Free Rather than Total Circulating Insulin-Like Growth Factor-I Determines the Feedback on Growth Hormone Release in Normal Subjects

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1 X/05/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 90(1): Printed in U.S.A. Copyright 2005 by The Endocrine Society doi: /jc Free Rather than Total Circulating Insulin-Like Growth Factor-I Determines the Feedback on Growth Hormone Release in Normal Subjects Jian-Wen Chen, Kurt Højlund, Henning Beck-Nielsen, Jens Sandahl Christiansen, Hans Ørskov, and Jan Frystyk Medical Research Laboratories and Medical Department M (J.-W.C., J.S.C., H.Ø., J.F.), Aarhus University Hospital, Nørrebrogade, and Laboratory of Biochemical Pathology (J.-W.C.), Aarhus University Hospital, DK-8000 Aarhus C, Denmark; and Diabetes Research Centre (K.H., H.B.-N.), Department of Endocrinology, Odense University Hospital, DK Odense C, Denmark Pituitary GH secretion is feedback regulated by circulating IGF-I. However, it remains to be determined whether the feedback control is mediated through circulating free or total IGF-I. To study this, we compared the temporal changes in circulating levels of GH vs. free and total IGF-I during fasting. Seventeen healthy normal-weight subjects (body mass index kg/m 2 ) were studied during 80 h of fasting. Serum was assayed for GH every 3 h; total, free, and bioactive IGF-I, IGF binding protein (IGFBP)-1, -2, and -3 as well as IGFBP-1 bound IGF-I were assayed every morning. During fasting, mean 24-h GH levels increased from to and g/liter (d 1 vs. d 2 and 3; P < 0.03). After 24 h of fasting, free and bioactive IGF-I had decreased by 40 5 and 17 5%, respectively (P < 0.02), and both concentrations remained suppressed for the rest of the study. In contrast, total IGF-I remained unchanged until the end of IN BOTH HUMANS and experimental animals, systemic administration of IGF-I inhibits GH secretion through negative feedback control. Based on the demonstration of IGF-I receptors (IGF-IRs), this feedback control appears to involve the hypothalamus and pituitary. In the hypothalamus, IGF-I increases the synthesis and secretion of somatostatin, whereas GHRH synthesis and secretion are suppressed. In the pituitary, IGF-I has been shown to inhibit GH synthesis and release (1 5). In normal-weight subjects, fasting for 2dissufficient to increase the secretory burst frequency and amplitude of GH, however, without any concomitant alteration in serum total (extractable) IGF-I (6 9). This finding has been interpreted as a condition of hepatic GH resistance (10), caused by a reduced hepatic insulin exposure because insulin has been shown to stimulate the hepatic GH receptor synthesis (11, 12). However, recent studies provided evidence for a role of free IGF-I in the feedback regulation of GH during fasting. First Published Online October 27, 2004 Abbreviations: BMI, Body mass index; CV, coefficient of variation; FFA, free fatty acid; IGFBP, IGF binding protein; IGF-IR, IGF-I receptor; KIRA, kinase receptor activation assay; rh, recombinant human; TR- IFMA, time-resolved immunofluorometric assay. JCEM is published monthly by The Endocrine Society ( endo-society.org), the foremost professional society serving the endocrine community. d 3, at which levels were slightly reduced (P < 0.007). IGFBP-1 increased from 38 2to137 24, , and g/liter (d 1 vs. d2,d3,andendofd3;p < ), and these changes closely paralleled those of IGFBP-1-bound IGF-I (P < ). IGFBP-2 increased only transiently at d 2 (P < 0.05), and IGFBP-3 remained unchanged. The increase in mean 24-h GH levels from d 1 to d 2 correlated inversely with the relative reduction in free IGF-I fromd1tod2(r 0.51; P 0.04), i.e. the larger the reduction in free IGF-I, the larger the increase in GH. None of the other IGF-related parameters correlated