help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hill, R. A.
Right arrow Articles by Pell, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hill, R. A.
Right arrow Articles by Pell, J. M.
Endocrinology Vol. 139, No. 3 1278-1287
Copyright © 1998 by The Endocrine Society

ARTICLES

Regulation of Insulin-Like Growth Factor I (IGF-I) Bioactivity in Vivo: Further Characterization of an IGF-I-Enhancing Antibody1

Rodney A. Hill2 and Jennifer M. Pell

The Babraham Institute, Cambridge, United Kingdom CB2 4AT

Address all correspondence and requests for reprints to: Dr. J. M. Pell, Babraham Institute, Cambridge, United Kingdom CB2 4AT. E-mail: jenny.pell{at}bbsrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously demonstrated the ability of a polyclonal antibody raised against human insulin-like growth factor I (IGF-I) to potentiate, rather than inhibit, the growth-promoting activity of IGF-I. The anti-IGF-I Ig had a modest affinity for IGF-I, protected IGF-I from degradation, and reduced the IGF-I clearance rate while allowing efficient transfer of peptide from the circulation, leading to the suggestion that the antiserum might be behaving in an analogous manner to enhancing IGF-binding proteins (IGFBPs). The purpose of these studies was to investigate further the characteristics of this antiserum as a means of assessing the importance of IGF-I associated with circulating high mol wt IGFBPs to serve as a bioavailable reservoir of IGF-I peptide.

1) Epitope scanning using sequential and overlapping peptides spanning the entire length of IGF-I revealed one major linear region of anti-IGF-I Ig binding to IGF-I comprising the C-terminal region of the C-domain and the N-terminal region of the A-domain (Arg36-Ile43), a region not associated with receptor or IGFBP binding. 2) The fact that the antibody could potentiate IGF-I whether administered as a preincubated complex or separately indicated that complex formation could occur in the presence of IGFBPs in vivo. 3) The ability of the antibody to attenuate the acute hypoglycemic actions of IGF-I and LR3IGF-I was assessed by pretreating dwarf rats with either anti-IGF-I Ig or nonimmune Ig; 1 h after sc administration of peptide, plasma glucose levels decreased by about 4 mM (P < 0.001) in rats pretreated with nonimmune Ig. The duration of hypoglycemia was more prolonged in the LR3IGF-I-treated rats (P < 0.01). Neither IGF-I or LR3IGF-I induced any decrease in circulating glucose concentrations in the rats pretreated with the anti-IGF-I Ig, suggesting that the antibody gave protection against inappropriate acute IGF-induced hypoglycemia. 4) The potentiating effects of the anti-IGF-I Ig on the anabolic actions of IGF-I and LR3IGF-I were compared in dwarf mice. The anti-IGF-I Ig potentiated the increase in whole body weight gain induced by IGF-I by over 3-fold (P < 0.001), but did not change the anabolic action of LR3IGF-I despite its ability to double circulating levels of both IGF peptides. It is, therefore, possible that part of the mechanism of action of the anti-IGF-I Ig involves transfer of IGF-I to smaller mol wt binding proteins. These data confirm the potential of IGFBP-associated IGF-I to act as a reservoir of peptide and to regulate IGF-I activity in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) is essential for normal growth and development (1, 2). Its potency is such that very little peptide exists in the free form in vivo; rather, it is bound to one of a family of at least six IGF-binding proteins (IGFBPs) that can inhibit or potentiate IGF-I activity depending in part on their affinity for IGF-I (for recent reviews, see Refs. 3 and 4). Further, the IGFs are unusual among growth factors, as they exist in high concentrations in blood; IGFBP-3 complexes in excess of 90% of this circulating IGF-I are present either as a binary complex of IGF-I and IGFBP-3 or as a ternary complex, after further interaction with an additional protein known as the acid-labile subunit (ALS) (5). The molecular masses of these complexes are approximately 40 and 150 kDa, respectively, and therefore, the binary complex may cross the capillary endothelium and gain access to tissue receptors, but the ternary complex probably cannot and is confined to the circulation. IGF-I is constitutively secreted as it is synthesized; no tissue stores exist, and therefore, one suggested function of the circulating ternary IGFBP-3 complex is to provide a pool of bioavailable IGF-I. Thus, the circulating levels and the form of IGFBP-3 will in large part determine body reserves of IGF-I (6), which will be important in conditions of reduced IGF-I biosynthesis, such as during trauma-induced catabolism or nutritional deficiency.

We have previously demonstrated the ability of a polyclonal antibody raised against human IGF-I to potentiate, rather than inhibit, the growth-promoting activity of IGF-I in vivo (7). A complex of IGF-I and anti-IGF-I Ig induced a proportional increase in whole body weight gain in dwarf mice, greater than that achievable by exogenous IGF-I alone, which was accompanied by increased circulating IGF-I concentrations. The anti-IGF-I Ig had a modest affinity for IGF-I, leading to the suggestion that the antiserum was protecting IGF-I from degradation while allowing effective competition for antibody-bound IGF-I by IGF type 1 receptors or receptor-targeting IGFBPs. The hypothesis that the anti-IGF-I Ig was maintaining IGF-I in a bioavailable form was pursued by examining the effects of the IGF-I-enhancing antibody on the pharmacokinetics and tissue distribution of IGF-I in vivo (8), and concurrent actions of the anti-IGF-I-enhancing antibody on IGF-I tracer kinetics were observed when IGF-I disappearance was defined by a two-pool model. One effect of the anti-IGF-I Ig was to bind circulating IGF-I, significantly decreasing its degradation rate in the plasma pool; additionally, the slower decaying pool (quantitatively the most important) had its half-life significantly reduced in the presence of anti-IGF-I Ig, implying that IGF-I can be transferred readily from plasma to tissues and is supportive of its enhancing properties.

An analogy can be drawn between the effects of this IGF-I-enhancing antiserum and those of potentiating IGFBPs; both have an affinity for IGF-I that is equivalent or less than that for IGF type 1 receptors, both probably protect IGF-I from proteolytic attack, and, hence, both could influence the bioavailability of IGF-I. The large amounts of binding protein- or antibody-associated IGF-I in the circulation could represent a significant latent reservoir of peptide. We therefore decided to investigate further the characteristics of the IGF-I-enhancing antiserum as a means of assessing the importance of circulating IGF-I in relation to IGF-I availability and the potential of blood IGFBPs to modify IGF-I action in vivo. Here we report specific properties of the anti-IGF-I Ig: a probable major site of its binding to IGF-I, its apparent ability to complex exogenous IGF-I in vivo, its effects on the acute hypoglycemic action of IGF-I, and its differential actions on IGF-I and the non-IGFBP-binding analog LR3IGF-I. The significance of these findings is considered in terms of our understanding of IGF-I action and its relationship with endogenous IGFBPs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibody production and partial purification of Ig
Polyclonal antibodies were raised against recombinant human IGF-I in adult sheep that had been conjugated to human {alpha}-globulin; anti-IGF-I Ig were purified from the resultant anti-IGF-I antiserum and also from nonimmune serum (NI Ig) using ammonium sulfate precipitation, as described previously (7). Protein concentrations of the Ig preparations were assayed by the method of Bradford (9), using BSA as the standard; the total protein concentration of the Ig preparations was adjusted to equivalent concentrations (8 mg/ml) using PBS (0.01 M sodium phosphate in 0.15 M saline, pH 7.4). Antibody titers for IGF-I were determined as described previously (7) using standard ELISA techniques and were 1:7000 and less than 100 for the anti-IGF-I Ig and NI Ig, respectively. When the ELISA plates were coated with LR3IGF-I instead of native sequence IGF-I, almost exactly the same titer was observed, indicating that the antibody could also bind readily to LR3IGF-I.

