0021-972x/92/7502-0603$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

Vol. 75, No. 2 Printed

in U.S.A.

Effects of Recombinant Insulin-Like Growth Factor-I (IGF-I) and Growth Hormone on Serum IGF-Binding Proteins in Calorically Restricted Adults* SIMON C. J. YOUNG, LOUIS AND DAVID R. CLEMMONS

E. UNDERWOOD,

ABBIE

CELNIKER,

Departments of Medicine (SC. J. Y., D.R.C.) and Pediatrics (L.E. U.), University Medicine, Chapel Hill, North Carolina 27599; and the Department of Medicinal Genentech, Inc., South San Francisco, California 94080

of North Carolina School of and Analytical Chemistry,

ABSTRACT To determine the effects of exogenous insulin-like growth factor-I (IGF-I) and GH on IGF-binding proteins (IGFBP)-1, -2, and -3, six healthy nonobese adult volunteers underwent two 2-week periods of diet restriction (20 Cal/kg.day), and during the last 6 days of the first period received either IGF-I (12 pg/kg. h by iv infusion over 16 h) or GH (0.05 mg/kg.day by SCinjection). During the second P-week study period, the alternate hormone was given. IGFBP-1 and -2 concentrations were determined by specific RIA, and changes in IGFBP-3 were assessed by ligand blotting. Free IGF-I concentrations were measured by size-exclusion high pressure liquid chromatography, followed by RIA. Diet restriction alone did not affect either IGFBP-1 or -2 significantly. IGF-I treatment increased IGFBP-1 from 78 + 46 ng/mL (mean pretreatment) to 137 f 64 ng/mL (P < 0.001; mean for the last 4 days of IGF-I). IGF-I also caused an increase in IGFBP-2 from 315 + 136 to 675 * 304 ng/mL (P < 0.001). GH injections caused a modest decline in IGFBP-1 concentrations but had no effect on IGFBP-2 concentra-

tions. By ligand blotting, both IGF-I and GH caused a modest increase in IGFBP-3 band intensity. In three subjects diet restriction alone caused a small decrease in IGFBP-3 hand intensity, and this was reversed by hormone treatment. Free IGF-I concentrations in serum were increased from 1.6% to 4.4% of the total IGF-I during IGF-I infusions. GH injections caused a smaller increase in free IGF-I concentrations. The results show significant increases in IGFBP-1 and -2 during IGF-I infusion. The change in IGFBP-3, while significant, is quantitatively less than that in experimental animals that have been given IGF-I while undergoing dietary restriction. The net effect of the changes in these three forms of IGFBPs is not sufficient to maintain a normal IGF-I-binding capacity in serum, because free IGF-I levels were increased disproportionately during the IGF-I infusions. Because hypoglycemia was noted in these subjects despite insulin suppression, these alterations in IGFBPs might have changed the tissue bioavailability of IGF-I and facilitated its hypoglycemic effects. (J Clin Endocrinol Metab ‘76: 603-608,1992)

I

such as hypopituitarism, diabetes, prolonged fasting, and extrapancreatic tumor hypoglycemia (8, 11, 19). Administration of IGF-I to hypophysectomized rats causesIGFBP-2 to rise (9), while GH replacement alone does not affect serum values. Diabetic rats that have elevated hepatic IGFBP-2 mRNA show normalization of values after 7 days of insulin therapy (20). Studies in humans are limited, bui Zapf et al. (8) showed that IGF-I induced a 34- to 36-kDa band assumed, but not proven, to be IGFBP-2 in two healthy adults. GH coadministration to one subject opposed this effect. We have carried out experiments in which the metabolic effects of iv infusions of IGF-I were compared with those of SCinjections of GH in diet-restricted normal adults (21). The present report details the effects of these manipulations on the serum concentrations of IGFBPs.

