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Mineral Metabolism Laboratory (D.J.B., S.M.), Jerry L. Pettis Memorial Veterans Administration Medical Center, Department of Nutrition (S.R.), School of Public Health, Departments of Medicine, Biochemistry, and Physiology, School of Medicine (D.J.B., S.M), Loma Linda University, Loma Linda, California 92357
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Abstract |
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 Top Abstract I. IntroductionII. Characteristics of the...III. Target Cell Actions...IV. IGF-IGFBP Complexes in...V. Assays for Circulating...VI. Relative Distribution of...VII. Regulation of Serum...VIII. IGFBP Proteases in...IX. Endocrine Functions of...X. ConclusionsReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Characteristics of the...III. Target Cell Actions...IV. IGF-IGFBP Complexes in...V. Assays for Circulating...VI. Relative Distribution of...VII. Regulation of Serum...VIII. IGFBP Proteases in...IX. Endocrine Functions of...X. ConclusionsReferences |
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In subsequent studies, Zapf et al. (14 ) incubated plasma with radiolabeled IGF and detected the somatomedin activity at 40 kDa. This binding was highly specific for the IGFs with no competition from insulin, suggesting the presence of specific high-affinity binding proteins for the IGFs. Furthermore, Kaufmann et al. (15 ) showed that the half-life of 125I-labeled NSILA was reduced when excess unlabeled NSILA was injected into normal rats, suggesting that NSILA is bound to carrier proteins. The binding of NSILAs to the carrier proteins in serum provided a possible explanation for the absence of insulin-like effects of endogenous NSILA in vivo.
The discovery of the six different binding proteins currently known to
exist did not occur all at the same time (1 2 3 4 5 6 7 16 ). The first three
IGFBPs were purified in the mid-1980s. The association of
[125I]IGF in rat serum initially with a small molecular
binding protein complex and a shift to a larger complex after a few
minutes confirmed the presence of two binding proteins (14 ). The first
was a major binding protein in serum identified to be a GH-regulated
acid-labile 150- to 200-kDa complex eventually designated as
IGF+IGFBP-3+acid labile subunit (ALS) complex. The second was a
GH-independent 50 kDa acid-stable protein (2 17 ). Later it was shown
that this 50-kDa small molecular binding protein consisted of IGFBP-1
and IGFBP-2. By the late 1980s and early 1990s, three additional
binding proteins, IGFBPs 4 through 6, were isolated and characterized.
All six binding proteins share 
35% sequence identity with each
other. Several excellent reviews on the structure, molecular and
cellular aspects, and biological actions of these binding proteins are
available (1 2 3 4 5 6 7 ) and therefore will not be the focus of this review.
The recent development of specific assays for measurement of various
IGFBPs in circulation and in other biological fluids has led to
significant new information on serum regulation of IGFBPs and their
functions. The main focus of this review is to present the data
regarding the characterization of the IGF-IGFBP complexes in serum and
other biological fluids and to evaluate their regulation and functions.
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II. Characteristics of the IGFBPs |
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TopAbstractI. IntroductionII. Characteristics of the...III. Target Cell Actions...IV. IGF-IGFBP Complexes in...V. Assays for Circulating...VI. Relative Distribution of...VII. Regulation of Serum...VIII. IGFBP Proteases in...IX. Endocrine Functions of...X. ConclusionsReferences |
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IGFBP-1, a nonglycosylated protein of 30 kDa, was first isolated from
mid-term amniotic fluid (20 ). This binding protein shares sequence
identity with the placental protein 12 (19 26 ), which is synthesized
by endometrium and decidua. It is present in the amniotic fluid in
concentrations 100–500 times higher than in serum. IGFBP-2, originally
isolated from rat liver (BRL)-3A cell line (27 ), is a nonglycosylated
protein of 31–36 kDa and is found in significant amounts both in serum
and cerebrospinal fluid (1 ). The major form of binding protein present
in human circulation is IGFBP-3; its molecular mass ranges from 38 kDa
to 43 kDa depending on the number of sites glycosylated (28 ). In
circulation, this glycoprotein is associated with an IGF molecule and
an 80-kDa acid-labile subunit (ALS) to form a 150- to 200-kDa complex
(2 29 30 ). This complex consists of IGFBP-3 and IGF-I + IGF-II in an
equimolar ratio, suggesting that most of the IGFBP-3 in serum is likely
to be saturated.
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Although the IGFBPs differ in their structure and binding specificity, it is not clear whether these differences contribute to functional differences among the various IGFBPs. For example, it is not known whether there is any functional significance for glycosylation of the IGFBPs and why some of the IGFBPs bind IGF-II with preferential affinity (39 40 ) compared with IGF-I. Interestingly, none of the IGFBPs bind IGF-I with preferential affinity. In any case, the differences in structure (glycosylation, number of cysteine, RGD sequence), binding affinity, and tissue-specific expression are consistent with the general idea that different IGFBPs have discrete functions. Based on the findings that extracellular fluids of certain tissues (described in Section IV) are enriched with specific IGFBPs and that tissues surrounding the body fluid express the same IGFBPs in high abundance (41 42 ), it is speculated that the IGFBPs may function locally to regulate IGF actions. However, there is no experimental data to demonstrate that this is in fact true.
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III. Target Cell Actions of the IGFBPs |
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TopAbstractI. IntroductionII. Characteristics of the...III. Target Cell Actions...IV. IGF-IGFBP Complexes in...V. Assays for Circulating...VI. Relative Distribution of...VII. Regulation of Serum...VIII. IGFBP Proteases in...IX. Endocrine Functions of...X. ConclusionsReferences |
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A. To modulate IGF actions
Studies in a number of laboratories including ours have shown that
IGFBPs are capable of modulating IGF-induced cell proliferation both in
a positive and negative manner (3 4 43 44 45 ). Several IGFBPs,
including IGFBP-1, IGFBP-2, IGFBP-4, and IGFBP-6, inhibit IGF
action by binding to IGFs and preventing the binding of IGFs to IGF
receptors (3 4 ). In contrast to the phosphorylated IGFBP-1 that
inhibits IGF actions, the nonphosphorylated form of IGFBP-1 potentiates
the effect of IGF-I on DNA synthesis in porcine smooth muscle cells
(4 ). Coincubation of human fibroblasts with IGF and IGFBP-3 showed an
inhibitory effect while preincubation with IGF had growth-potentiating
effect (43 ). It was suggested that binding of IGFBP-3 to the cell
surface reduces its affinity for IGF-I and results in a potentiating
effect (44 ). In contrast to other IGFBPs, IGFBP-5 is stimulatory for a
variety of cell types (45 46 47 48 ).