Epitope analysis of anti-IGF-I antiserum
Multiple pin peptide synthesis techniques (10) were used to construct two separate sets of overlapping and sequential octamers and hexamers onto plastic pins representing the entire sequence of human IGF-I, using an epitope-scanning kit and following the manufacturer’s instructions exactly (Cambridge Research Biochemicals, Northwich, UK). Briefly, for the octamers, the first plastic pin had a peptide corresponding to residues 1–8 of IGF-I, the second pin had residues 2–8, etc., through to the final octamer; hexamers were constructed in an analogous manner. Any antibody that bound to the pins was quantified using standard ELISA techniques, described below. The pins were assembled in plastic holders in an 8 x 12 configuration so that they could be placed in standard 96-well microtiter plates. The pin-bound peptides were incubated overnight at 4 C with the anti-IGF-I antiserum diluted 1:1000 in supercocktail [0.15 M NaCl and 0.01 M sodium phosphate buffer, pH 7.2, containing 0.05% (vol/vol) Tween-20] to which 0.1% (wt/vol) ovalbumin and 0.1% (wt/vol) BSA were added. The pins retaining any peptide-bound antibody were then rinsed using distilled water, washed in supercocktail (four times, 10 min each time, with agitation at room temperature) and incubated in new microtiter plates for 1 h at 37 C containing horseradish peroxidase-conjugated antisheep second antibody at a dilution of 1:1000 (rabbit antisheep antibodies, Dako, Glostrup, Denmark). The plates were then incubated at 37 C for 1 h and washed in supercocktail (three times, 10 min each time, as before). Substrate color reagent was added (100 µl 0.55 mg/ml 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid diammonium salt in 100 mM citrate-buffered saline, pH 4.3, containing 1.2 µl 30% hydrogen peroxide/20 ml); the absorbance at 405 nm was measured after 30 min.

Animals and treatments
Homozygous Snell dwarf mice (11) and homozygous dwarf rats (12) were bred at the Babraham Institute (Cambridge, UK) and fed high quality pelleted rodent feed (18% crude protein) and water ad libitum.

Exp 1: mode of administration of IGF-I and anti-IGF-I Ig on subsequent growth rate in dwarf mice
Thirty 10-week-old Snell dwarf mice (equal numbers of males and females) were weighed daily for 1 week (for acclimatization to handling) and then allocated to one of five treatments (n = 6/group) consisting of a daily sc (0.1 ml) and an ip (0.4 ml) injection for 7 days: group 1, saline sc and ip; groups 2 and 3, saline sc and 20 µg IGF-I preincubated with NI Ig or anti-IGF-I Ig ip; groups 4 and 5, pretreatment for 2 days with NI Ig or anti-IGF-I Ig ip followed by a similar ip treatment for 7 days in conjunction with 20 µg IGF-I sc in the intrascapular region. Preincubations were performed for 1 h at room temperature to allow complex formation. Thus, mice were treated with IGF-I that was either precomplexed to Ig before administration or noncomplexed but given to mice that had been loaded with Ig for 2 days. Mice were weighed daily; on the final day, mice were killed by decapitation exactly 2 h after the last injection. Trunk blood was collected, and liver and skeletal muscle (combined gastrocnemius, plantaris, and soleus) were dissected and weighed.

Exp 2: induction of hypoglycemia in the presence and absence of IGF-I-enhancing Ig in dwarf rats
Twenty 9-week-old female dwarf rats were weighed daily for 1 week for acclimatization to handling. At 10 weeks of age, they were housed individually and randomly allocated to one of four treatment groups (n = 5/group). They were weighed daily and given an ip injection (3 ml) of either anti-IGF-I Ig or NI Ig. On day 3, jugular catheters were inserted under halothane anesthesia, and 150 µl jugular blood were collected into heparinized syringes. The rats were fasted overnight. The following morning, an additional 150-µl blood sample was taken exactly 1 h after the administration of Ig; the rats were immediately administered either 100 µg IGF-I or LR3IGF-I (both in saline as a 100-µl sc injection; dose previously determined to induce moderate hypoglycemia in a pilot experiment). Treatment groups were, therefore, anti-IGF-I Ig plus IGF-I or LR3IGF-I and NI Ig plus IGF-I or LR3IGF-I. Jugular blood was sampled 15, 30, 60, 120, and 240 min after peptide administration. At 360 min, rats were killed by decapitation, and trunk blood was collected. Plasma was prepared from all samples and stored at -20 C until analysis of glucose concentrations.

Exp 3: IGF-I- and LR3IGF-I-induced growth in the presence and absence of IGF-I-enhancing Ig in dwarf mice
Twenty-four 10-week-old Snell dwarf mice were allocated to one of four treatment groups (n = 6/group): anti-IGF-I Ig (0.4 ml) plus 20 µg IGF-I or 20 µg LR3IGF-I, and NI Ig (0.4 ml) plus 20 µg IGF-I or LR3IGF-I. Doses of IGF peptide were selected to induce changes in weight gain that were well below the physiological maximum response expected for dwarf mice, to allow the potential observation of increased peptide activity. The Ig and peptide were preincubated at room temperature for 1 h before injection to allow complex formation. Injections continued for 10 days, when mice were killed by decapitation exactly 2 h after treatment. Trunk blood was collected, and liver and skeletal muscle (combined gastrocnemius, plantaris, and soleus) were dissected and weighed. Serum was prepared by centrifugation, and the samples were stored at -20 C until analysis.

Measurement of hormone and metabolite concentrations
Glucose concentrations were measured using a glucose oxidase kit (Sigma Chemical Co., Poole, UK). Total plasma or serum IGF-I concentrations were determined by RIA after a modified acid-ethanol extraction (13) using a polyclonal rabbit anti-IGF-I antiserum generated at the Babraham Institute. Previous studies using 125I-labeled IGF-I and size-exclusion chromatography demonstrated that the acid-ethanol treatment could dissociate any antibody-bound IGF-I in serum. This assay was used for the measurement of apparent total IGF-I concentrations in dwarf mice administered LR3IGF-I; as reported by Tomas et al. (14), it was found that the rabbit polyclonal anti-IGF-I antiserum did not completely cross-react with LR3IGF-I and, in this case, underestimated LR3IGF-I concentrations by about 50%.