NSULIN -like growth factor-I (IGF-I) and IGF-II are bound in blood to specific, high affinity binding proteins (IGFBPs) (1, 2), and only a small percentage of the IGFs circulate in the free form (3). Six distinct IGFBPs have been identified (4), and four of these(IGFBP-1 to -4) are detectable in adult human serum by ligand blotting. Most of the IGF-I and -11in the blood of normal adults is bound to the high affinity 150-kilodalton (kDa) complex, which consists of IGFBP-3, an acid-labile subunit of 88,000 mol wt, and IGFI or -11(5, 6). The serum concentration of IGFBP-3 is reduced in hypophysectomized animals and is raised by GH (7, 8) and IGF-I (B-10). IGFBP-3 is reduced in fasting (ll), protein restriction (12), and poorly controlled diabetes mellitus (13). IGFBP-1 is a 25,272-kDa protein that rises several-fold overnight and falls promptly postprandially (14, 15). These changes may be due to the suppressive effect of insulin on IGFBP-1 production or to enhanced clearance (16-18). Serum IGFBP-2 is increased in various pathological states, Received August 5, 1991. Address all correspondence and requests for reprints to: David R. Clemmons, M.D., Division of Endocrinology, Department of Medicine, CB 7170, MacNider, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7170. * This work was supported by Grants HD-26871 and HL26309 from the NIH.

Materials

and Methods

Study design Serum for these studies was obtained from six young adult normal volunteers (three men and three women) who underwent two studies of 2 weeks each in the General Clinical Research Center at the University of North Carolina at Chapel Hill, as described previously (21). The protocol was approved by the Committee for the Protection of the Rights of Human Subjects of the University of North Carolina School of

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YOUNG

604

Medicine, and all subjects gave informed consent. The subjects underwent caloric restriction (20 Cal/kg. day) for 2 periods of 2 weeks each. During the last 6 days of each period of diet restriction, each subject received either recombinant human IGF-1 (12 &kg.h by iv infusion over 16 h; 1600-0800 h; a gift from Genentech, Inc., South San Francisco, CA) or GH (0.05 mg/kg.day by SC injection; also provided by Genentech, Inc.). Between each of the study periods, the subjects received a normal diet for 2 weeks. In the second 2-week study period, each subject was given the alternate hormonal treatment. Serum BP determinations were performed on fasting blood samples drawn at 0700 h (GH arm) or 0600 h (IGF-I arm). On the last 3 days of the hormone treatments, samples were also taken 2 h after the evening meal,

Measurement

of

JCE & M -1992 Vol75.No2

TABLE

1. Changes

in free IGF-I

values

during

treatment

with

GH

or IGF-I IGF-I studY daY

8”

f rt f + zk + +

GH

% Free (free IGF-I/total IGFI x 100)

Free IGF-I bx/~L) 4.8 52.1 55.5 51.0 44.6 43.2 19.7

9 10 11 12 13 14

BPS by RIAs

Both IGFBP-1 and -2 were assayed by disequilibrium RIAs developed in this laboratory. The method for IGFBP-1 has been reported previously (15). The lower limit of detectability for IGFBP-1 is 2 ng/mL. The interassay coefficient of variation was 12%, and the intraassay coefficient of variation was 4%. In the RIA for IGFBP-2 (19), the lower limit of detectability was 0.2 ng/mL. The inter- and intraassay coefficients of variation were 11% and 5 %, respectively.

Ligand

ET AL.

2.1 24' 29* 33* 28' 26' 16.9’

1.7 4.5 4.1 3.9 3.8 3.9 3.3

Ftriea;z{i’

% Free (free IGF-I/total IGFI x 100)

2.7 + 1.4

1.1

11.3 + 6.7’~”

2.3

16.2 + 7.8'1'

3.2

’ The sample taken on day 8 preceded the IGF-I infusion. * P c 0.01 compared to the value from the IGF-I treatment for the corresponding day. ’ P C 0.01 compared to day 8 value.

group

250r

blots and immunoblots

One-microliter serum samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5% linear) under nonreducing conditions and transferred onto nitrocellulose, and the BPS were identified using either [‘251]IGF-I (ligand blot) or specific antibody (immunoblot). For the ligand blots the nitrocellulose was incubated for 16 h with 450,000 cpm [‘251]IGF-I, washed, and autoradiographed to identify the BPS according to the method of Hossenlopp et al. (22). For the immunoblots, the nitrocellulose was incubated with polyclonal antibodies at dilutions of between 1:500 and 1:2000 for 2 h. The IGFBPs were visualized using an alkaline phosphatase-linked antirabbit immunoglobulin G (Sigma, St. Louis, MO) and a phosphatase-dependent color development system (Promega, Madison, WI) (23).