Thus, the different binding proteins may modulate IGF action differently, and the same binding protein can have an IGF-inhibiting or potentiating role under different conditions. The factors that determine these differences include IGFBP phosphorylation, IGFBP proteolysis, and IGFBP cell surface association, among others. These variables may modulate IGF action in target tissues by altering the binding affinity of the IGFBPs to IGFs.
B. To facilitate storage of IGFs in extracellular matrices
Another important role of IGFBPs may be to help in the storage of
IGFs in the extracellular matrices of certain tissues. In this regard,
Jones et al. (45 ) provided evidence for fixation of IGFs via
IGFBP-5 binding to extracellular matrix proteins. We found evidence
that IGFBP-5 may help fix IGFs in bone since the complex of IGFBP-5 and
IGFs, but not IGFs alone, bind to hydroxyapatite (34 49 ). In terms of
the significance of fixation of IGFs in extracellular matrices such as
bone, it is speculated that the stored IGFs may be released during the
osteoclastic bone resorption phase of bone remodeling to stimulate
nearby osteoblasts during the bone formation phase of remodeling (50 ).
Similarly, IGFs stored in extracellular matrices of soft tissues may
have a role in wound healing.
C. To exert IGF-independent effects
Recent evidence suggests that some of the IGFBPs may mediate their
effects on target cells by an IGF-independent pathway. This concept has
evolved from a number of experimental studies, including the study by
Jones et al., which found that IGFBP-1 stimulated smooth
muscle cell migration by an IGF-independent mechanism involving
integrin receptors (51 ). IGFBP-3 has been shown to inhibit
proliferation of breast and prostate cancer cells by a cellular
signaling pathway independent of IGFs (52 53 ). In addition, Rajah
et al. (54 ) have recently shown that IGFBP-3 induces
apoptosis of the p53-negative prostate cancer cell line, PC3, through a
novel pathway independent of either p53 or the IGF-IGF
receptor-mediated cell survival pathway. Consistent with the idea that
IGFBP-3 may have IGF-independent effects on certain types of cells, two
recent reports have provided evidence for nuclear localization of
IGFBP-3 (55 56 ). The significance of this finding is not clear.
However, this exciting finding may clarify the direct intrinsic actions
of some of the IGFBPs on cells. We and others have found evidence that
IGFBP-5 may promote cell proliferation in osteoblasts, possibly through
putative cell surface-binding sites (46 47 ). Although studies from a
number of laboratories support the possibility that IGFBPs may have
IGF-independent effects in certain cell types, further experimental
evidence is needed to verify this mode of IGFBP action.
Thus, the explosion of IGFBP research during the past several years has provided evidence that IGFBPs may have both IGF-dependent and IGF-independent actions. Based on the complexity of IGFBP functions, it is clear that we cannot fully appreciate the significance of changes in IGFBP levels in serum and local body fluids until we know more about the functions of these IGFBPs.
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IV. IGF-IGFBP Complexes in Biological Fluids |
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TopAbstractI. IntroductionII. Characteristics of the...III. Target Cell Actions...IV. IGF-IGFBP Complexes in...V. Assays for Circulating...VI. Relative Distribution of...VII. Regulation of Serum...VIII. IGFBP Proteases in...IX. Endocrine Functions of...X. ConclusionsReferences |
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Figure 1 shows the relative distribution
of various IGF pools in human serum. In circulation, about 75–80% of
the IGFs are complexed to IGFBP-3 and the acid labile 
-subunit to
form the 150- to 200-kDa complex (57 59 63 ). This is possible because
under normal conditions, the total IGFs and IGFBP-3 in serum are in
equimolar concentrations (63 ). A smaller percentage (20–25%) of the
IGFs are associated with low molecular mass IGFBPs (57 ). Less than 1%
are found in the free form in circulation (64 65 ).
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).
Subsequently, the reconstitution of the 150- to 200-kDa complex was
achieved by using purified acid-labile subunit (ALS or -subunit),
IGFBP-3 (ß-subunit), and IGF-I or IGF-II (
-subunit). The role of
ALS appears to be to increase the molecular mass of the IGF+IGFBP-3
complex so that the access of the circulating IGF to the extracellular
fluid and thus to the various tissues is limited (57 58 ).
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The question of whether ALS can form a binary complex with IGFBP-3 in the absence of the IGF ligand is controversial at this time based on recent reports by Barreca and colleagues (71 72 73 ) and Lee et al. (74 ). Barreca et al. (71 ) demonstrated that incubation of recombinant human IGFBP-3 and ALS resulted in the appearance of a 150- to 200-kDa complex in the absence as well as in the presence of IGF. They also showed that ALS binding to IGFBP-3 increased the affinity of IGFBP-3 to IGF-I, possibly by inducing conformational changes in IGFBP-3. Based on these results, the authors speculate that ALS may play an important role in regulating the affinity of IGFBP-3 to IGF-I, thus regulating the levels of free IGFs. In addition, Yang et al. (75 ) observed that [125I]IGF-II readily bound to the 150-kDa fraction of adult rat serum to sites with a higher affinity for IGF-II than IGF-I. In subsequent studies, Lee and Rechler (76 ) demonstrated two different IGFBP-3 complexes in the 150- to 200-kDa fraction of the adult rat serum, one with similar affinity for IGF-I and -II and the other with greater affinity for IGF-II (77 ). The latter complex is formed from proteolytically nicked IGFBP-3 that is present in the native serum before acidification. The proteolytic cleavage in IGFBP-3 decreases the affinity of the IGFBP-3-ALS for IGF-I and increases the binding of IGF-II. Similar proteolytic nicking of IGFBP-3 occurs during human pregnancy, changing the binding specificity for IGFs (78 ). In contrast to these results, Baxter and co-workers (79 ) demonstrated that proteolyzed IGFBP-3 from maternal serum can bind to IGFs and form a ternary complex with ALS with normal affinity. Thus, they speculated that the altered binding affinity of the IGFBP-3 fragment during Western ligand blotting is an artifact resulting from breakage of a labile peptide bond after prolonged acidification or exposure to SDS.