Statistical analyses
Where variances within treatment groups were similar, differences between groups were assessed by one-way ANOVA (Exp 1) or two-way ANOVA (Exp 2) with Ig (anti-IGF-I Ig and NI Ig) and peptide (IGF-I and LR3IGF-I) as main effects, where appropriate. Where variances within treatment groups were markedly different between pairs of treatments (Exp 3), comparisons were made using a Welch test that does not assume equality of variance. Initial live weight was used as a covariate for the analysis of whole body and tissue weights. Where significant differences were obtained for main effects (defined as P < 0.05), these were examined in more detail using t tests to compare individual treatment groups. Pooled SEDs are presented for each analysis; individual group are also presented, but were not used in the statistical assessment unless specifically stated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Epitope scanning to determine the sites of binding of IGF-I to the enhancing antiserum
Figure 1Go shows the pin peptides to which the IGF-I-enhancing antiserum bound, as absorbances of the sequential ELISA assay wells. Even though a polyclonal antiserum was used, a single major site of binding to IGF-I was identified, comprising the five sequential octamer peptides beginning with residues 34–38 (Fig. 1Go, upper panel) and the three sequential hexamer peptides corresponding to residues 36, 37, and 38 (lower panel). An additional minor region of antibody binding to IGF-I was seen for peptides 51 to 53 (octamers) and 53 (hexamer). Further investigation of six additional sheep anti-hIGF-I antisera raised in a similar manner to this IGF-I-enhancing antiserum demonstrated that the epitope-scanning profiles described here are a consistent observation, with all six antisera yielding largely similar profiles, except that the major peak of absorbance sometimes extends to peptide 40; four of these antisera have been examined further in dwarf mouse bioassays, and all potentiate the growth-promoting activity of IGF-I (data not shown). These data, therefore, suggest that the IGF-I-enhancing antiserum defines one predominant, functionally continuous epitope on IGF-I; the maximum size of this region derived from the octamer peptides corresponds to residues Ser34 to Cys47 (SerSerArgArgAlaProGlnThrGlyIleValAspGluCys), and the minimum, from the hexamer peptides, corresponds to Arg36 to Ile43. Figure 2Go represents a structure of IGF-I derived from the Brookhaven database coordinates of Cooke et al. (15) using Rasmol (R. Sayle, Glaxo, Middlesex, UK; shareware available via the internet). The core region of IGF-I recognized by the anti-IGF-I Ig is highlighted in dark gray and spans the end of the C and the beginning of the A domains (the C domain is equivalent to residues 30–41, and the A domain is equivalent to residues 42–62). It appears to represent a loop region on the molecule. For orientation, the N- and C-terminal amino acids and residues thought to be important for IGFBP and type 1 receptor binding are highlighted in light gray. From this structure, the minor binding site at around residue 53 would appear to functionally distinct, as it is not in close proximity to residues 34–47 and, therefore, probably represents a distinct epitope region rather than part of a conformational epitope associated with the major site of binding. Thus, the IGF-I-enhancing antiserum, although polyclonal, binds to a region of restricted specificity of IGF-I as assessed by linear peptide analysis.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Scanning epitope analysis of binding of the anti-IGF-I Ig to linear regions of IGF-I. A, Octamer peptides; B, hexamer peptides. The pin number on the abscissa indicates the N-terminal residue of each pin-bound peptide. For details, see Materials and Methods.

 


View larger version (63K):
[in this window]
[in a new window]
  		Figure 2. The structure of IGF-I derived from the nuclear magnetic resonance study of Cooke et al. (15) using the molecular visualization program Rasmol (R. Sayle, Glaxo; shareware available via the internet). Residues important for IGFBP binding (Glu3, Thr4, Gln15, and Phe16), type 1 receptor binding (Tyr24, Tyr31, and Tyr60), and the N- and C-terminal residues (Gly1 and Ala70) are highlighted in pale gray; key residues identified by linear epitope scanning to be important for binding of the potentiating Ig preparation to IGF-I (Arg36-Ile43) are highlighted in dark gray. A, Orientation of IGF-I to display the site of Ig binding; B, rotation of the image in A by approximately 180° around the y-axis to display the putative type 1 receptor-binding regions.

 
IGF-I need not be precomplexed to the anti-IGF-I antiserum to potentiate the growth-promoting activity of IGF-I
To date, all studies on the modulation of IGF-I activity by the anti-IGF-I Ig had been performed with IGF-I that had been preincubated with an excess of antibody, giving the maximum opportunity for complex formation before administration; this did indeed occur, as assessed by the reduced ability of [125I]IGF-I to associate with high mol wt fractions of serum derived from mice treated with anti-IGF-I Ig and IGF-I vs. anti-IGF-I Ig plus saline (7). As IGFBP-3 and ALS are synthesized in different cell types in vivo, their ternary complex formation with IGF-I must occur in the pericellular or extracellular environment, unless translocation of proteins to different cells occurs. We, therefore, decided to examine the ability of the anti-IGF-I Ig to change the activity of a low dose of IGF-I when administered separately. Figure 3Go illustrates the average daily weight gain of dwarf mice treated with saline alone or with IGF-I that had been preincubated with NI Ig or anti-IGF-I Ig (to allow complex formation) or with IGF-I administered in a different site from NI Ig or anti-IGF-I Ig (noncomplexed). In the presence of NI Ig, IGF-I induced a modest increased weight gain that only achieved statistical significance (P < 0.05) for the mice administered "noncomplexed" IGF-I and NI Ig. Preincubation of the same dose of IGF-I with the anti-IGF-I Ig induced a significant increase in weight gain compared with that of equivalent animals given IGF-I and NI Ig (P < 0.05), confirming our previous observations (7). Administration of IGF-I to mice that had been pretreated for 2 days with the anti-IGF-I Ig also induced an increased weight gain compared with equivalent NI Ig only controls (P < 0.02), suggesting that prior formation of a complex between IGF-I and the anti-IGF-I Ig in vitro is not necessary for enhancement of IGF-I activity and demonstrating that it is possible to form IGF-I complexes in vivo in the presence of competing IGFBPs. The location for such complex formation is unknown, but blood and/or the extracellular environment would be likely candidates. It is interesting that the overall weight gain of the mice treated with IGF-I administered via the ip route is greater than that given via the sc route; however, the magnitude of the potentiation of IGF-I by the antibody (~2-fold) was similar for both routes of administration.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 3. The effect of administering IGF-I and anti-IGF-I Ig either as a preincubated complex or via two distinct sites on the bioactivity of IGF-I, as assessed by changes in whole body weight gain in dwarf mice in vivo. Appropriate controls were performed using IGF-I and NI Ig. For precise details, see Materials and Methods. Bars with different letters (a, b, c, and d) indicate significantly different responses (P < 0.05 or less). The pooled SED from the ANOVA and the individual group SEMs are presented. Liver and skeletal muscle (combined gastrocnemius, plantaris, and soleus) weights were also measured and demonstrated proportional changes in line with the increases in whole body weight gain.