with GH. In conclusion, the temporal relationship between the increase in GH and the reduction in free IGF-I supports the hypothesis that circulating free IGF-I mediates the feedback regulation of GH secretion. (J Clin Endocrinol Metab 90: , 2005) Within 24 h of fasting in lean subjects, i.e. before GH levels starts to increase, serum levels of free IGF-I become suppressed by 50%, whereas total IGF-I remains unchanged (9, 13). This reduction in free IGF-I is likely a result of the concomitant increase in IGF binding protein (IGFBP)-1, which becomes detectable a few hours after fasting has commenced (9, 14 16). Although the temporal relationship indicates that the reduction in free IGF-I triggers an increased GH release (9), it has not been possible to demonstrate a statistically significant inverse relationship between levels of free IGF-I and GH during fasting, most likely due to inclusion of too few subjects and samples. The aim of the present study was to test the hypothesis that the early changes in serum free IGF-I are indeed involved in the feedback control of GH secretion during fasting. Therefore, we studied 17 healthy, nonobese subjects during 80 h of fasting, collecting serum samples for determination of GH; free IGF-I; total IGF-I; IGFBP-1, -2, and -3; IGFBP-1 complexed IGF-I; and bioactive IGF-I. Subjects Subjects and Methods The study was approved by the local ethics committee and conducted in accordance with the Helsinki Declaration. Written informed consent was obtained from all participants. Seventeen healthy, normal-weight Caucasian volunteers were studied [male subjects with body mass index (BMI) 27 kg/m 2 and female 366

2 Chen et al. Serum Free IGF-I Controls GH Secretion in Fasting J Clin Endocrinol Metab, January 2005, 90(1): subjects with BMI 25 kg/m 2 ] (Table 1). Two to three weeks before the study, each subject underwent a routine medical examination, including clinical history, medication, blood pressure, electrocardiogram, and blood biochemical and hematological analyses. No history of hyperlipidemia, hypertension, ischemic heart disease, or liver or renal disorder was observed in these subjects, and all subjects had a normal oral glucose tolerance test. None of the subjects took medications except oral contraceptives. Experimental protocol All subjects started fasting at home from 2200 h the evening before admission. The next day, the subjects were hospitalized at 0830 h and stayed for 72 h. A cannula was inserted in a forearm vein, and blood samples were drawn every 3 h for determination of serum concentrations of GH. Serum levels of total, free, and bioactive IGF-I; IGFBP-1, -2 and 3; and the binary complex of IGF-I and IGFBP-1 were measured every morning at 0830 h and at the end of d 3 (after 80 h of fasting). During the study period, the participants were permitted to drink water. One and a half hours before blood sampling and between 2200 and 0700 h, subjects were required to rest. Assays Serum GH was measured by a commercial noncompetitive timeresolved immunofluorometric assay (TR-IFMA) (PerkinElmer Life Sciences, Turku, Finland). Mean within and in-between assay coefficients of variation (CVs) were less than 5%. Plasma glucose was determined by the glucose dehydrogenase oxidation method, and serum free fatty acids (FFAs) were determined by an enzymatic colorimetric assay from Wako Chemicals (Neuss, Germany). Serum insulin was assayed by a commercial TR-IFMA (PerkinElmer Life Sciences). A detailed description of the metabolic changes to fasting has previously been published (17). In this paper, we used 24-h mean levels (permission to reuse these data has been obtained from the American Physiological Society). Serum total (extractable) IGF-I was determined in acid ethanol serum extracts by an in-house TR-IFMA with mean within and in-between