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 Google Scholar
Google Scholar
Right arrow Articles by Hoeflich, A.
Right arrow Articles by Wolf, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoeflich, A.
Right arrow Articles by Wolf, E.
Endocrinology Vol. 140, No. 12 5488-5496
Copyright © 1999 by The Endocrine Society

ARTICLES

Overexpression of Insulin-Like Growth Factor-Binding Protein-2 in Transgenic Mice Reduces Postnatal Body Weight Gain

Andreas Hoeflich, Minyao Wu, Subburaman Mohan, Jürgen Föll, Rüdiger Wanke, Thomas Froehlich, Georg J. Arnold, Harald Lahm, Helmut J. Kolb and Eckhard Wolf

Institute of Molecular Animal Breeding (A.H., M.W., H.L., E.W.) and Laboratory of Molecular Biology (T.F., G.J.A.), Gene Center, and Institute of Veterinary Pathology (R.W.), Ludwig-Maximilian University, 81377 Munich, Germany; Musculoskeletal Diseases Center (S.M.), Loma Linda, California 92357; Endocrinology Laboratory (J.F.), University Child Hospital, 72070 Tübingen, Germany; and Institute of Clinical Chemistry (H.J.K.), Clinic Harlaching, 81545 Munich, Germany

Address all correspondence and requests for reprints to: Dr. Andreas Hoeflich, Institute of Molecular Animal Breeding/Gene Center, Ludwig-Maximilian University, Feodor-Lynen-Strasse 25, 81377 Munich, Germany. E-mail: hoeflich{at}lmb.uni-muenchen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) has been shown to inhibit IGF-dependent cell proliferation in a number of in vitro studies. However, no in vivo model of IGFBP-2 overexpression has been established so far. Therefore, we have generated transgenic mice, in which expression of a mouse IGFBP-2 complementary DNA is controlled by the cytomegalovirus (CMV) promoter. In two independent transgenic strains, transgene expression was highest in pancreas and stomach, followed by skeletal muscle, heart, colon, spleen, adipose tissue, brain, and kidney. Within the pancreas, IGFBP-2 expression was found in the islets but not in the exocrine part. Serum IGFBP-2 levels of CMV-IGFBP-2 transgenic mice were about 3-fold (P < 0.05) increased, compared with controls, whereas serum levels of IGF-I and IGF-II were unaffected by IGFBP-2 overexpression. Fasted serum glucose and fasted insulin levels were slightly reduced in transgenic mice, compared with controls. Postprandial serum glucose insulin levels were not affected by the genotype. At days later than 23, body weights of transgenic mice were significantly (P < 0.05) reduced in both sexes, compared with nontransgenic littermates. This reduction in body weight was mainly attributable to significantly (P < 0.05) lower carcass weights of CMV-IGFBP-2 transgenic vs. control mice. In contrast, absolute organ weights were not (or only as a tendency) reduced, except for the weight of the spleen, which was significantly (P < 0.05) lower in male transgenic than in control mice. Our data suggest that IGFBP-2 represents a negative regulator of postnatal growth in mice, potentially by reducing the bioavailability of IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor (IGF)-binding protein (IGFBP)-2 is among the predominant IGFBPs in serum of different species and binds IGF-II with several-fold higher affinity than IGF-I (1, 2). The murine gene for IGFBP-2 consists of four exons and spans more than 28 kb (3). IGFBP-2 expression is high in mouse embryos during midgestation (4). In adult mice, the highest IGFBP-2 messenger RNA (mRNA) expression was found in liver and kidney; and intermediate expression was detected in lung, spleen, brain, testis, and ovary (5). Increased expression of IGFBP-2 is found after fasting and in association with a number of pathological syndromes, including nonislet cell tumor hypoglycemia, diabetes, chronic renal failure, liver cirrhosis, and certain types of leukemia (6, 7). Elevated serum levels of IGFBP-2 after infusion of IGF-I (8), in patients suffering from IGF-II-secreting tumors (9) and in transgenic mice overexpressing IGF-II (10, 11), suggest positive regulation of IGFBP-2 expression by increased levels of free IGFs.