 
Prevention of acute IGF-induced hypoglycemia by anti-IGF-I Ig
Our previous studies have indicated that the kinetics of IGF-I exit from the circulating pool are different in the presence and absence of the enhancing anti-IGF-I Ig; the antibody apparently maintains a greater proportion of IGF-I in the circulation, at least in the short term (8). It was, therefore, of interest to determine whether the presence of the anti-IGF-I Ig could change the acute hypoglycemic response to a single dose of IGF peptide. Exp 1 of the current study demonstrated that pretreatment with anti-IGF-I Ig alone was efficacious for its enhancing properties; therefore, we pursued this approach in the current experiment. As the IGF-I analog LR3IGF-I can also bind to the anti-IGF-I Ig and induce a more severe hypoglycemia than IGF-I due to its reduced ability to associate with IGFBPs, its effects were also examined. Figure 4Go illustrates the changes in plasma glucose concentrations in dwarf rats pretreated for 4 days with NI Ig or anti-IGF-I Ig followed by a dose of IGF-I or LR3IGF-I sufficient to induce acute hypoglycemia when administered alone. Initial glucose concentrations (time zero) were slightly, although significantly (P < 0.01), lower in rats treated with anti-IGF-I Ig than in those given NI Ig, although only when considered as a main effect (NI Ig + IGF-I, 6.07; NI Ig + LR3IGF-I, 5.78; anti-IGF-I Ig + IGF-I, 4.77; anti-IGF-I Ig + LR3IGF-I, 5.78 mM; SED, 0.56 mM; n = 6). Both IGF-I and LR3IGF-I induced a significant decrease in plasma glucose concentrations within 30 min (P < 0.001 in both cases) when administered to control Ig rats, which were restored to normal values by 360 min. When decreases in glucose concentration at individual time points were compared in NI Ig-treated rats, LR3IGF-I caused a more rapid decline at 15 min (P < 0.01) and a more gradual return to normoglycemia at 240 min (P < 0.01) than did native sequence IGF-I. However, pretreatment of the dwarf rats for 4 days with the anti-IGF-I Ig completely abolished any IGF-I- or LR3IGF-I-induced hypoglycemia. The responses to both native and LR3IGF-I were equivalent (P > 0.05 for each time point), suggesting that the anti-IGF-I Ig could, in physiological terms, behave in a similar manner to both peptides. These data reinforce previous observations that the anti-IGF-I Ig and IGF peptide (both IGF-I and LR3IGF-I) can readily complex and suggest that the antibody initially binds free peptide, thus avoiding the hypoglycemia that can occur on administration of free IGF-I or LR3IGF-I. The effects of the anti-IGF-I Ig were confirmed (P < 0.001 for Ig as a main effect) when the area under (or over) the curve of change in glucose concentration vs. time was calculated (NI Ig + IGF-I, -480; NI Ig + LR3IGF-I, -743; anti-IGF-I Ig + IGF-I, +245; anti-IGF-I Ig + LR3IGF-I, +131 mM/min; SED, 152 mM/min; n = 6). Even though the area under the curve was 1.5-fold greater in rats treated with LR3IGF-I plus NI Ig than in those given IGF-I plus NI Ig, this was not statistically significant.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Change in plasma glucose concentration in dwarf rats pretreated with either anti-IGF-I Ig (solid symbols) or NI Ig (open symbols) for 4 days and then administered a sc single dose of either IGF-I (100 µg; square symbols) or LR3IGF-I (100 µg; circular symbols) at time zero (indicated on the abscissa). Treatment effects were assessed by comparison of the area under or over the curve of the change in glucose concentration vs. time (in Results) and for each time point by ANOVA, followed by t test (as described in Materials and Methods). Similar changes in glucose concentrations were obtained for rats treated with anti-IGF-I Ig plus either LR3IGF-I or IGF-I, but differences were obtained when NI Ig plus IGF-I or NI Ig plus LR3IGF-I groups were compared (*, P < 0.05 or less).

 
Anti-IGF-I Ig does not potentiate the growth-promoting activity of LR3IGF-I
The data generated in Exp 2 suggested that the anti-IGF-I Ig could bind acutely to the potent IGF-I analog LR3IGF-I in vivo; we, therefore, decided to investigate and compare the chronic effects of the Ig on the growth-promoting action of LR3IGF-I and native IGF-I. The average daily weight gain of dwarf mice treated for 10 days with IGF-I or LR3IGF-I in the presence of the anti-IGF-I Ig is shown in Fig. 5AGo together with the appropriate control values. Even though mice administered anti-IGF-I Ig and saline apparently exhibited increased weight gain compared with NI Ig plus saline controls, this was not a consistently observed effect and was not statistically significant (95% confidence limits for the difference of the means, -5 and 103 mg/day). In agreement with our previous observations (Exp 1) (7), mice treated with anti-IGF-I Ig plus IGF-I exhibited increased weight gain compared with the NI Ig plus IGF-I mice (P < 0.01). As expected, LR3IGF-I treatment in the presence of NI Ig tended to induce an increased weight gain compared with the effect of IGF-I plus anti-IGF-I Ig. Surprisingly, though, no evidence for potentiation of LR3IGF-I by the anti-IGF-I Ig could be found; both exhibited similar increases in weight gain during the 10-day period. However, the variance in the weight gain of the mice treated with LR3IGF-I and anti-IGF-I Ig was high (95% confidence limits around the difference in weight gain between the mice treated with LR3IGF-I with NI Ig and LR3IGF-I plus anti-IGF-I Ig, -113 and 152 mg/day). The small increase in daily gain for LR3IGF-I plus anti-IGF-I Ig mice vs. that in LR3IGF-I plus NI Ig mice was due in large part to a loss in weight of the latter group after their first set of injections; such a response is quite common in dwarf mice (average daily gain from day 2 to the end of the study: saline + NI Ig, 19; saline + anti-IGF-I Ig, 72; IGF-I + NI Ig, 62; IGF-I + anti-IGF-I Ig, 174; LR3IGF-I + NI Ig, 115; LR3IGF-I + anti-IGF-I Ig, 118 mg/day; SD, 34; n = 6/group).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 5. Responses of dwarf mice to exogenous IGF-I and LR3IGF-I preincubated with either anti-IGF-I Ig or NI Ig together with appropriate controls (see Materials and Methods for details). A, Whole body weight gain over 10 days; B, serum total IGF-I concentrations; C, serum glucose concentrations. Data were analyzed by Welch test where variances were markedly different and by t test where they were similar, as described in Materials and Methods. Differences in responses between NI Ig- or anti-IGF-I Ig-treated mice are indicated by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The pooled SEDs from the ANOVA and the individual group SEMs are presented. Liver and skeletal muscle (combined gastrocnemius, plantaris, and soleus) weights were also measured and demonstrated proportional changes in line with the increases in whole body weight gain.