If human IGFBP-3 must first bind to IGF-I before it can form the ternary complex, then the amount of IGF-I associated with the 150- to 200-kDa complex in rats injected with hIGFBP-3 should be twice that of normal rats. However, Lee et al. (74 ) did not observe an increase in the mobilized IGF and thus concluded that IGFBP-3 and ALS can form a binary complex independent of IGF both in vivo (80 ) and in vitro (71 76 ). Thus the question of whether IGF is required for the formation of a complex between IGFBP-3 and ALS remains controversial. One possibility is that there may be two IGFBP-3 pools, one with a higher binding affinity for ALS after first binding to IGF, and the second, which binds ALS even in the absence of IGF but with lower affinity. With differences in binding affinities, the functional roles of ternary complexes of ALS+IGFBP-3+IGF and binary complex of ALS+IGFBP-3 may also be different. Future studies are required to elucidate the extent to which IGFBP-3 forms a binary complex with ALS and if so, whether these binary complexes play a physiological role in modulating the actions of IGF.
One of the proposed functions of plasma IGFBPs is to increase the
half-life of IGFs in circulation. When IGF-I and IGF-II are injected
into normal rats, they bind to IGFBP-3, increasing their stability and
half-life to 4 h compared to 20 min in hypophysectomized rats
(81 ). Guler et al. (57 ) determined the half-lives of free
and IGFBP-bound [125I]IGF-I and -II after bolus injection
of the tracers in two normal adults. Apparent half-lives of
[125I]IGF-I and -II in each of the three IGF serum pools
(150 to 200 kDa, 50 kDa, and free IGF), calculated from the respective
disappearance rates of the tracer, are shown in Fig. 3. These results demonstrate that the
150- to 200-kDa complex is responsible for the relatively long
half-life of IGFs and that the 50-kDa and the free IGF pool have a
rapid turnover and account for most of the daily IGF production (82 ).
Another important role of IGFBP binding to IGF is in modulating IGF
action. The ternary complex does not permeate the capillary endothelial
barrier, but the smaller IGF-IGFBP complexes can easily do so and
facilitate tissue-specific IGF action (3 4 57 59 ). On the other
hand, the endocrine actions of IGFs bound to IGFBP-3 may be achieved by
specific proteolytic enzymes that dissociate the ALS+IGFBP-3+IGF
complex, and it is suggested that this may increase the bioavailability
of IGFs. An additional mechanism altering IGF bioavailability has been
proposed by Yamamoto and Murphy (83 ). They identified the presence of a
protease in rat serum that cleaves IGF-I into des(1 2 3 )IGF-I. Since
this form dissociates from the binding proteins easily, it may serve to
increase IGF bioavailablity. The role of proteases in modulating IGF
bioavailability is discussed in detail in Sections VIII and
IX.
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B. Milk
Human milk contains IGFBP-1, -2, and -3 (84 ), the functions of
which remain unclear. In addition, IGFBP-4 has been identified in rat,
porcine, and bovine milk (85 86 ). As with serum, IGFBP-3 is the major
binding protein of IGFs in milk. Although maternal serum is the source
of rat milk IGFBP-3 (21 ), the 150- to 200-kDa complex is not
translocated from serum into milk. It is suggested that IGFBP-3 may
enter milk from circulation in the free form or complexed to IGF-I. On
the other hand, IGFBP-2 and -4 are produced locally by the mammary
gland as shown by expression of their respective mRNA in the mammary
tissue (21 ). IGFBP-1 in human milk (87 ) parallels the level of IGF-I in
that immediately after birth, both milk IGF-I and IGFBP-1 decline. The
exact role of IGFBPs in milk remains to be explored, but it is
possible that they protect against the degradation of milk IGF-I or
that they modulate the local mitogenic activity of the IGFs.
C. Urine
IGFBP-1, -2, and -3 have been detected in healthy adult urine by
Western ligand blot analysis (25 ), and the concentration is
approximately 3 orders of magnitude less than that in serum (88 ).
Previously, IGFBP-2 was shown to be the predominant form in dialyzed
adult urine, but when urine is not dialyzed, IGFBP-3 is the major
binding protein (88 ). The reason for this discrepancy is not known. It
appears that urinary IGFBP-3 originates mainly from the kidney and/or
the urinary tract and (89 ), unlike the serum, is not found as a 150- to
200-kDa complex in the urine. Quantification by RIA show that urinary
IGFBP-3 is age dependent, with an increase at approximately age 9–11
yr that corresponds to the pubertal rise in serum IGFBP-3 and is
followed by a decline until it plateaus at approximately age 26 yr
(89 ). Although the antiserum used in the RIA recognizes both intact and
proteolyzed fragments of IGFBP-3, there is no evidence for the presence
of urinary protease in normal individuals from age 4–45 yr (25 89 ).
It is suggested that urinary IGFBPs may have diagnostic utility, but
this has not been established.
D. Cerebrospinal fluid (CSF)
Although several different binding proteins have been identified
in human CSF, IGFBP-2 is the major form present (22 ). The CSF contains
high concentrations of IGF-II (90 ), which binds IGFBP-2 with 10- to-
20-fold greater affinity than IGF-I (91 ). Also purified from the CSF is
IGFBP-6, which is present at slightly lower concentrations in the CSF
than serum (92 ), but also has a preferential affinity for IGF-II over
IGF-I. Both IGFBP-3 and -5 have been identified in the CSF at lower
concentrations than IGFBP-2 and -6. It is suggested that the IGFBPs
found in the CSF may be synthesized locally by glial cells and neurons
and not derived from plasma by crossing the blood-brain barrier (93 ). A
30-kDa IGFBP, corresponding to IGFBP-2, was demonstrated in rat CSF
(94 ). By analogy with other transport proteins synthesized by the
choroid plexus, it is suggested that this IGFBP may facilitate the
secretion of IGF-II to the CSF and modulate its biological action at
distant sites within the brain (94 ). In a number of disease states
related to the central nervous system (CNS) (95 96 ), changes in IGFBP
concentrations have been documented, suggesting a possible diagnostic
utility for the measurement of these binding proteins. Thus IGFBPs are
thought to modulate the biological actions of IGFs (39 94 97 )
including a possible regulatory role in growth and differentiation of
the CNS. However, the mechanisms remain to be investigated.
E. Follicular fluid
IGFBPs 1–4 (41 98 99 100 101 102 103 104 ) were identified by Western ligand
blotting in human follicular fluid, suggesting that the regulation of
IGF action in the ovary is probably under the control of regulatory
binding proteins (98 ). In women with normal menstrual cycles, after
ovulation, progesterone stimulates the endometrium to release IGFBP-1
(105 ), which mediates the cell differentiation effects of IGF-I on the
endometrium. IGFBP-1 is synthesized by granulosa cells and is secreted
into the follicular fluid (101 103 106 107 108 109 ), where the concentration
is 4- to 5 times higher than that found in the serum (110 ). The
preovulatory rise in serum IGFBP-1 is not regulated by insulin or
ingestion of a meal, nor is it associated with diurnal variation (111 ).