Measurement

of free

IGF-I

in serum

L

To determine free (unbound) IGF-I values, 0.2 mL serum was clarified by centrifugation, and 0.1 mL supernatant was injected onto a 30 X 2.5cm size-exclusion HPLC column (TSK model G2000SW, Tosotthas, Montgomeryville, PA) that had been equilibrated in 0.2 M sodium phosphate and 0.05% Tween-20, pH 6.5. This column has been shown to separate four forms of serum IGFBPs from IGF-I and -II. The column was run using a flow rate of 1.0 mL/min, and 0.5-mL fractions corresponding to the elution time of IGF-I were collected and analyzed by RIA (24, 25). There was an 85-100% recovery of free IGF-I from the eluates. Analysis of the column fractions that contained IGF-I detected no BPS by ligand blotting or RIA for IGFBP-3. Recombinant IGFBP-3 was cross-linked to IGF-I using disuccinyl suberate and passed across the same column. Results from this study were compared to those where equivalent amounts of the noncross-linked peptides were chromatographed, and it was found that there was no significant dissociation of IGF-I from IGFBP-3 during passage.

Statistics The comparisons

for Table

1 were

made

using

Student’s

t test.

Results

The serum IGFBP-1 concentrations did not change significantly during either period of diet restriction (Fig. 1). Infusions of IGF-I causedIGFBP-1 to rise slowly from a pretreatment concentration of 89 + 56 ng/mL (mean f 1 SD) to 126 + 31 ng/mL after 5 days of administration. Over the next 24

I

I1

1 2

3

I

4

5

I

I

I

-Treatment I I

I

6 7 8 9 IO II STUDY DAYS

PeriadI I,

12 13 14

1. The effect of GH injections (0.05 mg/kg.day; 0) or IGF-I infusions (12 pg/kg.h, 0) on daily fasting serum IGFBP-1 (mean + SEM) in six normal adults who were ingesting a diet containing 20 Cal/ kg.day. The results shown were obtained by RIA (see Muter& and Methods). Each data point shows the mean from all six subjects. The effect of diet restriction alone on days 1-8 and the effect of diet and hormone administration from days 9-14 are shown. Fasting blood for RIA was drawn either 15 h after each GH injection or after the 14th hour of each IGF-I infusion. FIG.

h, concentrations rose by 60% to 201 + 36 ng/mL after six dosesof IGF-I. In contrast, treatment with GH causedIGFBP1 concentrations to decline from 82 + 27 ng/mL on the last pretreatment day to 45 + 27 ng/mL after the last injection of GH (Fig. 1). Pooled data from the last 4 days of diet restriction and the last 4 days of the two hormonal therapies showed that IGF-I caused an increase from 78 + 46 (mean + 1 SD) to 137 f 67 ng/mL (P < O.OOl),while GH injections causedno significant decreasefrom 77 f 46 to 57 f 37 ng/ mL (P = NS). There was a consistent 55-60% fall in IGFBP-1 concentrations from the fasting state compared to samplestaken 2 h after the evening meal. This was associatedwith increased insulin concentrations with both hormonal treatments compared with fasting morning concentrations. Ligand blots of

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INDUCTION

OF IGFBPs

serum samples from three typical subjects during the diet restriction and IGF-I treatment periods showed a marked increase in IGFBP-1 band intensity as a result of IGF-I treatment (Fig. 2). Identification of the 28,000 mol wt IGFBPI band was confiied by an immunoblot using the IGFBP-1 antibody (data not shown). There was no evidence on immunoblot for formation of proteolytic fragments of IGFBP1. The ligand blots of GH-treated subjects had too little IGFBP-1 under basal conditions to discern GH-induced changes. IGFBP-2 did not change during the week of diet restriction. From a preinfusion concentration (day 8) of 387 +- 135 ng/ mL, infusion of IGF-I produced an impressive rise in serum IGFBP-2 concentrations that was apparent after the first day of IGF-I infusion (Fig. 3). IGFBP-2 concentrations rose to a peak of 827 f 400 ng/mL after 6 days of IGF-I infusions, a mean increase of 2.14-fold. The pooled data from the last 4 days of IGF-I treatment compared to the last 4 days of diet alone also showed a 2.1-fold increase from 315 & 136 to 675 + 304 ng/mL (P < 0.001). In contrast, GH injections caused no significant change in IGFBP-2 concentrations (Fig. 3). By ligand blot, there was a significant increase in IGFBP2 band intensity during IGF-I treatment (Fig. 2), but no significant changes could be detected during GH therapy (data not shown). The 34,000 mol wt IGFBP-2 band could be easily detected by immunoblotting in five of six subjects (data not shown), and a faint proteolytic fragment at or below 14-15 kDa was detected. Neither diet nor GH therapy altered the intensity of the fragment band. Changes in IGFBP-3 band intensity on ligand blot were less than those noted for IGFBP-1 or -2 and differed among the subjects. Three subjects showed no change in IGFBP-3 band intensity during diet restriction, and three subjects had a small decline (Fig. 4 shows a typical case). The decrease in IGFBP-3 in two calorically restricted subjects was reversed by IGF-I treatment (Fig. 4), while GH reversed the decrease in the other case. Overall, increases in IGFBP-3 band intensity were noted in four of six subjects as a result of JGF-I