 
This apparent lack of response of the activity of LR3IGF-I to the IGF-I-enhancing antiserum in the dwarf mice was investigated in more detail by measurement of their serum IGF-I and glucose concentrations (Fig. 5Go, B and C). In mice treated with saline plus NI Ig or anti-IGF-I Ig, serum IGF-I concentrations were less than 30 ng/ml, as expected. Treatment with IGF-I and NI Ig induced a predictable increase in circulating IGF-I concentrations (P < 0.002), but when IGF-I was administered in combination with the anti-IGF-I Ig, a further increase in serum concentrations was observed of almost 86% (P < 0.01), confirming previous studies. Even though exogenous LR3IGF-I and NI Ig did induce an increase in apparent IGF-I concentrations compared with levels in NI Ig plus saline mice (from 25 to 41 ng/ml), these values cannot really be compared, as the rabbit antibody used for the assay does not cross-react equivalently with LR3IGF-I and native sequence IGF-I. The combination of LR3IGF-I and anti-IGF-I Ig further augmented apparent IGF-I concentrations by 1.8-fold to a mean of 116 ng/ml; using a simple t test and comparing the LR3IGF-I plus NI Ig and LR3IGF-I plus anti-IGF-I Ig groups, this increase was highly significant (P < 0.001). It is concluded, therefore, that the anti-IGF-I Ig complexed to LR3IGF-I in the mice and increased the circulating pool of IGF peptide over the 10-day period of the study.

Serum glucose concentrations were about 8 mM in control dwarf mice (Fig. 5CGo). Administration of IGF-I plus NI Ig decreased serum glucose concentrations by 50% (P < 0.02), and this was completely abolished when IGF-I was given with the anti-IGF-I Ig, confirming our earlier observations. Mice treated with LR3IGF-I and NI Ig exhibited a decrease in glucose concentrations to as low as 2.1 mM. This was significantly increased when LR3IGF-I was given with the anti-IGF-I Ig (P < 0.05), although not to control levels; however, a mean glucose concentration of 5 mM was achieved, which is probably sufficient to support growth in dwarf mice. In contrast to these data, the anti-IGF-I Ig completely abolished the acute hypoglycemic action of LR3IGF-I in Exp 2, even though this might be due to the use of dwarf rats vs. dwarf mice. In Exp 2, the rats had been pretreated with antibody for 2 days, rather than coinjected as in Exp 3. There was no correlation between circulating glucose concentrations and daily gain or circulating IGF-I concentrations and daily gain within treatment groups in Exp 3.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented here confirm and extend our initial observations on the actions of a novel IGF-I-enhancing antibody (7, 8), providing further information on the functional properties of the IGF-I molecule and the potential of IGFBP-associated IGF-I to act as a mobilizable reservoir of bioactive peptide. The animals models used in this investigation are GH-deficient dwarf mice and dwarf rats; the former are panhypopituitary and, therefore, lack other pituitary hormones in addition to GH, whereas dwarf rats have a more specific lesion and exhibit only diminished GH secretion. Both have proportionately decreased circulating IGFBP-3 and increased IGFBP-2 levels as expected for GH-deficient animals and have always responded in a similar manner to treatment with peptides of the GH axis and the anti-IGF-I Ig preparation when compared.

To try and localize motifs within the IGF-I molecule to which the anti-IGF-I antibody preparation binds, epitope scanning was performed to identify linear sequences. One clear region, spanning at least residues Arg36 to Ile43, comprising the C-terminal region of the C domain and the proximal N-terminal region of the A domain (C7 to A2), was strongly and consistently observed to be present in antibody preparations derived from several different host animals. These residues are highly conserved across IGF-I derived from different mammalian species, but not with IGF-I from chicken, Xenopus, and salmon, which differ in the residues equivalent to C9 to C12. This region also has only 25% homology with human insulin (mature insulin lacks a C-domain) and 50% homology with human IGF-II (which lacks residues in the C11 and C12 positions). The key central four residues are therefore unique to IGF-I and its analogs such as LR3IGF-I, explaining the specificity of the Ig preparation for IGF-I (negligible cross-reactivity with insulin or IGF-II). Comparison with the structure of insulin suggests that residues 30–41, comprising the C-peptide region of IGF-I, easily span the last residue of the B domain (Thr29) and the first of the A domain (Gly42); this distance probably only needs about three residues. Indeed, the hydrophilicity of the residues Ser33 to Arg37 and their rare occurrence in helical or sheet structures have lead to the suggestion that this is a highly hydrophillic region and is almost certainly loose on the surface of the molecule (15, 16), although it is possible that coupling of IGF-I to the carrier protein, {alpha}-globulins, induces a conformational change and "presents" this region. The residues Arg36 to Ile43 are, therefore, likely to define an area of potentially high antigenicity.

In terms of IGF-I physiology, the region Arg36 to Ile43 of IGF-I has not been attributed with any specific function to date. It is well established that the IGFBPs interact primarily with residues within the N-terminal region of the A domain. Glu3, Thr4, Leu15, and Gln16 are particularly important (17, 18), with a more minor contribution from residues Phe49, Arg50, and Ser51. These are all on the opposite side of IGF-I to the C-peptide and proximal A domain. This latter region, 49–51, has also been associated with type II receptor binding (19). The aromatic residues Phe23, Tyr24, and Phe257 from the B domain as well as Tyr31 from the proximal N-terminal C domain have been identified as particularly important for the interaction of IGF-I with the type 1 IGF receptor (20, 21), and these also define a region of IGF-I that is oriented away from Arg36 to Ile43. Thus, provided no steric hindrance occurred, it is theoretically possible that IGF-I could bind to specific antibodies from the enhancing Ig preparation while also binding to either IGFBPs or the type 1 receptor. Such interactions could be related to its potentiating properties. Antibodies raised against a synthetic peptide comprising the region of IGF-I defined by the enhancing Ig preparation also potentiate IGF-I activity (Westbrook, S., and J. M. Pell, unpublished observations) (22). Manes et al. (23) generated a panel of mouse monoclonal antibodies to IGF-I that defines eight epitopic clusters; however, none can be identified unequivocally as recognizing the region 36–43, although one (termed group VI) might at least overlap.

In our previous studies on the anabolic actions of the anti-IGF-I Ig preparation and IGF-I, antibody and peptide were always preincubated before administration, allowing maximum opportunity for complex formation (7). In vivo, IGFs and IGFBPs are not necessarily synthesized in the same cell. For example, IGF-I and the ALS are synthesized in parenchymal cells, whereas IGFBP-3 is produced by nonparenchymal cells in the liver (24, 25), and therefore, ternary complex formation must occur extracellularly, presumably primarily in the circulation. It was thus important to demonstrate that the anti-IGF-I Ig and the IGF peptides used in this study could form a bioactive complex in vivo. This was shown functionally by the ability of the anti-IGF-I Ig to potentiate the growth-promoting activity of exogenous IGF-I when they were administered via different routes. The extent to which the antibody, either alone or as a complex, is confined to the circulation or the ip area or whether it gains widespread access to tissues, for example via Fc receptors, is unknown at present. Additionally, the ability of the antibody to interact with endogenous IGF-I is unknown; our earlier studies indicate that this might occur (7).