assay CVs less than 5 and 10%, respectively (18). Serum free IGF-I was determined after ultrafiltration by centrifugation at conditions approaching those in vivo (8). The lower detection limit of free IGF-I in the ultrafiltrates was g/liter. Mean within and in-between assay CVs of free IGF-I was 15 and 20%. Serum bioactive IGF-I was measured by a novel IGF-I kinase receptor activation assay (KIRA), based on human embryonic renal cells (EBNA 293) transfected with the human IGF-IR gene (19). In this assay, cultured cells are stimulated with either IGF-I standards or unknown serum samples. After 15 min, samples are removed and the cells lysed. Then the crude cell lysates are transferred to an assay that detects the concentration of phosphorylated (i.e. activated) IGF-IRs. This assay uses a monoclonal antibody against the extracellular IGF-IR for coating and a Europium-labeled monoclonal antiphosphotyrosine antibody (PY20) as tracer. The assay is sensitive (detection limit 0.08 g/liter), specific (IGF-II cross-reactivity 12%; proinsulin, insulin, and insulin analogs have a cross-reactivity 1%), and precise (mean within and in-between assay CVs were 7 and 15%). Serum IGFBP-1 was assessed by an in-house RIA and IGFBP-2 by an in-house TR-IFMA (20). Mean within and in-between assay CVs were less than 6 and 12%, respectively. IGFBP-3 was analyzed by a commercial immunoradiometric assay from Diagnostic Systems Laboratories (Webster, TX). IGFBP-1-associated IGF-I (binary complex of IGF-I and IGFBP-1) was determined by an in-house TR-IFMA using an IGFBP-1 antibody for coating, and a Europium-labeled IGF-I antibody as tracer. Mean within and in-between assay CVs were 5 and 15%, respectively (9). FIG. 1. Twenty-four-hour mean serum GH levels during 3 d of fasting.d1(open column),d2(light gray column),andd3(dark gray column). *, P 0.05, compared with d 1. Results are mean and SEM. Statistics Continuous variables were logarithmically (log 10 ) transformed to obtain a normal distribution of data. Parameters were compared by ANOVA for repeated-measures or one-way ANOVA. Bonferroni s tests for multiple comparisons were employed if a significant ANOVA was obtained. Linear regression analysis was used to estimate correlations between parameters. Results were expressed as mean sem, except where otherwise stated. Statistical analyses were performed using SPSS for Windows (SPSS, Chicago, IL). P 5% or less was considered significant. Results The clinical characteristics and baseline levels are shown in Table 1. During fasting, mean 24-h GH concentrations increased significantly from d1tod2( vs g/liter, P 0.001), and levels remained significantly elevated at d 3 ( g/liter; P as compared with d 1; Fig. 1). Fasting decreased 24-h mean levels of plasma glucose at d 2 and 3, when compared with d 1 ( vs and mmol/liter, P , d 1 vs. d 2 and 3), and similar changes were observed for 24-h mean levels of serum insulin ( vs and pmol/liter, P , d 1 vs. d 2 and 3). In contrast, FFAs showed a steady increase in 24-h mean levels, when comparing the 3 d ( vs vs mmol/liter, P , d 1 vs. d2vs. d 3). Serum free IGF-I was significantly reduced by % at the morning ofd2(p 0.001, Fig. 2), and levels remained suppressed for the rest of the study (reduced by % at the morning of d 3, and by % at the end of d 3 as compared with the morning of d 1, P 0.001, Fig. 2). TABLE 1. Clinical characteristics and baseline levels of the circulating IGF system in 17 normal-weight subjects Age (yr) Sex (male/female) BMI (kg/m 2 ) Mean 24-h GH a Total IGF-I Free IGF-I Bioactive IGF-I IGFBP-1 IGFBP-2 IGFBP-3 IGFBP-1 bound IGF-I 33.7 (3.4) 9/ (0.6) 1.41 (0.20) 232 (25) 0.98 (0.14) 2.01 (0.16) 38.2 (3.4) 151 (26) 3801 (21) 16.6 (1.6) Data are expressed as mean (SEM) except for sex (numbers). a Mean GH level during the first 24 h of fasting (d 1).