Subject

1 2 3 LDay8---‘-DayII-J

1

2

3

[ 3daysIGF-I 1 FIG. 2. Autoradiograph of a ligand blot, showing the effect of IGF-I on serum IGFBPs. Blots were probed for IGFBPs using [1251]IGF-I, as described in Materials and Methods. Fasting sera from three subjects obtained on the last pretreatment day (day 8) were compared to those obtained after the third IGF-I infusion (day 11). The vertical ads on the left shows the mol wt (M,; ~10~~) of standards (ovalbumin, carbonic anhydrase, and trypsin inhibitor at 46, 30, and 21.5, respectively). Arrows at the right denote BP species. The two top arrows corresponding to 43,000 and 39,000 mol wt designate IGFBP-3. The arrow at 34,000 mol wt marks IGFBP-2, and that at 28,000 mol wt shows IGFBP-1. The bottom arrow at 24,000 mol wt designates IGFBP-4.

BY IGF-I

1

, 2

605

I 3

I 4

I 5

I 6

, 7

I 8

c-Treatment , , I 9 IO II

Period-t , I, 12 13 14

STUDY DAYS FIG. 3. The effect of GH (0.05 mg/kg.day; 0) or IGF-I (12 pg/kg.h; 0) on daily fasting serum IGFBP-2 (mean f SEM) in six normal adults fed a diet containing 20 Cal/kg-day. IGFBP-2 concentrations were measured by specific RIA (see Materials and Methods), with each data point showing the mean result from all six subjects. The effect of diet restriction alone on days l-8 and the effect of diet and hormone administration on days 9-14 are shown. Serum was collected as indicated in Fig. 1.

Day 1

2

5

6

8

II 12 13 -lGF-IFIG. 4. Autoradiograph of a ligand blot probed with [1261]IGF-I, showing the effect of diet and IGF-I infusion on IGFBPs. The lanes contain serum from a single individual on 8 separate study days. The effect of diet alone is shown on days 1, 2, 5, 6, and 8, and the effect of IGF-I plus diet is shown on days 11-13. The vertical axis on the left shows mol wt (M.; X10v3) standards, and the arrows at the right designate BP species, indicated in Fig. 2. Note that IGFBP-3 band intensity is reduced on days 6 and 8 of diet restriction and is restored by the infusion of IGF-I (days 11,12, and 13).

infusions (Fig. 2) and five of six suibjects who received GH injections (Fig. 5). During IGF-I treatment, the rise in IGFBP3 was apparent by 4 days and did not increase thereafter (data not shown), but two GH-treated subjects showed further increases in IGFBP-3 band intensity at 7 days. Immunoblot of the serum of four subjects revealed no evidence of immunoreactive fragments of IGFBP-3 during diet restriction or hormone treatment. Free (unbound) IGF-I concentrations changed in parallel to the total IGF-I concentration (Table 1). During the first day of IGF-I infusion, free IGF-I rose significantly (-lofold). While total IGF-I levels rose approximately 3-fold, the free IGF-I level was disproportionately increased, leading to

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YOUNG ET AL.

606 M,xl()-3.

46-

1 -Day

23123 l--‘--Day

1

61L

2 3 Day 12--’ [ 4 daysGH ]

5. Autoradiographof a ligandblot probedwith [iz51]IGF-I,showingtheeffectsof GH on serumIGFBPs.Serafromthreesubjects taken on days1 and6 of diet restrictionare compared with serataken after 4 daysof GH injections(day 12). The vertical axis on the left shows molwt (M,; x10e3)standards, andthe arrowsat the right designate BP species, indicatedin Fig. 2. Note that injectionsof GH producedonly a smallincreasein the bandintensityof IGFBP-3.