Acute administration of a bolus of IGF-I-induced short term hypoglycemia in dwarf rats. However, pretreatment of dwarf rats with the anti-IGF-I Ig preparation completely abolished the acute hypoglycemic action of a single sc dose of either IGF-I or even LR3IGF-I, implying that the antibody could prevent inappropriate acute interaction of free IGF peptide with receptors and could achieve this as effectively for IGF-I or more mobile non-IGFBP-binding analogs (26). Formation of binary IGF-I/IGFBP-3 complexes may afford some protection against hypoglycemia (27), and the degree to which administered IGF peptide can induce short term hypoglycemia probably depends on the ability of free peptide to leave the circulation; the formation of high molecular mass ternary complexes that are confined to the circulation would, therefore, be expected to inhibit such hypoglycemia. This hypothesis has also been examined using hypophysectomized rats, which have reduced circulating IGFBP-3 and ALS concentrations and became hypoglycemic when administered IGF-I in the form of a 40-kDa IGF-I/IGFBP-3 binary complex. Normal rats, which had the ability to form a 150-kDa ternary complex with ALS, did not (28). In the short term, therefore, the anti-IGF-I Ig is equivalent to a ternary complex "sink," preventing free IGF-I from leaving the circulation and exerting inappropriate acute actions.

Recent evidence suggests that the ternary complex of IGF-I, IGFBP-3, and ALS has a regulatory function in IGF-I action rather than merely to prevent inappropriate actions of IGF-I; the data presented in this study support this hypothesis. When ternary complex formation is limited, IGF-I action is impaired. Both diabetic (6) and dwarf (29) rats have a reduced ability to form ternary complexes due to decreased circulating IGF-I concentrations, resulting in elevated IGFBP-3 degradation. Administration of IGF-I alone to calorie-restricted subjects is not as effective as coadministration of IGF-I and GH in terms of both inappropriate hypoglycemia and the reversal of nitrogen loss (30). This is supported by studies in severely GH-deficient rodent models (13, 31) in which coadministration of GH and IGF-I is more effective than treatment with either peptide alone. GH induces ALS synthesis to which the exogenous IGF-I can form a ternary complex; IGFBP-3 synthesis is induced by both GH and IGF-I (31). Minimal effects have been observed on growth rate in IGFBP-3 transgenic mice, in which the majority of IGFBP-3 is in the 50-kDa form (32).

The transfer of IGF-I from the circulating ternary complex to tissue receptors must be a highly regulated process. Proteolytically modified IGFBP-3, either within or before ternary complex formation, is known to result in a less stable complex and could, therefore, favor IGF-I transfer out of the circulation or its increased susceptibility to degradation (33). Additionally, the existence of binary complexes of IGFBP-3 and ALS has been demonstrated, although the physiological significance of this is unclear at present (34). Bar et al. (35) have shown that IGF-I transfer across the capillary endothelium is regulated by IGFBP-1 and -2, and it is possible that the smaller molecular mass binding proteins have a specific function in the regulated transport of the IGFs to tissues. Non-IGFBP-binding analogs, such as LR3IGF-I, are more potent than native sequence IGF-I when administered continuously via a sc osmotic minipump (36). This apparent increased activity is related to its rapid clearance from blood and, therefore, its ease of access to tissue receptors rather than to an increased activity of the analog itself at the receptor level. LR3IGF-I has a slightly lower affinity than IGF-I for the type 1 receptor (~4.3-fold in L6 myoblasts), but appears to use similar signaling mechanisms (37). One disadvantage of nonassociation with IGFBPs is the increased degradation and clearance of non-IGFBP-binding IGF analogs (26); therefore, for maximal benefit, IGFBPs must be administered by continuous infusion (36). Guan et al. (38) demonstrated that IGF-I-induced neuronal rescue after hypoxic-ischemic brain injury requires IGFBP binding; des(1, 2, 3)IGF-I is ineffective other than at pharmacologically high doses. Thus, the ability for normal association with IGFBPs may be required for IGF-I to manifest all of its actions optimally. In the current study, no evidence could be found to support the potentiation of LR3IGF-I activity by the anti-IGF-I Ig, at least in terms of whole body, liver, and skeletal muscle weight gain over 10 days, despite the Ig preparation binding readily to LR3IGF-I and maintaining an increased circulating pool. Additionally, the affinity of the IGF type 1 receptor is still an order of magnitude greater than that for anti-IGF-I Ig; therefore, the receptor would still have been able to compete very effectively for antibody-bound LR3IGF-I (if antibody-bound IGF gains access to tissue receptors). This suggests that the transfer of IGF-I to tissue compartments via other IGFBPs may be important and, indeed, Bar et al. (39) demonstrated that localization of endothelial IGF-I may be dependent on IGFBP association.

The precise mechanism(s) of action of the IGF-I-potentiating antiserum are unknown at present; however, it is noteworthy that it can stimulate an increase in weight gain in dwarf mice that could probably not be achieved by a high dose of peptide alone (7). The enhancement of peptide hormone activity by antibodies has been demonstrated previously for other hormones of the GH axis (40, 41) and was reviewed in Pell and Aston (42). Our previous studies in vivo have demonstrated that the antiserum can prevent IGF-I degradation and change the kinetics of transfer of IGF-I from plasma to tissue compartments, decreasing the clearance rate of IGF-I by maintaining an increased bioavailable serum pool of peptide that is readily transferred out of the circulating compartment (7). A more complex question is whether the antibody in some way facilitates transfer and targeting of IGF-I from the circulating antibody-bound reservoir via smaller molecular mass IGFBPs, allowing them to compete effectively for IGF peptide by virtue of the antibody’s relatively modest affinity for IGF-I; the lack of potentiation of LR3IGF-I by the antibody supports this suggestion. For a mechanism of enhancement only involving a change in circulating IGF-I kinetics, the site of binding of antibody to IGF-I would not be important, as the function of the antibody preparation is merely to provide a reservoir of IGF-I peptide. Of key relevance is whether the antibody can in some manner directly influence hormone-receptor interaction, for example by an antibody-induced change in peptide hormone conformation (43), increasing its affinity for the receptor or by prolonging hormone-receptor interaction in some manner. In support of this, GRF activity has been enhanced by site-directed antibodies in vitro (40). Obviously, any antibody-mediated change in hormone-receptor function will depend on the site of binding of the antibody to peptide hormone and will depend on maintained access of IGF-I to type 1 IGF receptors.

The use of molecules that bind IGF-I with properties similar to those of the IGF-I-enhancing antibody described here but that are nonimmunogenic in man may be of potential therapeutic benefit. Acute and chronic illnesses, such as infection, burns, tissue repair, nutritional deprivation, and diabetes, are often accompanied by nitrogen wasting and muscle catabolism that cannot be reversed by increased nutritional support, as the factors inducing protein degradation override normal control mechanisms. GH therapy has been used in some of these situations, but is often ineffective because GH receptor function may be refractory (44); additionally, GH administration may exacerbate or induce hyperinsulinemia and glucose intolerance (45) due to its well established diabetogenic action. As circulating IGF-I concentrations usually decrease during catabolism (46), and IGF type 1 receptor levels may increase (47), exogenous IGF-I has been investigated as a possible therapeutic agent; experimentally induced muscle catabolism has been arrested or partially reversed by IGF-I in rodent models (48). However, the potential benefit of exogenous IGF-I on whole body nitrogen balance in catabolic conditions in man is limited by two main factors: the negative actions induced by free IGF-I peptide (for example, acute hypoglycemia, down-regulation of the endogenous GH axis, and resultant restriction of ternary complex formation) and the increased IGFBP protease activity that accompanies catabolism (33). We suggest that administration of IGF-I as a complex that is initially confined to the circulation but can be transferred readily to endogenous IGFBPs may form the basis for efficient IGF-I therapy.