3 368 J Clin Endocrinol Metab, January 2005, 90(1): Chen et al. Serum Free IGF-I Controls GH Secretion in Fasting Bioactive IGF-I changed in parallel with free IGF-I, being reduced by % at the morning of d 2, by % at the morning of d 3, and by % at the end of d 3, compared with d1(p 0.05, Fig. 2). In contrast to free and bioactive IGF-I, serum total IGF-I showed a blunted response to fasting. Thus, 3 d of fasting was necessary before total IGF-I was significantly reduced, compared with d 1 (P 0.006, Fig. 2). IGFBP-1 was markedly increased by fasting (P , Fig. 3), and the same was true for the binary complex of IGF-I and IGFBP-1. In contrast, IGFBP-2 was only transiently increased at d2(p 0.01, Fig. 3), and IGFBP-3 remained unchanged during the fasting period. Mean 72-h GH levels correlated inversely with BMI (r 0.74, P 0.001). In addition, the increase in 24-h mean GH levels fromd1tod2(i.e. d2 d 1) correlated inversely with the relative reductions in free IGF-I from the morning of d 1 to the morning of d 2 (d 2 divided by d 1) (r 0.51, P 0.04, Fig. 4B). This correlation remained significant after adjusting for BMI. In contrast to free IGF-I, neither serum total nor bioactive IGF-I showed any significant relationships to changes in GH (Figs. 4, A and C). Finally, no correlations between levels of GH and IGFBP-1 were observed (data not shown). FIG. 2. Changes in serum levels of total IGF-I, free IGF-I, and bioactive IGF-I during 3 d of fasting. Baseline (morning of d 1, open column), morning of d 2(light gray column), morning of d 3(dark gray column),andendofd3(black column). *, P 0.05, compared with d 1. Results are mean and SEM. Discussion Previous studies using systemic administration of IGF-I convincingly demonstrated that circulating IGF-I plays a very important role in regulating pituitary GH secretion (1 5). However, they also raised the question whether the GH-suppressive effect of IGF-I was exerted via its proteinbound and/or unbound component because both bound and free IGF-I is increased during IGF-I infusion (2, 5). This issue was investigated by Chapman et al. (2), who studied changes in fasting-stimulated GH hypersecretion before and after a 4-h iv infusion of recombinant human (rh)igf-i (3 g/kg h). As expected, infusion of rhigf-i increased levels of free as well as total IGF-I and suppressed spontaneous GH secre tion. After cessation of rhigf-i infusion, circulating free IGF-I declined to baseline levels within 2 h, whereas serum total IGF-I remained elevated. Concomitant with the fall in serum free IGF-I, the spontaneous GH secretion recovered, despite the fact that total IGF-I was still highly elevated. Based on these findings, the authors suggested that free IGF-I was the major IGF-I component responsible for feedback inhibition of GH secretion (2). In contrast to Chapman et al. (2), who studied the effect of artificially elevated serum levels of free and total IGF-I on fasting-induced GH hypersecretion, we investigated the relationship between changes in GH and the endogenous serum IGF-I levels. We observed significant reductions in serum free and bioactive IGF-I after the first day of fasting, and both levels remained suppressed during the next 2 d, whereas total IGF-I was much more stable. Furthermore, a statistically significant association was observed between the increase in 24-h mean GH levels from d1tod2andthe decrease in free IGF-I, whereas no such relationship was observed for bioactive and total IGF-I. Thus, our study strongly supports that physiological changes in the endogenous serum levels of free IGF-I are able to feedback regulate GH release. It is widely accepted that free, unbound IGF-I is biologically active. However, loosely bound IGF-I (often referred to as readily dissociable IGF-I) being released from the IGFBPs at the site of the IGF-IR has also been suggested to contribute to overall IGF-I bioactivity (21). In the present study, the circulating IGF-I bioactivity was estimated by measurement of free IGF-I, obtained after ultrafiltration of serum (8) as well as by a recently developed highly specific IGF-I bioassay (KIRA), which determines the serum concentration of IGF-I