This implies that during the preovulatory phase, IGFBP-1 detected in
serum is primarily of follicular origin. IGFBP-1 is thought to inhibit
the biological activity of free IGF on androgen-producing theca cells,
since that might lead to atresia and anovulation (112 ).
Changes in the various IGFBPs during atresia and follicular growth have been reported. The levels of IGFBP-2 and -4 are higher in atretic follicles (99 113 ) compared with healthy developing follicles of serum, suggesting a role for them in inducing atresia. A decrease in proteolytic activity degrading IGFBP-3 and an increase in IGFBP-2, -4, and -5 protease were observed during follicular growth in ovine follicular fluid, and an increase in IGFBP-3 protease and a decrease in IGFBP-4 and -5 protease were observed during atresia (100 101 114 115 ). These observations suggest that changes in intrafollicular IGFBP proteolytic activity could be responsible in part for the changes in IGFBP levels seen during growth and atresia (114 ). Since the expression of various IGFBPs is altered during follicular development and atresia, it is speculated that the changes in IGFBP levels may regulate follicular growth by modulating the local IGF bioavailability.
F. Amniotic fluid
The binding protein isolated from amniotic fluid (AFBP) is a small
molecular mass binding protein that is both heat and acid stable (42 116 ) and was later identified to be the same as IGFBP-1. It is the
major IGFBP in the amniotic fluid and is present in concentrations
100–500 times higher than that found in the serum (117 ). During
pregnancy, a surge in the concentration of amniotic fluid IGFBP-1
reflects its local production in decidual tissues (42 118 ). The
amniotic fluid IGFBP-1 contributes to the increase in serum IGFBP-1
levels during the second trimester of pregnancy (117 ). IGFBP-1 is twice
as high in preterm amniotic fluid as in term amniotic fluid, suggesting
a role for this binding protein in growth and development.
Immunoreactive IGFBP-3 is present in amniotic fluid but at a much lower concentration than that in serum. Western ligand blot analysis of amniotic fluid failed to reveal evidence for the presence of IGFBP-3 in amniotic fluid. This could be due to the presence of IGFBP-3 protease capable of degrading intact IGFBP-3 into fragments that do not bind [125I]IGF tracer. Consistent with this interpretation, incubation of amniotic fluid with radiolabeled IGFBP revealed the presence of a protease(s) specific for IGFBP-3, -4, and -5 (119 ). These proteases alter the binding affinity of the IGFs for their binding proteins and thereby could modulate the bioactivity of IGFs (119 ). The role of IGFBP-6 in the amniotic fluid is not yet known, although the levels present are similar to those seen in serum (92 ).
G. Lymph
The concentration of both IGFs and IGFBPs in lymph are lower than
that found in serum (120 121 ). Using gel filtration chromatography, it
was shown that the IGFBPs present in lymph eluted in the 40- to 50-kDa
size range. The finding that little IGF activity eluted as a 150- to
200-kDa complex from lymph is consistent with the fact that the
IGF+IGFBP-3 complex does not cross the capillary endothelial barrier.
IGFBP-2 is believed to be one of the major binding proteins in the
lymph tissue and may originate from both the serum and surrounding
local tissues (3 ).
H. Seminal fluid
IGFBP-1-like immunoreactivity was detected in human seminal plasma
(122 ), with levels similar to those found in human adult serum. Intact
IGFBP-3 could not be detected in seminal fluid by Western ligand blot
analysis, but Western immunoblot analysis using IGFBP-3 antiserum
revealed the presence of immunoreactive IGFBP-3 fragments (122 ). Since
the amounts and ratio of these binding proteins do not correlate with
those present in the serum, it is likely that the source of these
proteins are specific to the cell population within the local tissues,
such as Sertoli cells. Human seminal plasma also contains intact
IGFBP-2 and IGFBP-4, while IGFBP-3 is present in the fragmented
form (123 124 125 126 ). The prostate-specific antigen (PSA) has proteolytic
activity for not only IGFBP-3, but also for IGFBPs -4 and -5. In
addition to the PSA protease, an IGFBP-5-specific protease has been
identified in seminal plasma (125 126 127 ). Since IGFBP proteolytic
activities in seminal fluid from normal volunteers, vasectomized
patients, or patients with idiopathic azoospermia were not
significantly different, the role of IGFBPs and IGFBP proteases in the
male reproductive system and male infertility remains to be
established.
I. Other biological fluids
The presence of IGFBPs 1 through 4 has been detected in
interstitial fluid obtained from human skin blisters caused by high
negative pressure in healthy volunteers (128 ). The IGFBP-3
concentration was lower than that present in the circulation and was
due to increased IGFBP-3 protease activity. Several IGFBPs have been
identified in vitreous and aqueous humors (129 ), but the predominant
serum carrier protein IGFBP-3 was not detected in these fluids. This
may be due to the presence of increased amounts of IGFBP-3 protease
activity in vitreous and aqueous humors (129 ). Vitreous humor from
diabetics had a higher amount of IGFBP-3 proteolytic fragment compared
with healthy controls, suggesting that the rate of IGFBP-3 proteolysis
is different in vitreous humor of normal and diabetic individuals.
Western ligand blotting and immunoprecipitation of normal synovial
fluid revealed the presence of IGFBPs 1 through 4, with levels higher
in synovial fluid of patients with rheumatoid arthritis (130 ) compared
with controls. These findings suggest that understanding the normal
IGF/IGFBP axis in physiological states and the alterations that occur
in pathological conditions may provide clues to our understanding of
the pathophysiology of different disease states.
Although the enrichment of certain biological fluids with one or more
IGFBPs, together with the increased expression of the same IGFBP in the
local tissues surrounding the body fluid (Table 2), suggests that these IGFBPs may
function locally to regulate IGF actions, more work is required to
understand the specific role of these binding proteins in modulating
IGF action in various biological fluids.