FIG.

an increase in the percentage of free hormone from 1.6% to 4.4%. The percentage of free IGF-I remained relatively constant thereafter until the infusion was stopped. GH caused an increase in free IGF-I, but the extent of the increase was far lessthan that causedby the IGF-I infusion. Discussion The results of this study show that infusion of IGF-I causes induction of serum IGFBP-1, while GH suppressesit. The induction of IGFBP-1 was associated with a prompt 77% suppressionof fasting insulin and a 37% fall in fasting blood glucose(seeFigs. 4 and 5 in Ref. 21). These observations are consistent with previous reports that suggestthat insulin is a major regulator of IGFBP-1. Patients with type I diabetes have elevated IGFBP-1 concentrations (16, 17, 26), and this elevation declines with insulin infusion (17). Prolonged exercise also results in elevated IGFBP-1 and reduced insulin levels (27). After glucoseloading of normal subjects, there is a strong inverse correlation between insulin and IGFBP-1 concentrations (r = 0.68; P < 0.001) (28). At the cellular level (29) insulin inhibits IGFBP-1 release from cultured HepG2 cells, supporting the conclusion that this is a direct effect. Our conclusion that the increased IGFBP-1 in our subjects during IGF-I infusions is due to suppression of insulin has to be reconciled with the observation that infusion of IGF-I mimicks almost all of insulin’s actions on carbohydrate metabolism (30), suggesting that IGF-I is activating the insulin receptor. Rechler (31) has shown that insulin-mediated suppression of IGFBP-1 in rat hepatoma cells occurs in associationwith insulin receptor activation. Therefore, either the IGF-I is not exerting its hypoglycemic effect through the insulin receptor or it stimulates IGFBP-1 secretion by a different mechanism. In contrast to IGF-I, injections of GH increased insulin levels (21) and decreasedIGFBP-1. These results support the conclusion that the GH-related decrease in IGFBP-1 is secondary to augmentation of insulin secretion. However, on the last day of our study IGFBP-1 showed the greatest increment from the previous day in responseto IGFI infusion. This value was obtained during fasting 8 h after

JCE & M -1992 Vol75.No2

the IGF-I infusion had been discontinued. It is possiblethat the response to IGF-I is biphasic, and at high serum IGF-I concentrations it has a direct insulin-like suppressive effect on IGFBP- 1. IGF-I caused a quantitatively greater increase in IGFBP-2 than in IGFBP-1, while GH had no apparent suppressive effect on IGFBP-2. Zapf et al. (8) infused IGF-I into two normal human subjects and observed an increaseby ligand blotting in a 34-kDa band assumed to be IGFBP-2. Coadministration of GH opposed this effect. Our findings establish that IGF-I induces IGFBP-2 and suggestthat this BP has the potential to modify the effects of infused IGF-I. The mechanism(s) accounting for the IGF-I-induced increase in IGFBP-2 is not entirely clear. Diabetic rats have increasesin hepatic IGFBP-2 mRNA (32, 33) and in a 32kDa band in serum by ligand blotting presumed to be IGFBP2 (34). Another study that used immunoprecipitation, however, showed no increase in IGFBP-2 in diabetic rats (35). Treatment of diabetic rats with insulin for 4 days failed to suppresselevated IGFBP-2 mRNA (33), but treatment of less severely diabetic animals for 7 days normalized the IGFBP2 mRNA (33). Taken together these animal studies suggest that insulin regulates IGFBP-2 mRNA expressionin rats. The effects of insulin on serum IGFBP-2 concentrations, however, are less well defined. Our results showing that IGFBP-2 is increased at a time when insulin is suppressedby IGF-I are consistent with the hypothesis that IGF-I is working through insulin suppression,but doesnot exclude a direct IGF-I effect. In another study (19) we showed that serum IGFBP-2 concentrations were not suppressedin humans in responseto food intake that resulted in a 2.6-fold increasein C-peptide and a 61% suppression of IGFBP-1. Likewise, IGFBP-2 did not increase significantly during overnight fasting. In contrast, 9 days of fasting in humans causeda 1.6-fold increase in IGFBP-2. We conclude, therefore, that acute fluctuations in insulin secretion in humans do not change IGFBP-2, but chronic alterations in insulin secretion may have an effect. The early increasesin IGFBP-2 after IGF-I treatment that we observed, therefore, may be independent of insulin, whereas the effect noted on day 6 could be due in part to changesin insulin sensitivity or secretion. The role of GH in regulating IGFBP-2 is even less clear. Hypophysectomized animals have increasesin IGFBP-2 concentrations by ligand blotting (11) or immunoprecipitation (34). Likewise, IGFBP-2 mRNA is elevated in hypophysectomized animals (32,33). GH treatment alone, however, fails to suppress IGFBP-2 mRNA levels in hypophysectomized rats, and combined treatment with Tl, testosterone, cortisol, and GH is required for suppression(32). In continuously fed cattle, GH causessuppression of serum IGFBP-2 concentrations by RIA (36). In calorically restricted obesehumans (19) treated for 7 days with GH, however, no suppression of IGFBP-2 has been noted. This is consistent with our findings in the present study. Taken together, these results suggest that GH-mediated suppression of IGFBP-2 is predicated upon adequate nutritional intake. Alternatively, the variability of the effect of GH on IGFBP-2 in our study compared with those cited could be due to dosage or interspecies