   Acknowledgments
 
We thank Ms. Helen Flick-Smith for her excellent technical assistance, Dr. Simon Westbrook for critical evaluation of the manuscript, and Mr. David Brown for statistical advice.


   Footnotes
 
1 Part of this work falls within the scope of international patent application PCT/GB93/01774. Funding was provided by a Rees Jones Fellowship (to R.A.H.) and the Babraham Institute BBSRC Competitive Strategic Grant (to J.M.P.). Back

2 Present address: Tropical Beef Center, P.O. Box 5545, Rockhampton Mail Center, Queensland 4702, Australia. Back

Received July 14, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Liu J-P, Baker J, Perkins AS, Robertson EJ, Efstratiadis A 1993 Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75:59–72[Medline]
  2. Powell-Braxton L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, Gillett N, Stewart TA 1993 IGF-I is required for normal embryonic growth in mice. Genes Dev 7:2609–2717[Abstract/Free Full Text]
  3. Rechler MM 1994 Insulin-like growth factor binding proteins. Vitam Horm 47:1–114
  4. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[CrossRef][Medline]
  5. 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 Natl Acad Sci USA 86:6878–6902[Abstract/Free Full Text]
  6. Lewitt MS, Saunders H, Baxter RC 1993 Bioavailability of insulin-like growth factors (IGFs) in rats determined by the molecular distribution of human IGF-binding protein-3. Endocrinology 133:1797–1802[Abstract]
  7. Stewart CEH, Bates PC, Calder TA, Woodall SM, Pell JM 1993 Potentiation of insulin-like growth factor-I (IGF-I) activity by an antibody: supportive evidence for enhancement of IGF-I bioavailability in vivo by IGF binding proteins. Endocrinology 133:1462–1465[Abstract]
  8. Hill RA, Flick-Smith HC, Dye S, Pell JM 1997 Actions of an IGF-I-enhancing antibody on IGF-I pharmacokinetics and tissue distribution: increased IGF-I bioavailability. J Endocrinol 152:123–130[Abstract]
  9. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantitites of protein using the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  10. Geyson HM, Meloen RH, Bartling SH 1984 Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc Natl Acad Sci USA 81:3998–4002[Abstract/Free Full Text]
  11. Van Buul-Offers SC, Ueda I, Van den Brande JL 1986 Biosynthestic somatomedin-C (SM-C/IGF-I) increases the length and weight of Snell dwarf mice. Pediatr Res 20:825–827[Medline]
  12. Charlton HM, Clark RG, Robinson ICAF, Porter Goff AE, Cos BS, Bugnon C, Bloch BA 1988 Growth hormone-deficient dwarfism in the rat: a new mutation. J Endocrinol 119:51–58[Abstract]
  13. Pell JM, Bates PC 1992 Differential actions of growth hormone and insulin-like growth factor-I on tissue protein metabolism in dwarf mice. Endocrinology 130:1942–1950[Abstract]
  14. Tomas FM, Knowles SE, Chandler CS, Francis GL, Owens PC, Ballard FJ 1993 Anabolic effects of insulin-like growth factor-I (IGF-I) and an IGF-I variant in normal female rats. J Endocrinol 137:413–421[Abstract]
  15. Cooke RM, Harvey TS, Campbell ID 1991 Solution structure of human insulin-like growth factor I: a nuclear magnetic resonance and restrained molecular dynamics study. Biochemistry 30:5484–5491[CrossRef][Medline]
  16. Blundell TL, Bedarkar S, Rinderknecht E, Humbel RE 1978 Insulin-like growth factor: a model for tertiary structure accounting for immunoreactivity and receptor binding. Proc Natl Acad Sci USA 75:180–184[Abstract/Free Full Text]
  17. Baxter RM, Bayne ML, Cascieri MA 1992 Structural determinants for binary and ternary complex formation between insulin-like growth factor-I (IGF-I) and IGF binding protein-3. J Biol Chem 267:60–65[Abstract/Free Full Text]
  18. Clemmons DR, Dehoff ML, Busby WH, Bayne ML, Cascieri MA 1992 Competition for binding to insulin-like growth factor (IGF) binding protein-2, 3, 4, and 5 by the IGFs and IGF analogs. Endocrinology 131:890–895[Abstract]
  19. Cascieri MA, Chicchi GG, Applebaum J, Green BG, Hayes NS, Bayne ML 1989 Structural analogs of human insulin-like growth factor (IGF) I with altered affinity for type 2 IGF receptors. J Biol Chem 264:2199–2202[Abstract/Free Full Text]
  20. Cascieri MA, Chicchi GG, Allpelbaum J, Hayes NS, Green BG, Bayne ML 1988 Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor. Biochemistry 27:3229–3233[CrossRef][Medline]
  21. Bayne ML, Applebaum J, Underwood D, Chicchi GG, Miller RE, Cascieri MA 1990 The roles of tyrosines 24, 31 and 60 in the high affinity binding of insulin-like growth factor-I to the type 1 insulin-like growth factor receptor. J Biol Chem 26:15648–15652
  22. Pell JM, Flick-Smith HC, Stewart CEH, Hill RA Localization of a major binding site on IGF-I for an IGF-I-enhancing antibody. 10th International Congress of Endocrinology, San Francisco, CA, 1996 (Abstract P1–547)
  23. Manes S, Kremer L, Albar J P, Mark C, Llopis R, Martinez-A C 1997 Functional mapping of insulin-like growth factor I (IGF-I) by anti-IGF-I monoclonal antibodies. Endocrinology 138:905–915[Abstract/Free Full Text]
  24. Arany E, Afford S, Strain AJ, Winwood PJ, Arthur MJP, Hill DJ 1994 Differential cellular synthesis of insulin-like growth factor binding protein-1 (IGFBP-1) and IGFBP-3 within human liver. J Clin Endocrinol Metab 79:1871–1876[Abstract]
  25. Chin E, Zhou J, Baxter RC, Bondy CA 1994 Cellular localization and regulation of gene expression for componants of the insulin-like growth factor ternary binding protein complex. Endocrinology 134:2498–2504[Abstract]
  26. Bastian SEP, Walton PE, Wallace JC, Ballard FJ 1993 Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I) and an analogue LR3IGF-I in pregnant rats. J Endocrinol 138:327–336[Abstract]
  27. Sommer A, Maack CA, Spratt SK, Mascarenhas D, Tressel TJ, Rhodes ET, Lee R, Roumas M, Tatsuno GP, Flynn JA, Gerber N, Taylor J, Cudney H, Nanney L, Hunt TK, Spencer EM 1991 Molecular genetics and actions of recombinant insulin-like growth factor binding protein-3. In: Spencer EM (ed) Modern Concepts of Insulin-Like Growth Factors. Elsevier, New York, pp 715–728
  28. Zapf J, Hauri C, Futo E, Hussain M, Rutishauser J, Maack CA, Froesch ER 1995 Intravenously injected insulin-like growth factor (IGF) I/GF binding protein-3 complex exerts insulin-like effects in hypophysectomized, but not in normal rats. J Clin Invest 95:179–186
  29. Lewitt MS, Saunders H, Phuyal JL, Baxter RC 1994 Complex formtation by human insulin-like growth factor-binding protein-3 and acid-labile subunit in growth hormone-deficient rats. Endocrinology 134:2404–2409[Abstract]
  30. Kupfer SR, Underwood LE, Baxter RC, Clemmons DR 1993 Enhancement of the anabolic effects of growth hormone and insulin-like growth factor I by use of both agents simultaneously. J Clin Invest 91:391–396
  31. Fielder PJ, Mortensen DL, Mallet P, Carlsson B, Baxter GC, Clark RG 1996 Differential long-term effects of insulin-like growth factor-I (IGF-I), growth hormone (GH), and IGF-I plus GH on body growth and IGF binding proteins in hypohpysectomized rats. Endocrinology 137:1913–1920[Abstract]
  32. Murphy LJ, Molnar P, Lu X, Huang H 1996 Expression of human insulin-like growth factor binding protein-3 in transgenic mice. J Mol Endocrinol 15:293–303
  33. Holly JMP, Claffey DCP, Cwyfan-Hughes SC, Frost VJ, Yateman ME 1993 Proteases acting on IGFBPs; their occurance and physiological significance. Growth Regul 3:88–91[Medline]
  34. Lee CY, Wu HB, Suh DS, Rechler MM 1997 Insulin-like growth factor-I (IGF-I) concentration in 150 kilodalton complexes containing human IGF-binding protein-3 (hIGFBP-3) after intravenous injection of adult rats with hIGFBP-3. Endocrinology 138:1649–1657[Abstract/Free Full Text]
  35. Bar RS, Boes M, Clemmons DR, Busby WH, Sandra A, Dake BL, Booth BA 1990 Insulin differentially alters transcapillary movement of intravascular IGFBP-1, IGFBP-2 and endothelial-cell IGF-binding proteins in the rat heart. Endocrinology 127:497–499[Abstract]
  36. Tomas FM, Lemmey AB, Read LC, Ballard FJ 1996 Superior potency of infused IGF analogs which bind poorly to IGF-binding proteins is maintained when administered by injection. J Endocrinol 150:77–84[Abstract]
  37. Francis GL, Ross M, Ballard FJ, Milner SJ, Senn C, McNeil KA, Wallace JC, King R, Wells JRE 1992 Novel recombinant fusion-protein alaogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency. J Mol Endocrinol 8:213–223[Abstract]
  38. Guan J, Williams CE, Skinner SJM, Mallard EC, Gluckman PD 1996 The effects of insulin-like growth-factor (IGF)-1, IGF-2, and des-IGF-1 on neuronal loss after hypoxic-ischemic brain injury in adult rats: evidence for a role for IGF binding-proteins. Endocrinology 137:893–898[Abstract]
  39. Bar RS, Boes M, Dake BL, Sandra A, Bayne M, Cascieri M, Booth BA 1990 Tissue localization of perfused endothelial cell IGF binding protein is markedly altered by association with IGF-I. Endocrinology 127:3243–3245[Abstract]
  40. Pell JM, James S 1995 Immuno-enhancement and -inhibition of GH-releasing factor by site-directed anti peptide antibodies in vivo and in vitro. J Endocrinol 146:535–541[Abstract]
  41. Pell JM, Aston R 1991 Active immunization with a specific peptide region of growth hormone: increased lean tissue growth. J Endocrinol 131:R1–R4
  42. Pell JM, Aston R 1995 Principles of immunomodulation. Livestock Prod Sci 42:123–133[CrossRef]
  43. Mazza MM, Retigui LA 1989 Monoclonal antibodies to human growth hormone induce an allosteric conformational change in the antigen. Immunology 67:148–153[Medline]
  44. Belcher HJ, Mercer CRD, Judkins KC, Shalaby S, Wise S, Marks V, Tanner NSB 1989 Biosynthetic growth hormone in burned patients: a pilot study. Burns 15:99–107[CrossRef][Medline]
  45. Sherwin RS, Shulman GA, Hendler R, Walesky AB, Belous A, Tamborlane W 1983 Effect of growth hormone on oral glucose tolerance and circulating metabolic fuels in man. Diabetologia 24:155–161[Medline]
  46. Timmins AC, Cotterill AM, Hughes SCC, Holly JMP, Ross RJM, Blum W, Hinds CJ 1996 Critical illness is associated with low circulating concentrations of insulin-like growth factor-I and growth factor-II, alterations in insulin-like growth factor binding proteins, and induction of an insulin-like growth factor binding protein-3 protease. Crit Care Med 24:1460–1466[CrossRef][Medline]
  47. Eshet R, Werner H, Klinger B, Silbergeld A, Laron Z, LeRoith D, Roberts Jr CT 1993 Up-regulation of insulin-like growth factor-I (IGF-I) receptor gene expression in patients with reduced serum IGF-I levels. J Mol Endocrinol 10:115–120[Abstract]
  48. Tomas FM, Knowles SE, Owens PC, Read LC, Chandler CS, Gargosky SE, Ballard FJ 1991 Effects of full-length and truncated insulin-like growth factor-I on nitrogen balance and protein metabolism in nitrogen-restricted rats. J Endocrinol 128:97–105[Abstract]