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V. Assays for Circulating Levels of IGFBP |
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TopAbstractI. IntroductionII. Characteristics of the...III. Target Cell Actions...IV. IGF-IGFBP Complexes in...V. Assays for Circulating...VI. Relative Distribution of...VII. Regulation of Serum...VIII. IGFBP Proteases in...IX. Endocrine Functions of...X. ConclusionsReferences |
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B. Western immunoblotting
Antibodies specific for a IGFBP can be used to quantitate IGFBPs
in a conventional Western immunoblotting, after size separation by
SDS-PAGE. Western immunoblotting can be improved by optimizing protein
transfer, antibody binding, and detection systems (e.g.,
chemiluminescence). In general, immunoblot analysis using polyclonal
antiserum usually detects both intact and fragmented forms of the
IGFBPs (25 ). For example, when pregnancy sera were analyzed using the
Western ligand blot technique (25 135 ), there was no evidence of
IGFBP-3, while both immunoblot and RIA detected the presence of
fragmented IGFBP-3 that arose from protease activity. In subsequent
studies, Baxter and co-workers (79 ) demonstrated that the proteolyzed
IGFBP-3 fragment from maternal serum can bind IGF with normal affinity
and that the lack of detection by Western ligand blot analysis is an
analytical artifact resulting from using [125I]IGF-I for
binding. Another example of the usefulness of the immunoblot assay was
shown in diabetics. Patients with untreated insulin-dependent diabetes
mellitus (IDDM) showed lower levels of IGFBP-3 compared with healthy
controls (136 137 ). While the Western ligand blot could only detect
the intact fragment, immunoblot assay was able to show a decrease in
intact IGFBP-3 and also an increase in fragmented IGFBP-3 compared with
controls. This finding led to subsequent investigation of proteolytic
activity in these patients. IGFBP-3 protease activity was found to be
higher in the serum of untreated IDDM patients compared with
age-matched controls (137 ). The usefulness of Western immunoblotting in
the identification of IGFBP fragments has fostered studies on
characterization of IGFBP proteases in a variety of biological fluids.
C. RIA
One of the major problems with Western ligand blot and Western
immunoblot analysis was the lack of precision, which was overcome by
the development of RIA (92 138 139 140 141 142 143 144 ). At present RIAs for IGFBP-1, -2,
and -3 are commercially available. Recent success in purifying IGFBPs
from a variety of sources to homogeneity and recombinant expression of
various IGFBPs has led to the development of specific antibodies
suitable for establishment of RIAs for accurate measurement of the
various IGFBPs. These RIAs have already provided important new
information on the physiological and hormonal regulation of the various
IGFBPs. The RIAs for the various IGFBPs do not require an extraction
procedure as in the case of the IGFs since endogenous IGFs do not
interfere with the assay (138 139 140 141 142 143 144 ). Thus, the various biological
fluids can be assayed directly for IGFBPs. The majority of IGFBP RIAs
thus far developed utilize polyclonal antiserum, which reacts with both
intact and fragment forms of IGFBPs. Although measurement of both
intact and fragmented forms of IGFBPs may provide useful information in
various clinical settings, one of the disadvantages with the use of
polyclonal antisera is that the antisera developed in various
laboratories may recognize different fragments. This could lead to
inconsistent quantitative results using antisera that recognize
dissimilar epitopes for similar biological samples. For example, it is
known that serum from children with end-stage renal disease contains
increased amounts of fragmented forms of various IGFBPs (145 146 ). The
quantitative measurements of various IGFBPs in serum from children with
end-stage renal disease may depend on whether a particular antiserum
used for measurement of a given IGFBP recognizes only selected
fragments or all of the forms of that particular IGFBP.
D. Immunoradiometric assay
The immunoradiometric assay (IRMA) is a noncompetitive assay in
which the IGFBP to be measured is "sandwiched" between two
antibodies. The first antibody, which needs to be specific, is
immobilized to the inside wall of the tubes. The second antibody is
used as a capture antibody (radiolabeled or enzyme conjugated). Since
the two antibodies used for IRMA are typically developed against
amino-terminal and carboxy-terminal ends of the molecule, the advantage
of this assay is that it is often more specific than RIA (147 148 ) and
more likely to measure the intact molecule. The disadvantage with IRMA
is that it may not reflect production rate as well as RIA since the
analyte measured may be degraded during storage or during experimental
conditions, resulting in artifactually lower values than that actually
present. At present, IRMA is commercially available only for IGFBP-3.
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VI. Relative Distribution of IGFBPs in Serum |
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TopAbstractI. IntroductionII. Characteristics of the...III. Target Cell Actions...IV. IGF-IGFBP Complexes in...V. Assays for Circulating...VI. Relative Distribution of...VII. Regulation of Serum...VIII. IGFBP Proteases in...IX. Endocrine Functions of...X. ConclusionsReferences |
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). IGFBP-3 is the
predominant form present in serum with levels more than 10-fold
higher than the other IGFBPs (59 149 ). The concentration of the small
molecular mass binding proteins are found in increasing order
(IGFBP-4 > IGFBP-5 > IGFBP-2 > IGFBP-6 >
IGFBP-1) in human serum (92 138 139 140 141 142 143 144 149 150 ). There is a 50% molar
excess of IGFBPs over IGFs in serum, which implies that a very small
percentage of the IGFs remain in the free form. Since the antisera used
for measurement of various IGFBPs recognize both intact and fragment
forms of IGFBPs, the relative abundance of intact forms of various
IGFBPs in adult human serum is not known at this time.
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) between IGFs and various IGFBPs reveal
that only IGFBP-5, in addition to IGFBP-3, showed positive correlation
with IGF concentration in normal adult human serum (142 ). In contrast,
serum IGFBP-1 and IGFBP-2 levels showed negative correlation while
IGFBP-4 levels did not correlate with IGF concentration. These data
suggest that different mechanisms may regulate the amounts of various
IGFBPs in serum.
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Regarding the MCR, it is known that IGFBPs play a major role in extending the half-life of IGFs in the circulation (see Section IX). In this regard, the half-life of IGF-II in serum may be longer than that of IGF-I since human serum contains IGFBPs with selective affinity for IGF-II over IGF-I (1 3 ). For example, human IGFBP-6 has 50- to 100-fold higher affinity for IGF-II over IGF-I. IGFBP-2 and IGFBP-5 have slightly higher affinity for IGF-II than IGF-I. In addition, Lee and Rechler (76 ) showed that the 150- to 200-kDa protein complexes in the rat serum have higher affinity for IGF-II than IGF-I. They propose that these 150- to 200-kDa complexes in the adult rat serum contain proteolytically nicked IGFBP-3 and ALS that bind to IGF-II preferentially. Based on these data, it is speculated that the presence of IGFBPs with higher affinity for IGF-II over IGF-I could contribute to a greater half-life of IGF-II over IGF-I. This explains some of the observed differences in the greater abundance of IGF-II vs. IGF-I in human serum. However, it appears likely that the differences in IGF-binding affinity of IGFBPs is not the only mechanism that contributes to the greater abundance of IGF-II over IGF-I, since 75% of IGF-II is bound to IGFBP-3 in the form of ternary complex and the intact IGFBP-3 binds IGF-I and IGF-II with similar affinity (3 ).