4 Chen et al. Serum Free IGF-I Controls GH Secretion in Fasting J Clin Endocrinol Metab, January 2005, 90(1): FIG. 3. Changes in serum levels of IGFBP-1, -2, and -3 and the binary complex of IGF-I and IGFBP-1 during 3 d offasting. Baseline (morning of d 1, open column), morning of d 2(light gray column), morning of d 3(dark gray column), and end ofd3(black column). *, P 0.05, compared with d 1. Results are mean and SEM. that is able to activate (i.e. autophosphorylate) the IGF-IR in vitro (19). In contrast to the ultrafiltration procedure, which is designed to isolate free IGF-I only (8), the KIRA is believed to measure the sum of truly free IGF-I plus IGF-I being released from the IGFBPs during incubation with the IGF-IR transfected cells. Accordingly, the serum level of bioactive IGF-I was higher than that of free IGF-I throughout the study. However, whereas free IGF-I was reduced by approximately 50% during the fast, bioactive IGF-I was less affected, being reduced by approximately 20% only, when compared with baseline levels. Furthermore, only free IGF-I showed a significant inverse relationship with the increase in 24-h mean GH serum levels. Bioactive IGF-I was relatively less affected by fasting than ultrafiltered free IGF-I, and we speculate this finding to be associated with the concomitant increase in IGFBP-1-complexed IGF-I. Thus, an increased complex formation between IGF-I and IGFBP-1 may serve to increase levels of readily dissociable IGF-I, which is detected by the KIRA but not by the free IGF-I assay. Supportive of this view, we have previously shown that addition of physiological concentrations of IGFBP-1 to serum markedly reduced levels of ultrafiltered free IGF-I, whereas levels of free plus readily dissociable IGF-I as determined by a commercial immunoradiometric assay remained much less affected (22). Alternatively, it may be speculated that the cell-based KIRA is generally less responsive to changes in the IGF system than the ultrafiltration method. Our data demonstrated the elevation in circulating GH during fasting was preceded by parallel reductions in ultrafiltered and bioactive IGF-I as determined by the KIRA, whereas total IGF-I remained unaffected. However, only free IGF-I showed a statistically significant inverse association with changes in GH levels. At present the reason that bioactive IGF-I did not correlate statistically with changes in GH remains unknown, and further studies are needed to compare and validate the two estimates of the endogenous circulating IGF-I bioactivity. Multiple factors, including other circulating hormones than IGF-I, neurotransmitters, neuropeptides, and metabolic substrates, participate in the regulation of GH secretion (for review please refer to Ref. 3). Thus, circulating IGF-I is unlikely to be the only factor responsible for the observed increase in serum GH, and accordingly, the correlation coefficient (r 2 ) indicated that changes in free IGF-I could explain about 25% only of the observed increase in GH secretion during fasting. In the context of fasting, it is obvious to focus on metabolic substrates such as glucose and FFAs, which are both well-known regulators of GH secretion. Acute hypoglycemia is a potent stimulator of GH secretion, and it may therefore be speculated that the sustained decline in plasma glucose during the fast may serve to stimulate the somatotrope cells. On the other hand, FFA levels increased. Elevated FFA levels is known to block GH secretion provoked by virtually all stimuli (3), and this may therefore counteract the stimulatory effects caused by low glucose and free IGF-I levels. Finally, injections of GH to normal subjects have indicated that GH is able to control its own secretion through autofeedback (3). Hence, several factors besides cir-