Disruption of the IGFBP-2 gene in mice resulted in only minor phenotypical changes and increased expression of other IGFBPs, suggesting functional redundancy of the IGFBPs (12, 13). However, there is at least indirect evidence for an inhibitory effect of increased levels of IGFBP-2 on IGF actions. Transgenic rabbits expressing high levels of recombinant human IGF-I in their mammary glands did, unexpectedly, not show any phenotypic alterations, such as increased milk yield, changes of milk composition, hyperplasia, or even tumors of the mammary gland. Ligand blot analysis of milk from these transgenic rabbits revealed a marked increase in the activity of IGFBP-2, which might have buffered effects of excess IGF-I (14). In addition, reduced growth of mice selected for low body weight was associated with increased hepatic IGFBP-2 mRNA expression and elevated serum IGFBP-2 levels (15), further suggesting IGFBP-2 as a negative growth regulator in vivo. Increased IGFBP-2 expression was also found in several experimental models of growth retardation in rat and swine (16, 17, 18).

Recently, we have shown that IGFBP-2 overexpression in transfected embryonic kidney fibroblasts (293 cells) inhibits cell proliferation. Furthermore, conditioned media of these cells inhibited IGF-dependent growth of several colon carcinoma cell lines (19).

To evaluate the specific role of IGFBP-2 in vivo, we generated transgenic mice overexpressing homologous IGFBP-2 under the control of the cytomegalovirus (CMV)-promoter. The present study evaluates level and tissue-specificity of transgene expression, effects on other components of the IGF system, and consequences for body and organ growth. IGFBP-2 transgenic mice displayed reduced postweaning body weight gain, which was mainly explained by reduced carcass weights. Increased serum and tissue levels of IGFBP-2 are therefore likely to reduce the bioavailability of IGF-I.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of pCMV-int-mouse (m)IGFBP-2
The full-length mouse IGFBP-2 complementary DNA (cDNA) (5), kindly donated by Dr. S. Drop (Rotterdam, The Netherlands) was cloned into the mammalian expression vector pCMV-int, as described previously (19) (Fig. 1Go).



View larger version (108K):
[in this window]
[in a new window]
  		Figure 1. Identification of two IGFBP-2 transgenic strains by Southern blot analysis (upper panel) and schematic representation of pCMV-int-mIGFBP-2 (lower panel). Five micrograms of genomic DNA from six transgenic individuals of strain 1, four nontransgenic (Co), and five transgenic individuals from strain 2 were EcoRI-digested and analyzed, by Southern blot hybridization, using a fluorescein labeled IGFBP-2 cRNA probe, as described in Materials and Methods. The SpeI/XhoI microinjection fragment (3.2 kb) includes the CMV promoter (550 bp), the rat insulin II intron A sequences (int, 120 bp), the mouse IGFBP-2 cDNA (mIGFBP-2, 1480 bp), the terminating sequences of the human GH gene (hGH-term, 624 bp), and 391 bp including SV40 origin of replication. M, Molecular weight marker.

 
Generation of transgenic mice
All mice were purchased from Charles River Laboratories, Inc.-Wiga (Sulzfeld, Germany) and were maintained under standard (nonbarrier) conditions. Transgenic mice were generated by microinjection of the 3.2 kb SpeI/XhoI fragment from the expression vector pCMV-int-mIGFBP-2 into pronuclei of fertilized eggs from superovulated donors (B6D2F1 x B6D2F1). The injected embryos were transferred to pseudopregnant female mice (CD-1). Genomic DNA from tail tips of 3-week-old offspring was isolated using the Puregene genomic DNA purification system (Biozym, Hess; Oldendorf, Germany). Transgenic offspring of two founder mice were produced by backcrossing with C57BL/6 mice and identified by Southern blot hybridization. Five micrograms of genomic DNA were hydrolyzed with EcoRI, separated by 1% Tris-acetate/EDTA buffered agarose gel electrophoresis, and blotted onto Nytran membranes (Schleicher & Schuell, Inc., Dassel, Germany) by capillary transfer. A fluorescein-labeled IGFBP-2 riboprobe (riboprobe length, 1.5 kb) was synthesized using the Riboprobe Gemini Transcription System (Promega Corp., Mannheim, Germany) and fluorescein-12-UTP (Roche Molecular Biochemicals, Mannheim, Germany). The riboprobe was purified as described previously (15). Hybridization and detection were carried out using the fluorescein Gene Images labeling system (Amersham Pharmacia Biotech Buchler, Braunschweig, Germany). The number of integrated construct copies was quantified using the TaqMan SYBR Green technology (Perkin-Elmer Corp., Weiterstadt, Germany) and internal standards. The standard curve was derived from serially diluted plasmids 40–10(40–10,000 copies) containing the IGFBP-2 cDNA added to genomic DNA from a nontransgenic control. PCR conditions were according to the manufacture’s instructions and were identical for the standards and the samples from the transgenic strains (n = 4). Primers used in the TaqMan Assay were as follows: mIGFBP-2 sense: 5' GCG CGG GTA CCT GTG AAA 3'; mIGFBP-2 antisense: 5' TCC CTC AGA GTG GTC GTC ATC 3'. Transgenic offspring were also identified by PCR analysis (CMV-specific sense primer: 5' GTG TAC GGT GGG AGG TC 3'; IGFBP-2-specific antisense primer: 5' TCG GCA GCA TGT TGG CTT GT 3') according to standard protocols from 100 ng of genomic DNA from tail tips.