Indeed, inasmuch as the differences in production rate and IGFBP affinities to IGFs cannot account for the observed differences in the serum levels of IGF-I and IGF-II, it would seem that some aspect of metabolic clearance, such as the degradation rate, is higher for IGF-I than for IGF-II, thereby contributing to the lower level of serum IGF-I compared with IGF-II in adults. Based on the above analysis, it seems reasonable to conclude that three mechanisms may contribute to the greater serum level of IGF-II than IGF-I in humans: 1) greater production rate of IGF-II than IGF-I; 2) the preferential binding of minor IGFBPs for IGF-II as compared with IGF-I; and 3) a lower degradation rate of IGF-II than IGF-I (the latter two mechanisms would lead to a greater MCR for IGF-I than IGF-II).
If we assume that the actions of IGF-I and IGF-II are similar (i.e., both act via the Type I IGF receptor), then the structural differences between IGF-I and IGF-II would serve some other mechanism than functional activity. This raises the possibility that the differential structure of the two IGFs could lead to differences in MCR. Accordingly, we can speculate that serum contains two pools of reserve IGFs — a smaller IGF-I pool, which is rapidly turning over, and a larger IGF-II pool, which is slowly turning over. If so, the differential structure of the IGFs may produce differential three-dimensional structures with the IGFBPs and, therefore, could lead to a lower proteolysis rate of IGF-II than IGF-I. In this regard, recent studies have shown that exogenous addition of IGF-II to cell-free conditioned medium derived from a number of cell types, including human osteoblasts and fibroblasts, increases the rate of IGFBP-4 proteolysis (157 158 159 160 ). Since IGFBP-4 proteolysis is not induced by the addition of insulin, des(1 2 3 )IGF-I, or des(1 2 3 4 5 6 )IGF-II, all of which bind IGFBP-4 with extremely low affinity, it is speculated that the binding of IGF-II to IGFBP-4 may alter the conformation of the protein and enhance the susceptibility of IGFBP-4 to proteolytic degradation (159 ). Although these data are consistent with the possibility that the binding of ligand to binding protein may result in altered proteolysis of the ligand and/or the binding protein due to conformation changes, further studies are needed to establish whether or not there are, in fact, different MCRs for the serum IGF-I pool and the serum IGF-II pool. If this proved to be the case, this would open the possibility that the two reserve serum IGF pools provide a metabolic advantage to maintain overall body economy in the face of dramatic changes in functional demands, such as during growth, pregnancy, and starvation. Regardless of whether or not this concept has merit, the findings that IGF-II circulates in greater abundance than IGF-I in human serum, and that IGF-II is produced by several adult tissues in large amounts, are consistent with an important role for IGF-II in human physiology.
The finding that IGFBP-3 is the most abundant IGFBP present in adult human serum does not necessarily mean that the production rate of IGFBP-3 is more than that of other IGFBPs. In this regard, the higher abundance of IGFBP-3 in serum may be due to the fact that the half-life of IGFBP-3 is considerably longer (15–20 h) since it is bound to the 80- to 85-kDa ALS. In contrast, the half-lives of IGFBP-1 and IGFBP-2 have been estimated to be on the order of 1–2 h (161 ), which suggests that these binding proteins must be produced at a higher rate than that of IGFBP-3 to achieve similar serum levels based on the differences in their half-lives. Thus, it is essential to understand not only the regulation of IGFBP-3 and other IGFBPs in serum, but it is also necessary to know the regulation of different IGFBPs in various extracellular body fluids since the relative levels of these IGFBPs and their corresponding proteases in local body fluids may play a role in regulating the local actions of IGFs depending on the needs of local tissues.
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VII. Regulation of Serum IGFBPs |
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TopAbstractI. IntroductionII. Characteristics of the...III. Target Cell Actions...IV. IGF-IGFBP Complexes in...V. Assays for Circulating...VI. Relative Distribution of...VII. Regulation of Serum...VIII. IGFBP Proteases in...IX. Endocrine Functions of...X. ConclusionsReferences |
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A. Physiological conditions
1. Diurnal variation. Plasma IGFBP-1 values are subject to
diurnal variation with the levels reaching the lowest during the
afternoon and midnight, and highest in the morning (61 ). In
circulation, IGFBP-1 has a free IGF-binding site, suggesting that it is
unsaturated in contrast to GH-dependent IGFBP-3, which is normally
saturated. It appears that the increase in IGFBP-1 during the morning
hours coincides with an increase in IGF-I level, thus reducing
insulin-like activity (20 ). However, this is independent of both IGF
and GH (61 ). In contrast to IGFBP-1, IGFBP-2 and -3 are more stable and
do not exhibit diurnal variation nor are they subject to postprandial
changes (143 144 ). Diurnal variations have not yet been studied for
IGFBPs 4–6.
2. Nutrition. Nutritional regulation of IGFBP-1, -2, and -3 has been discussed briefly in a recent review by Thissen et al. (162 ), but little is known regarding the nutritional regulation of IGFBP-4, -5, and -6 (163 164 ). The metabolic state of an individual is reflected by insulin level, which influences the circulating concentration of IGFBP-1. Insulin-dependent diabetic patients have higher serum IGFBP-1 levels than nondiabetic controls (136 165 ). Further, acute steady state hyperinsulinemia reduces the serum IGFBP-1 concentration to values that are 40–70% lower than baseline values in normal individuals and also in diabetic and insulinoma patients (166 ), suggesting that insulin is involved in the regulation of serum IGFBP-1 levels (167 ). Serum IGFBP-1 levels fluctuate acutely in response to dietary food intake, with a marked increase (3- to 4-fold higher than baseline) after an overnight fast (168 ) or long-term dietary restriction (169 ), and a decline immediately after a meal. This decline in serum IGFBP-1 may be attributed to the direct effect of insulin or insulin-induced changes in glucose transport.