This article has been cited by other articles:


Home page
 Mol. Endocrinol.Home page
T. C. Voss, M. P. Flynn, and D. L. Hurley
IGF-I Causes an Ultrasensitive Reduction in GH mRNA Levels via an Extracellular Mechanism Involving IGF Binding Proteins
Mol. Endocrinol., September 1, 2001; 15(9): 1549 - 1558.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. J. Fedele, C. H. Lang, and P. A. Farrell
Immunization against IGF-I prevents increases in protein synthesis in diabetic rats after resistance exercise
Am J Physiol Endocrinol Metab, June 1, 2001; 280(6): E877 - E885.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. M. Pell, R. A. Hill, C. E. H. Stewart, C. R. Weston, and H. C. Flick-Smith
Enhancement of Insulin-Like Growth Factor I Activity by Novel Antisera: Potential Structure/Function Interactions
Endocrinology, February 1, 2000; 141(2): 741 - 751.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
L. P. Koziris, R. C. Hickson, R. T. Chatterton Jr., R. T. Groseth, J. M. Christie, D. G. Goldflies, and T. G. Unterman
Serum levels of total and free IGF-I and IGFBP-3 are increased and maintained in long-term training
J Appl Physiol, April 1, 1999; 86(4): 1436 - 1442.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hill, R. A.
Right arrow Articles by Pell, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hill, R. A.
Right arrow Articles by Pell, J. M.

HOME HELP