T₄ or free T₄ index. This included patient 1, whose tumor weakly immunostained for TSH. None of the patients was taking exogenous sex steroids, and none had clinical or biochemical evidence for adrenal insufficiency. Meals were served at 0730, 1200, and 1730 h, standardized as previously described (15), and caffeinated beverages or between-meal snacks were not allowed. Lights were turned on at 0700 h and off at 2300 h, and daytime napping and tobacco use were not allowed. The subjects refrained from strenuous exertion throughout each study period.

Protocol 2 studied the effects of IGF-I on GH secretion. The protocol was identical to that previously reported for normal subjects. In brief, five men with acromegaly were administered a 28-h infusion (0800–1200 h) of normal saline (NS), followed by a 48-h iv infusion (1200–1200 h) of rhIGF-I (Genentech, Inc., South San Francisco, CA; 10 µg/kg·h). Venous blood was sampled every 10 min throughout the saline infusion and for 24 h during the second half of the rhIGF-I infusion. Blood samples for plasma IGF-I were obtained every 4 h throughout the study. On d 2 and 4 during saline and rhIGF-I infusions, respectively, the subjects were given iv boluses of TRH (Thypinone, Abbott Laboratories, North Chicago, IL; 50 µg) at 0800 h and human GHRH (Bachem, Torrance, CA; 0.33 µg/kg) at 1000 h.

All plasma GH samples for a patient were measured in duplicate in the same chemiluminometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) as previously described (15). The mean intraassay coefficient of variation (CV) was less than 10%, and the detection limit was 10 ng/liter. Samples with plasma GH concentrations above 20 µg/liter were diluted with the zero standard before being assayed. Total plasma IGF-I was measured by immunoradiometric assay in extracted plasma using commercial kits (Diagnostics Systems Laboratories, Inc., Webster, TX). All IGF-I samples for a specific patient were analyzed in the same assay. The mean IGF-I intraassay CV was less than 6%. Plasma IGF-binding protein-3 was measured by immunoradiometric assay, and PRL was determined by ELISA using commercial kits (Diagostics Systems Laboratories, Inc.). For each of these analytes, all samples were run in a single assay, and the mean CV was less than 6%.

Discreet parameters of GH pulsatility were analyzed by Cluster using parameters previously described (16). The 24-h integrated GH concentration (IGHC) was measured by the trapezoidal rule, and mean IGF-I was defined as the average of the seven daily samples during the first and the second 0800–0800 h periods of the NS or IGF-I infusions. The GH response to GHRH was defined as the maximum increase in GH concentration over the 1000 h baseline during the first hour following the GHRH bolus. The GH and TSH responses to TRH were calculated in an identical manner.

GH concentrations were grouped into six 4-h blocks, and the average of the log_e GH concentration was calculated for each block. Two-way ANOVA was used to determine whether the pattern of GH concentration was similar between the 2 d of study in the protocol 1 (NS/NS) and protocol 2 (NS/IGF-I) datasets. A three-way ANOVA with random effect model using treatment group as the third factor was calculated to determine whether the protocol 1 and protocol 2 groups were similar between days. A potential effect of IGF-I on GH was also determined by comparing day to day variability in GH between protocol 1 and protocol 2. Variability was calculated as the CV of IGHC for d 1 and 2. The mean and SD of the mean for the IGHC was calculated for protocol 1, and the 95% confidence limits were calculated as the mean ± 2 SD. The probability of one or more IGHC CV in protocol 2 exceeding this limit was calculated using binomial expansion.

Concordance between d 1 and 2 was further investigated by calculating the cross-correlation for GH between these 2 d, lagging one dataset in relation to the other set -100 to +100 min at 10-min intervals. A correlation coefficient between d 1 and 2 of each subject for log-transformed 1-h average data were also calculated.

To detect a time of maximum GH level, the log-transformed 1-h average data were regressed on sin(wt) and cos(wt) for each subject and each day, where w = 2p/24 and t = 1,2 ... , 24. As this analysis assumed that the daily maximum and minimums were 12 h apart, acrophase (time of maximum GH) was estimated in a separate analysis by regressing the log-transformed 1-h average data on the higher order harmonic transformation, sin(nwt), cos(nwt), where n = 1,2,3.