5 370 J Clin Endocrinol Metab, January 2005, 90(1): Chen et al. Serum Free IGF-I Controls GH Secretion in Fasting culating free IGF-I are likely to be responsible for the fastinginduced GH hypersecretion. At the third day of fasting, GH levels were numerically lower, but statistically insignificant from d 2 (Fig. 1), whereas levels of free (and bioactive) IGF-I remained at the same level as during d 2. This finding could be argued to contradict with the hypothesis that the increase in GH is partly driven by the reduction in free IGF-I. On the other hand, FFAs continued to increase from the second to the third day of fasting, and this may explain our findings. In healthy subjects, the main part of the IGFs circulates as ternary complexes with IGFBP-3 and the acid-labile subunit, whereas IGFBP-1 on a molar basis accounts only for a small proportion of the circulating IGFBP pool (23). It has been suggested that the ternary complex plays a major role in long-term regulation of IGF-I bioactivity, whereas short-term regulation of IGF-I bioactivity not sequestered in the ternary complex is dependent on variations in IGFBP-1 (24). As shown in many other studies in humans as well as rats (14, 25 27), the present study demonstrated that 3 d of fasting induced a significant increase in serum levels of IGFBP-1. This was also the case for IGF-I complexed to IGFBP-1. We previously investigated the concomitant changes in serum levels of free IGF-I, IGFBP-1, and IGFBP-1-bound IGF-I in a subgroup of subjects (n 7) from the present cohort. That study demonstrated that the increase in IGFBP-1 during fasting could explain more than 50% of the reduction in free IGF-I (9). In conjunction with the present study, it appears that the fasting-induced increase in IGFBP-1, caused by portal hypoinsulinemia (28 30), is likely to be directly involved in the concomitant reduction in free IGF-I and hence indirectly involved in changes in GH secretion. However, we could not observe any relationship between changes in IGFBP-1 and GH. IGFBP-2 is considered to be less affected by nutritional changes than IGFBP-1 (31, 32), and previous studies indicated that at least 2 d of fasting are required to increase levels in obese subjects (32). However, in the present study, we observed an early transient increase in IGFBP-2, indicating that it may indeed respond to short-term nutritional deprivation. In conclusion, in normal-weight subjects the fastinginduced stimulation of GH secretion was preceded by a reduction in free IGF-I and bioactive IGF-I. Of note, the reduction in serum free IGF-I correlated positively with the increase in GH (i.e. the larger the reduction in free IGF-I, the larger the increase in GH). In contrast, total IGF-I showed no association with GH. This observation provides evidence that circulating free IGF-I is the most important component of the circulating IGF system in the feedback regulation of GH. FIG. 4. Relationship between changes in 24-h mean GH levels from d1tod2vs. the morning level of IGF-I at d 2, compared with the morning level at d 1. The y-axes show absolute differences calculated asd2 d 1, the x-axes show relative changes in percent calculated as the ratio between d 2 and d 1. Acknowledgments We are indebted to Mrs. K. Nyborg Rasmussen, Mrs. S. Sørensen, Mrs. Joan Hansen, and Mrs. I. Bisgaard for skilled technical assistance. Dr. Mette Wildner-Christensen (Odense University Hospital, Odense, Denmark) is thanked for helping with recruitment and clinical examination of the study group. We are grateful to Professor Thomas Ledet (Laboratory of Biochemical Pathology, Aarhus University Hospital, Aarhus, Denmark) for helping us with the IGF-I bioassay.

6 Chen et al. Serum Free IGF-I Controls GH Secretion in Fasting J Clin Endocrinol Metab, January 2005, 90(1): Received January 9, Accepted October 13, Address all correspondence and requests for reprints to: Dr. Jan Frystyk, Medical Research Laboratories and Medical Department M, Aarhus Kommune Hospital, Norrebrogade 44, DK-8000 Aarhus C, Denmark. jan@frystyk.dk. This work was supported by Grant from The Danish Healthy Research Council (Aarhus University-Novo Nordisk Center for Research in Growth and Regeneration), Grant from the Institute of Experimental Clinical Research, and grants from the University of Aarhus, The Hørslev Foundation, the Novo Nordisk Foundation, and the Family Hede Nielsen Foundation. References 1. 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