The effect of calorie and protein restriction on the concentrations of the serum IGFBPs is different for adults and children. A 50% calorie reduction for 6 days increased IGFBP-1 levels in healthy adults but not in children. Levels returned to normal after refeeding (170 171 ). The differences in these responses were not due to differences in insulin secretion, since both adults and children had a significant decline in fasting C peptide levels. Although insulin is the major regulator of IGFBP-1 concentration (165 172 173 ), this study showed that IGFBP-1 changes in children may not be linked to changes in insulin secretion.
Long-term dietary deprivation decreases plasma IGF-I and IGFBP-1 and may modify the tissue response to IGF by increasing IGF receptor synthesis (174 ). Another interesting finding is the role of glucagon as a stimulator of plasma IGFBP-1 independent of insulin levels (175 ). This is evident in healthy subjects, patients with GHD, and IDDM patients who have increased levels of IGFBP-1 when glucagon is administered in spite of an increase in plasma glucose and insulin levels. Based on these data, it is speculated that the nutritional regulation of serum IGFBP-1 level is complex and may be dependent on changes in the level of hormones such as insulin and glucagon, in addition to metabolic changes.
The serum IGFBP-2 level is more stable than IGFBP-1 level and is not influenced by postprandial changes (141 ). However, serum IGFBP-2 increased markedly in both adults and children on protein restriction (170 ). Similar observations have been made in patients with anorexia nervosa (171 ), chronic protein-calorie malnutrition (170 ), and in prolonged fasting that lasted more than 1 week (172 ). This increase in serum IGFBP-2 follows a cellular increase in the expression of the IGFBP-2 mRNA in rat liver (174 176 ). Although protein refeeding normalized the serum IGFBP-2 levels of undernourished children, high-protein intake is required to achieve complete normalization (177 ). Thus, nutrition-induced changes in serum IGFBP-2 level appear to be the direct effect of dietary protein on IGFBP-2 expression in liver.
Serum IGFBP-3 levels declined slightly but significantly with calorie restriction in both children and adults (170 ), but protein restriction caused a decrease in IGFBP-3 only in adults. However, this was normalized after protein refeeding (177 ). Although serum IGFBP-3 levels are regulated by IGF-I and GH under normal conditions (140 ), the decrease seen in undernourished children is more likely due to the presence of IGFBP-3-specific protease levels (177 ). The presence of similar proteolytic activity accompanying a low IGFBP-3 level is seen in other catabolic states (135 178 ), pregnancy (135 179 ), and in postsurgical patients (180 ). Age appears to influence changes observed in IGFBPs as a result of dietary modification. The response to calorie restriction among children and adults differs more so than during protein restriction, which may be due, in part, to an overestimation of the energy requirements for children (169 ).
Weight loss or long-term moderate energy restriction does not alter IGFBP-3 (181 ). Serum IGFBP-3 was not influenced by a very low calorie diet (VLCD) consumed by normal and obese subjects, while IGFBP-1 increased markedly in controls on VLCD and not in obese subjects (182 ). The increase in IGFBP-1 is suggested to inhibit the IGF-I feedback regulation of GH secretion, while a similar response is absent in obese individuals. Thus, GH secretion is increased in normal subjects on VLCD, but this response is abolished in obese individuals. The impaired GH secretion in obese subjects resulted in a lowered IGF/IGFBP-3 molar ratio, but this was reversed after weight loss by these subjects (183 ). Thus, changes in serum levels of IGFBPs induced by VLCD appear to be different between normal and obese subjects.
Based on the observations that serum levels of IGFBPs change depending on the nutritional status, two general conclusions can be made: 1) serum IGFBP-1 and IGFBP-2 levels are regulated differently than IGFBP-3 by nutrition, and 2) the decrease in serum IGFBP-3 with corresponding increases in serum IGFBP-1 and IGFBP-2 levels during malnutrition would decrease the half-lives of IGFs but tend to increase the transport of IGFs across the vascular endothelium and thereby could modulate the bioavailability of IGFs to target tissues (see Section IX). Further studies are needed to determine whether nutrition regulates IGF action by altering the ratio of IGFs bound to the 150- to 200-kDa and 50-kDa complexes.
3. Exercise. Exercise increased IGF-I and IGF-II in human adults (184 185 ) with the degree of response influenced by the intensity of exercise (186 ). This increase in IGF level in serum appears to be GH independent, since the increase in serum IGF-I occurred earlier than the increase in serum GH. In addition, GH secretion increased only in high-intensity exercise while IGF increased under both low and high intensities. Circulating IGF-I levels are also influenced differently by different types of exercise. Weight-bearing exercise caused no change in the IGF system (186 187 ), while endurance-type exercise induced significant increases in serum IGF-I level (185 ). Exercise is also accompanied by changes in some of the IGFBPs (increase in IGFBP-1 and IGFBP-3) with or without changes in IGFs, with an overall change in the IGF to IGFBP ratio.
Prolonged exercise increased the need for plasma glucose because of depleted muscle glycogen or increased hepatic glucose output. Prolonged exercise increased serum IGFBP-1 (188 189 ), and this was inversely related to serum insulin and IGF-I levels (188 ). This raised the possibility that serum insulin was the main regulator of IGFBP-1 in circulation during exercise. However, another study (189 ) showed that serum IGFBP-1 increased in response to prolonged exercise even when normal plasma glucose and insulin levels were maintained, suggesting that factors other than insulin levels, e.g., muscle glycogen, may be involved in the regulation of serum IGFBP-1 during exercise.
Increased serum IGFBP-3 levels have been reported in adults after exercise (184 190 ). This increase paralleled an increase in IGFBP-3 proteolytic activity. It is speculated that the proteolysis was induced by activation of calcium-dependent protease (191 ) and might be responsible for the IGF-induced anabolic effect of exercise on muscle tissue. These changes in the IGF system components are observed immediately after exercise and are of short duration (<1 h). It is not known at this time whether the alterations in the IGF system components in circulation play a role in mediating the anabolic effects of physical activity or whether the exercise-associated changes in circulating levels of IGFs and IGFBPs reflect processes that occur in the exercising tissue itself (191 ).
4. Glucocorticoid. Glucocorticoids inhibit somatic growth in humans in part by suppressing GH secretion and IGF activity. When dexamethasone was administered to healthy male volunteers, it suppressed IGFBP-1 and IGFBP-2 levels while increasing IGF-I and IGFBP-3 levels (192 193 ). The mean IGF bioactivity was reduced by 60% over the sampling period (192 ). This decrease in bioactivity could be due to the induction of serum inhibitors, alteration in IGFBP activity, and/or alteration in secretory profiles of GH. Recently it was shown that the negative effects exerted by glucocorticoid on bone formation may be mediated, in part, via changes in endocrine and local action of IGFs (194 ). In this study, the reduction in bone formation after glucocorticoid therapy of chronic obstructive pulmonary disease patients was accompanied by a decrease in stimulatory IGF system components including IGFBP-3. Whether or not other stimulatory and inhibitory IGFBPs are also affected remains to be determined.