Mean pulse amplitude, interpulse GH concentration, and average nadir as well as GH responses to GHRH and TRH and TSH response to TRH were compared between saline and IGF-I treatment days in protocol 2 by paired t tests. When appropriate, the data were analyzed after logarithmic transformation. Plasma IGFBP-3 concentrations at 0800 h on d 2 and 4 (before and near the completion of the rhIGF-I infusion) were compared by paired t test.

Results

Twenty-four-hour GH concentration profiles for two subjects in protocol 1 (NS/NS) are shown in Fig. 1. There are obvious similarities in the daily GH profiles for these two subjects. The mean (±SE) plasma GH for each day and time (block) in protocol 1 is shown in Fig. 2. These data are presented as the logarithm of the block area under the curve for all time blocks. By two-way ANOVA there was no day effect (P = 0.13). There was, however, a strong time effect (P < 0.0001), but no day-time interaction (P = 0.20). Therefore, there was a daily pattern in GH secretion that was maintained both across subjects and day to day within subjects.

View larger version (35K): [in this window] [in a new window]   Figure 1. Individual GH concentration profiles from patient 2 (top panel) and patient 6 (bottom panel) during 48 h of continuous every 10 min blood sampling in protocol 1. Obvious similarities in the pattern of GH secretion are seen between the 2 d.

View larger version (47K): [in this window] [in a new window]   Figure 2. Mean (±SE) plasma GH for each day and time (block) in protocol 1 (top panel) and protocol 2 (bottom panel). Data are presented as the logarithm of the GH area under the curve over the 4-h time blocks. In protocol 1 there was a time effect, no day effect, and no day-time interaction, indicating that the daily GH secretion pattern was maintained across subjects and day to day within subjects. In protocol 2 there was a strong time effect and a day effect, but no day-time interaction. Therefore, the rhIGF-I infusion suppressed average daily GH secretion, but did not affect the GH secretion pattern.

View larger version (30K): [in this window] [in a new window]   Figure 3. Mean (±SE) plasma IGF for the five subjects in protocol 2 who received rhIGF-I (10 µg/kg·h).

View larger version (38K): [in this window] [in a new window]   Figure 4. Individual GH concentration profiles for patient 9 (top panel) and patient 10 (bottom panel) during the infusion of saline () or rhIGF-I (10 µg/kg·h; •). Daily IGHC during rhIGF-I infusion was suppressed to a degree not expected by day to day variability in three of five subjects (see Results).

Concordance between the GH concentrations on the 2 study days in the two protocols was further tested by cross-correlation and lag analyses. Assuming that GH values 30 min apart were independent, there were 48 independent observations for each day. Therefore, a correlation coefficient greater than 0.4 was significant at the 0.01 level. Cross-correlations with 10-min lags for protocols 1 and 2 are shown in the top and bottom panels of Fig. 5, respectively. In protocol 1, all six subjects had high cross-correlation when the lag was 0 min, and four of five of the protocol 2 subjects during saline infusion similarly had a high cross-correlation. Wide GH pulses accounted for the relatively gradual decline in correlation around the maximum at a lag of 0 min.

View larger version (20K): [in this window] [in a new window]   Figure 5. Cross-correlation profile for subjects with acromegaly in protocol 1 (top panel) and protocol 2 (bottom panel). The critical value for significance was conservatively calculated to be 0.4, and 10 of 11 subjects showed a positive cross-correlation at a lag of 0 min at the 0.01 level.

Sine/cosine fitting to logarithm-transformed 1-h average GH data were performed for the two 24-h profiles and demonstrated 24-h periodicity in most of the hormone series. Fifteen of the 22 regressions were significant at the 1% level, and 3 were significant between the 1% and 5% levels. The fit in three profiles was significant between the 5% and 10% levels. One regression was nonsignificant. The latter data were from a protocol 1 patient and were excluded from subsequent calculations of acrophase. Acrophases in d 1 and 2 in protocol 1 were 19:56 ± 2:14 and 22:02 ± 2:25 h. For protocol 2, the mean acrophases were 00:33 ± 2:42 and 00:07 ± 1:36 h for the 2 d, respectively. Overall, using saline studies alone, the acrophase was 23:14 h.