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INDUCTION

OF IGFBPs BY IGF-I

differences. We were surprised that the changes in IGFBP-3 in our subjects during diet restriction were heterogenous and less than those observed in diet-restricted rats. These rat studies, however, involve extreme dietary restriction, such as fasting (11) or a 5% protein diet (10). Likewise, the induction of IGFBP-3 by IGF-I was less than that observed in rodents (9, 10, 37). While we have no definitive explanation for this, one possible reason is that GH secretion is decreased in dietrestricted rodents, whereas it is increased in humans. The changes noted in IGFBP-3 may have some relevance to understanding the role of IGF-BPS in mediating IGF-I accessibility to the extravascular tissues. In our study the degree of increase in total IGF-I levels exceeded the increase in IGFBP-3. Although the increases in IGFBP-1 and -2 may have compensated partially for this relative lack of a change in IGFBP-3, they were not sufficient to compensate for the increase in total IGF-I values, because a disproportionate change in free IGF-I was noted. The magnitude of changes in all forms of IGFBPs, therefore, was not adequate to maintain the normal percentage of IGF-I binding. The metabolic consequences of increased free IGF-I may be significant, particularly when it is sufficient to produce the hypoglycemic response that was noted in our subjects. Baxter and Martin (38) have proposed that IGFBP-3 in the circulation is present in amounts that are equimolar with the sum of IGF-I plus IGF-II concentrations. Therefore, a disproportionate increase in total IGF-I that perturbs this relationship would be expected to result in an increase in free IGF-I unless the change in IGFBP-1 and -2 were sufficient to compensate for this alteration. Because IGFBP-1 and -2 cross intact capillary barriers (39), whereas IGFBP-3 bound to the 150kDa complex does not, the IGF-I that is bound to IGFBP1 and -2 may still equilibrate freely with IGF-I in the extravascular space. Thus, increases in free IGF-I as well as changes in the distribution of IGF-I among the various types of IGFBPs that are present in blood could alter IGF tissue accessibility. Such alterations could result in hypoglycemia as well as modification of other IGF-I-mediated metabolic effects. This suggests that understanding the alterations in IGFBP profiles produced by the administration of IGF-I and/ or GH will be critical to determining why these proteins modify the metabolic responses to IGF-I and GH.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15

16

17.

18.

Acknowledgments The authors gratefully and GH from Genentech, prepared the manuscript.

acknowledge Inc. We

the also

gifts thank

of recombinant Jennifer O’Lear,

IGF-I who

19.

20.

References 1, Zapf J, Waldvogel M, Froesch ER. 1975 Binding of nonsuppressible insulin-like activity to human serum: evidence for a carrier protein. Arch Biochem Biophys. 168638-45. 2. Hintz RL, Liu F. 1977 Demonstration of specific plasma protein binding sites for somatomedin. J Clin Endocrinol Metab. 45:988-95. 3. Guler H-P, Zapf J, Schmid C, Froesch ER. 1989 Insulin-like growth factors I and II in healthy man. Estimations of halflives and production rates. Acta Endocrinol (Copenh). 121:753-8. 4. Shimasaki S, Shimonaka M, Zhang H-P, Ling N. 1991 Isolation

21.