B. Development and aging
1. Fetal and neonatal development. It is possible that IGFBPs
play a major role in regulating the mitogenic and
differentiation-promoting effects of IGFs in fetal tissues. IGFBPs 1 to
6 are expressed in the different organ systems of the developing fetus
(12–16 weeks) as shown by Northern blot analyses (195 196 ). Of the
IGFBPs seen in the serum of a human fetus, IGFBPs 1, -2, and -3
originate predominantly from the liver, while only small amounts of
IGFBPs 4, -5, and -6 are expressed in the liver. IGFBP-5 mRNA was
detected in several cell types during early postimplantation stages of
the developing rat, suggesting that IGFBP-5 has a role in the
development of different organ systems (197 ). These binding proteins
either cause inhibitory or stimulatory effects on IGF action, depending
on the amount of IGFs bound to each of the IGFBPs and the pattern of
distribution of these binding proteins in the various fetal tissues.
In order for optimal fetal growth, a constant interaction between the maternal host and the developing embryo/fetus is required. The presence of IGFs, IGFBPs, and fragments of IGFBP-3 in human extraembryonic cavities provide support for maternal-fetal exchange of IGF system components (115 ). It is suggested that the altered affinities of the proteolyzed IGFBP-3 for IGF-II in extraembryonic cavities may play a role in regulating the bioavailability of IGF-II in the chorion and/or the amnion (115 ). However, the role of IGFBP-3 protease in modulating IGF bioavailability remains controversial.
There is also a developmental switch during transition from fetal to neonatal life in the IGFBPs present in circulation. In a fetus of less than 27 weeks of gestation, serum contains IGFBP-1, while cord serum contains mainly IGFBP-3 (198 ). The level of IGFBP-1 is higher in the fetal and cord blood than in adult plasma. In normal weight fetuses, the IGFBP-3 and IGFBP-1 concentration in serum is 15% and 50% of maternal serum levels, respectively (199 ). Low cord serum IGFBP-1 and elevated IGFBP-3 concentrations were reported in large-for-gestational age fetuses at term birth (200 ). Since these infants also had elevated cord serum insulin levels, it was suggested that the changes in IGFBP-1 were mediated by insulin, mainly by directing greater delivery of the IGF/IGFBP complex to the target tissue, resulting in the accelerated growth as seen in large-for-gestational age fetuses. IGFBP-3 levels increase significantly during the last trimester of intrauterine life. This is supported by a study (201 ) that showed an increase in serum IGFBP-3 in preterm infants from birth (3 months preterm) to 2 months past appropriate term age. Thus IGFBP-1 and IGFBP-2 are the predominant binding proteins during fetal life, but they decline during the early neonatal period, with IGFBP-3 becoming the predominant binding protein.
In intrauterine growth-retarded (IUGR) fetuses, there is a marked elevation in cord serum IGFBP-1 and -2 compared with normal fetuses (199 200 202 ). Giudice et al. (200 ) showed that serum IGFBP-3 levels were decreased in IUGR fetuses, but this is in contrast to the results of Lassarre et al. (203 ), who showed that fetal cord serum had higher IGFBP-3 than normal cord serum. Typically, serum IGFBP-3 declines in cord serum due to a specific protease that degrades this protein to increase the amount of IGFs available for stimulation of growth in the target tissues. Thus, in fetal circulation, the increased availability of IGF is due to a molar excess of IGF-I and -II, an increase in IGFBP-2, and a decrease in the ternary IGFBP-3 complex formation. It is therefore speculated that IGFBP-3 protease is less likely to play a significant role in fetal serum in contrast to maternal or neonatal serum (204 ).
During fetal life, not only is the total plasma IGFBP-3 lower than in the adult circulation, but the amounts of IGF-I and IGF-II bound to this binding protein are also low compared with amounts of IGFs bound to IGFBP-3 in adults. IGF-I levels are depressed in infants with IUGR, suggesting that IGFs play a significant role in promoting growth. However, the majority of IGFs are bound to IGFBP-3 as a 50-kDa ALS-independent complex in infant serum, which is capable of crossing the endothelial barrier, thus increasing the bioavailability of the IGFs (203 ). The role of IGFBP-4 in neonatal development has not yet been explored, but given the inhibitory effect of IGFBP-4 on IGF action, it is possible that serum IGFBP-4 levels are higher in children with slow growth compared with normally growing children (205 ). However, future studies need to confirm these speculations.
2. Puberty. Although low at birth, the serum IGFBP-3
concentration rapidly increases during the first years of infancy
(206 207 208 ), reaches a peak at puberty (207 ), and declines during
adulthood (208 ). Girls have higher serum IGFBP-3 levels than boys of
comparable age throughout childhood, and levels peak a year earlier
than boys during puberty (207 208 ). In addition, IGFBP-3 increases
with the increasing stage of pubertal maturation. Both the height and
body mass index of an individual correlates positively with IGFBP-3
levels independent of age, sex, and pubertal stage. The molar ratio
between IGF-I and IGFBP-3 is increased during puberty, suggesting that
more biologically active IGF is available in the free form during the
pubertal growth spurt. Serum levels of IGFBP-2 show marked
age-dependence with high levels at birth and senescence and low levels
during puberty (144 ). Serum IGFBP-1 declines progressively with age,
with the lowest concentration observed during puberty (207 208 ). The
height variability seen among pubertal children correlates with the
concentration of IGF-I and IGFBP-3, with lower values in short stature
children and higher values in tall children (209 ). Also, in patients
with acromegaly and those with high serum GH levels, circulating
IGFBP-3 and IGF-I are increased. Under normal physiological conditions,
most of the serum IGFs are bound to IGFBP-3, with an approximate 1:1
molar ratio of total IGF (IGF-I + IGF-II) and IGFBP-3 (60 63 ).
However, this ratio seems to vary with developmental age, with a
greater increase in circulating IGF-I than IGFBP-3 during puberty
(208 ). Changes in serum concentrations of the various IGFBPs during
puberty (Table 4) have been proposed to
play a role in inducing the growth spurt during puberty.
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