Twenty-four-hour GH profiles in protocol 2 were also analyzed by Cluster. There was no effect of rIGF-I on either GH pulse frequency (14.2 ± 2.0 vs. 12.6 ± 1.6 pulses/24 h; P = 0.37) or mean GH pulse amplitude (10.0 ± 4.6 vs. 7.5 ± 2.9 µg/liter; P = 0.44). There were weak trends to lower 24-h GH nadir (23.8 ± vs. 17.3 ± 6.9 µg/liter; P = 0.14) and interpulse GH concentration (27.7 ± 13.2 vs. 19.4 ± 7.9 µg/liter; P = 0.13) during rhIGF-I infusion.

GH responses to exogenous GHRH in protocol 2 are shown in Fig. 6. There was a trend for smaller GH responses to GHRH during rhIGF-I treatment (32.7 ± 17.3 vs. 18.7± 8.3 µg/liter; P = 0.11), with two subjects having nearly identical responses during the 2 d and three subjects having GH increases that were 45–72% below control responses. The mean GH response to TRH is shown in Fig. 7. Only two of the five subjects had clear increases in GH after the TRH bolus during NS infusion. In one of these patients, the GH response during rhIGF-I was approximately half the baseline response, whereas the GH responses on both days were nearly identical for the second subject. TSH responses to TRH are shown in Fig. 8. There was a trend for a lower baseline TSH (1.38 ± 0.40 vs. 1.04 ± 0.39 mU/liter; P = 0.06) during rhIGF-I infusion. The TSH response to TRH tended to be lower during IGF-I infusion, but this difference did not reach statistical significance (4.68 ± 0.57 vs. 2.75 ± 0.49 mU/liter; P = 0.11). Serum total T₄ (74 ± 5 vs. 86 ± 7 µg/liter; P = 0.02) and free T₄ index (69 ± 2 vs. 79 ± 5; P = 0.04) were higher during IGF-I infusion. Serum total T₃ was within the normal range for all subjects before any treatment and was modestly higher during IGF-I infusion than during saline treatment (1.39 ± 0.09 vs. 1.59 ± 0.11 µg/liter; P = 0.02). IGFBP-3 also increased during the IGF-I infusion (5.9 ± 0.6 vs. 8.7 ± 1.3 mg/liter; P = 0.03).

View larger version (30K): [in this window] [in a new window]   Figure 6. GH responses to GHRH (0.33 µg/kg, iv) in subjects with acromegaly during saline () and rhIGF-I (•) infusions.

View larger version (27K): [in this window] [in a new window]   Figure 7. GH responses to TRH (50 µg, iv) in the subjects during saline () and rhIGF-I (•) infusions.

View larger version (24K): [in this window] [in a new window]   Figure 8. TSH responses to TRH (50 µg, iv) in the subjects during saline () and rhIGF-I (•) infusions.

The present study, which was performed in two groups of newly diagnosed acromegalics, examined the regulation of spontaneous GH secretion as well as the integrity of the negative IGF-I feedback in this disease. Based on visual inspection of 24-h GH profiles, it had previously been suggested that GH secretion profiles were reproducible on a day to day basis in acromegalic patients (7). We have demonstrated that GH secretion in acromegaly is not random; rather, it is remarkably constant in terms of both mean daily GH output and GH secretion profile. In most instances, 24-h periodicity with preserved nocturnal augmentation in GH secretion pattern was maintained. In addition, this is the first study to directly investigate IGF-I negative feedback in acromegaly in vivo. We demonstrate that this feedback is present in approximately half the subjects, although it is attenuated compared with that in normal subjects.