22.

and molecular characterization of three novel insulin-like growth factor binding proteins (IGFBP-4, 5 and 6). In: Spencer EM, ed. Modern conceuts of insulin-like growth factors. New York: Elsevier; pp 343-59. * Baxter RC. 1988 Characterization of the acid labile subunit of the growth hormone dependent insulin like growth factor binding protein complex. J Clin Endocrinol Metab. 67:265-72. Baxter RC, Martin JL. 1989 Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity labeling. Proc Nat1 Acad Sci USA. 86:6898-902. Grant MB, Schmetz I, Russell B, Harwood Jr HJ, Silverstein J, Merimee TJ. 1986 Changes in insulin-like growth factors I and II and their binding protein after a single intramuscular injection of growth hormone. J Clin Endocrinol Metab. 63:981-4. Zapf J, Schmid C, Guler HP, et al. 1990 Regulation of binding proteins for insulin-like growth factors in humans: increased expression of IGF bindine: uroteins during treatment of healthv adults and in patients with extiapancreatic tumor hypoglycemia. J’Clin Invest. 86:952-61. Zapf J, Hauri C, Waldvogel M, et al. 1989 Recombinant human insulin-like growth factor I induces its own specific carrier protein in hypophysectomized and diabetic rats, Proc Nat1 Acad Sci USA. 86:3813-7. Clemmons DR, Thissen JP, Maes M, Ketelslegers JM, Underwood LE. 1989 Insulin-like growth factor-I (IGF-I) infusion into hypophysectomized or protein-deprived rats induces specific IGF binding proteins in serum. Endocrinology. 125:2967-72. McCusker RH, Campion DR, Jones WK, Clemmons DR. 1989 The insulin-like growth factor-binding proteins of porcine serum: endocrine and nuitritional regulation. Endocrinology. 125:501-9. Thissen JP, Underwood LE, Maiter DM, Maes M, Clemmons DR, Ketelslegers JM. 1991 Failure of IGF-I infusion to promote growth in protein-restricted rats despite normalization of serum IGF-I concentrations. Endocrinology. 128:885-90. Baxter RC, Martin JL. 1986 Radioimmunoassay of growth hormone dependent insulin-like growth factor binding protein in human plasma. J Clin Invest. 78:1504-12. Baxter RC, Cowell CT. 1987 Diurnal rhythm of growth hormoneindependent binding protein for insulin-like growth factors in human plasma. 1 Clin Endocrinol Metab. 65:432-40. Busby WH, Snyder DK, Clemmons DR. 1988 Radioimmunoassay of a 26,000 dalton plasma insulin like growth factor binding protein: control by nutritional variables. J Clin Endocrinol Metab. 67:122530. Brismar K, Gutniak M, Povoa G, Werner S, Hall K. 1988 Insulin regulates the 35 kDa IGF binding protein in patients with diabetes mellitus. J Endocrinol Invest. 11:599-602. Suikkari A-M, Koivisto VA, Rutanen E-M, Yki-Jarvinen H, Karonen SL, Seppala M. 1988 Insulin regulates the serum levels of low molecular weight insulin-like growth factor binding protein. J Clin Endocrinol Metab. 661266-72. Bar RS, Boes M, Clemmons DR, et al. 1990 Insulin differentially alters transcapillary movement of intravascular IGFBP-I, IGFBP-2 and endothelial cell IGF binding proteins in rat heart. Endocrinology. 127:497-9. Clemmons DR, Busby WH, Snyder DK. 1991 Variables controlling the secretion of insulin-like growth factor binding protein-2 in normal human subjects. J Clin Endocrinol Metab. 73:727-33. Wallace JC, Bagley CJ, May BL, Ross M, Francis GL, Ballard FJ. 1989 The use of IGF analogs to determine ligand specificity of different IGF binding protein; In: Drop SLS, Hintz RL, eds. Insulinlike erowth factor bindine uroteins. Excerpt Med Int Conar Ser 881. AmGerdam: Elsevier; pp?.25-30. ’ Clemmons DR, Smith-Banks A, Underwood LE. 1992 Reversal of diet-induced catabolism by infusion of recombinant insulinlike growth factor-1 (IGF-I) in humans. J Clin Endocrinol Metab. 75:234-B. Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M. 1986 Analysis of serum insulin-like growth factor binding proteins using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem.

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YOUNG ETAL.

608 154:138-43.

32.

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