In normal subjects, IGF-I plays a central role in the negative feedback regulation of GH secretion. The importance of IGF-I feedback is clearly established by the suppression of GH secretion in GH-resistant children treated with rhIGF-I (17). The negative feedback mechanisms are probably complex. IGF-I has direct effects at the pituitary and suppresses both basal and GHRH-stimulated release of GH from primary pituitary culture (18, 19). In addition, IGF-I probably inhibits GH secretion through a hypothalamic effect, as IGF-I treatment of primary hypothalamic cells stimulated somatostatin secretion in vitro, and intracerebroventricular administration of IGF-I to rats inhibited GH release (20). In humans, fasting or GH receptor abnormalities are associated with low circulating IGF-I and elevated plasma GH, and the latter is suppressed by systemic IGF-I administration (17, 21). We have previously shown that the dose of rhIGF-I used in this study (10 µg/kg·h) suppressed GH secretion in normal men and women by approximately 80% and 60%, respectively (15, 16). IGF-I infusion also suppressed the GH response to GHRH and the TSH response to TRH (15, 16). Although it is conceivable that TSH suppression is the result of altered thyroid hormone levels, these data are consistent with IGF-I-enhanced hypothalamic somatostatin secretion.

rhIGF-I administration to acromegalic patients led to clear suppression of GH in three of five patients. Although the number of subjects was small, these data suggest that over 50% of somatotrophic adenomas respond to negative feedback by IGF-I, albeit to a lesser degree than in normal subjects. In normal subjects, the increase in plasma IGF-I was approximately 1000 ng/ml during the infusion, and total plasma IGF-I concentrations were roughly 5 times the baseline value. In the acromegalic subjects, although the absolute increase in IGF-I was similar, the relative rise in plasma IGF-I concentrations was only 2–2.5 times the baseline levels. It is conceivable that higher concentrations of IGF-I would have suppressed GH further. If this were true, it would be similar to the original data reported by Liddle (8), demonstrating dose-dependent suppression of the hypothalamic-pituitary-adrenal axis by glucocorticoids in patients with Cushing’s disease.

Whether the IGF-I-mediated GH suppression in the patients was a hypothalamic or a direct pituitary effect is not clear. In contrast to what we observed in normal adults (15, 16), there was no clear decrease in GH pulse amplitude. There were, however, trends toward lower interpulse GH concentrations and smaller GH responses to GHRH. Either an increase in somatostatin or direct pituitary suppression could mediate this suppression. Similarly, the TSH response to TRH was blunted. Again, this is consistent with an increase in hypothalamic somatostatin output. Serum T₃ was normal before IGF-I treatment, and as we previously reported in normal subjects (16), IGF-I infusion increased serum T₃ in these patients with acromegaly. In contrast to an IGF-I infusion having no effect in normal subjects (16), the infusion resulted in a modest, but statistically significant, increase in T₄ in our patients. The reason for the difference in T₄ economy in normal and acromegalic subjects is not apparent.

The possibility that loss of IGF-I feedback at the level of the pituitary could play a role in GH hypersecretion in acromegaly is supported by in vitro data. In primary rat pituitary cell cultures, IGF-I suppressed basal and GHRH-stimulated GH transcription and release (19, 22). Most in vitro data on the direct pituitary effects of IGF-I on human somatotrophs have come from either primary culture of somatotroph adenomas or pituitary cell lines. IGF-I inhibited both basal and GHRH-stimulated GH secretion (23, 24) and GH transcription (24) in primary culture of somatotroph adenomas. Of interest, rhIGF-I also attenuated GH secretion from the GC cell line, but failed to suppress GH release from GH₃ cells (11). The affinity for IGF-I binding to receptors on the two cell types is similar; however, GC cells contain 3-fold more binding sites. Moreover, overexpression of IGF-I receptors in GC cells increases responsiveness to inhibition by IGF-I (25). These data suggest that a decrease in the number of IGF-I receptors on somatotroph adenoma cells could contribute to GH hypersecretion in acromegaly. Alternatively, receptor mutations or postreceptor abnormalities might be present. However, analysis of a submembrane domain in the IGF-I receptor that is important for ligand-mediated signaling did not find any mutations in 19 GH-secreting tumors (26). More studies are needed to define the pathophysiology behind the attenuated IGF-I feedback in acromegaly.

In both the NS/NS and the NS/IGF-I protocols, GH secretion was predictable. Multiple analyses demonstrated concordance between the pattern of secretion on the 2 d and that GH secretion in acromegaly has a 24-h periodicity. Whereas there was considerable variability in the timing of maximum daily GH secretion, the acrophase for most patients was in the late evening or early night hours, similar to the nocturnal augmentation observed in normal adults (16). These data confirm our earlier observations of nocturnal augmentation of GH in acromegaly (6). They also confirm observations by Christensen and colleagues (7), who reported that six of seven acromegalics had reproducible 24-h profiles measured by hourly samples on up to three separate occasions, and four of seven had nocturnal GH elevation. Murialdo et al. (27), using Fourier analysis, also found 24-h periodicity in most subjects with acromegaly, although they did not find nocturnal GH augmentation. Only one of our patients (patient 8) did not have a significant correlation between GH output on the 2 d. Of interest, his tumor was shown by immunohistochemical staining to be a mammosomatotroph.

The genesis of either reproducible mean GH or daily periodicity in GH secretion is not known. In normal subjects, the CV for mean daily GH secretion is approximately 30% (28), and the CV for GH responses to pharmacological stimuli is as high as 100% (29). In contrast, the mean CV for the six patients studied on two occasions during normal saline infusion was only 6%. Predictability in the pattern of daily GH secretion is unlikely to be an intrinsic function of the pituitary itself. More likely, hypothalamic or extrahypothalamic neuropeptides or other molecules are modulating GH secretion. The presence of physiological nocturnal augmentation of GH secretion, as observed in this and other (6, 7) studies, strongly suggests that somatotroph adenomas are under at least some degree of central nervous system control.

What peptide(s) is involved in this regulation is uncertain. In normal subjects, the persistence of pulsatile GH secretion during continuous infusion of GHRH has been used as an argument supporting the existence of a non-GHRH mechanism, such as a fall in hypothalamic somatostatin, as the timing mechanism for GH pulses (30). In addition, increased pituitary sensitivity to GHRH at night suggested a non-GHRH mechanism for nocturnal augmentation of GH secretion (31). Yet, nocturnal GH augmentation persisted during the infusion of a supraphysiological dose of octreotide, so that the nocturnal decline in somatostatin is unlikely to be the cause of the augmented GH secretion (32). Similarly, a nocturnal rise in GH secretion persisted despite continuous infusion of GH-releasing peptide-6 (33). This suggests that a nighttime increase in GH secretagogue is also unlikely to be the driving force for nocturnal GH release. In contrast, a continuous infusion of GHRH antagonist eliminated nearly all of the nocturnal GH rise in normal subjects (2). Therefore, the role of central GHRH as a driving force in normal and, conceivably, in acromegalic subjects needs to be considered.

In conclusion, we have shown that GH secretion in acromegaly is characterized by a high degree of day to day reproducibility, persistence of nocturnal GH augmentation, and preserved (albeit attenuated) negative feedback regulation by IGF-I. Thus, human somatotroph adenomas are not entirely autonomous, but remain under a considerable degree of central and peripheral physiological regulatory control.

Acknowledgments

We thank the General Clinical Research Center staff for their excellent nursing support and Genentech, Inc., for providing recombinant human IGF-I.

Footnotes

This work was supported in part by V.A. Merit Review Awards (to C.A.J. and A.L.B.), RO1-DK-38449 (to A.L.B.), MO1-RR-0043-34S3 (Clinical Associate Physician Award to C.A.J.), MO1-RR-0042 (General Clinical Research Center), P60-DK-20572 (University of Michigan Diabetes Research Training Center), and the Research Service of the Department of Veterans Affairs.

Abbreviations: CV, Coefficient of variation; IGHC, integrated GH concentration; NS, normal saline; rhIGF-I, recombinant human IGF-I.

Received December 20, 2000.

Accepted May 17, 2001.

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