Analysis of IGFBP expression
IGFBP-2 mRNA expression was analyzed by Northern blot hybridization, as described previously (19). In brief, tissues were homogenized in guanidinium thiocyanate, and RNA was pelleted in 5.7 M CsCl by ultracentrifugation. Ten micrograms of total RNA were separated by formaldehyde gel electrophoresis. For hybridization, fluorescein-labeled IGFBP-2 cRNA probes were used as for Southern blot hybridization. Serum samples and extracts from different tissues were analyzed by Western ligand blot analysis, as previously described (19), to demonstrate the molecular weight of the transgene product and its capacity to bind human IGF-II. Briefly, tissue samples were homogenized in extraction buffer [10 mM Na2HPO4, pH 7.0; 0.2% (wt/vol) SDS; 10% (wt/vol) glycerin] using a cell homogenizer (ART, Mühlheim, Germany). Fifty micrograms of protein were boiled (5 min) and electrophoresed on a 5% stacking/12% separating SDS-polyacrylamide gel using the Mini Protean II system (Bio-Rad Laboratories, Inc., Munich, Germany). Separated proteins were transferred to a nitrocellulose membrane (Millipore Corp., Eschborn, Germany). The blots were blocked with 1% fish gelatin and incubated with [125I]-IGF-II (106 cpm per blot). Binding proteins were visualized and quantified on Phosphor-Imager Storm (Molecular Dynamics, Inc., Krefeld, Germany). All incubations and washing steps were performed at 4 C.

An IGFBP-2 specific antiserum was generated by immunization of rabbits using a synthetic peptide (amino acids 117–132: KRRVGTTPQQVADSDD) of mouse IGFBP-2. Specificity of the antiserum was analyzed by two-dimensional Western blotting of pancreatic protein extracts from IGFBP-2 transgenic mice. Mass spectrometry of the spot detected by two-dimensional Western blotting revealed a complete identity with murine IGFBP-2 (data not shown). For Western immunoblotting, membranes were prepared, as described above, with the only exception being that the proteins were separated under reducing conditions. Membranes were incubated with peptide-induced antibodies (dilution 1:1000) for 1 h, and bound antibodies were detected with peroxidase-coupled antibodies against rabbit IgG [Dianova (Germany), Hamburg, Germany] and subsequent addition of 3,3‘-diaminobenzidine tetrahydrochloride (Sigma, Munich, Germany).

Histology and immunohistochemistry
Pancreata of two IGFBP-2 transgenic mice (one male, one female) and two nontransgenic littermate controls (one male, one female) were used for histological and immunohistochemical investigations. The animals were killed by cervical dislocation, under ether anesthesia, at an age of 5.5 months. The entire pancreas (with attached spleen, stomach, and intestine) was rapidly excised and fixed by immersion in 4% formaldehyde in PBS (pH 7.4), for 48 h, at room temperature. After fixation, the pancreas was trimmed free of surrounding tissues, placed in a tissue capsule, routinely processed, and embedded in paraffin wax. From each organ, several serial paraffin sections were cut, at a nominal thickness of 3 µm, and were mounted on aminopropyltriethoxysilane-treated glass slides. The first two sections from each series were routinely stained with hematoxylin and eosin. Subsequent sections were taken for IGFBP-2 immunohistochemistry using an indirect immunoperoxidase technique (20).

For immunohistochemistry, sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. After blocking of endogenous peroxidase, by treatment with 1% hydrogen peroxide in PBS for 15 min and rinsing in PBS (2 x 10 min), sections were treated with normal swine serum for 30 min. Subsequently, sections were incubated with the primary antibodies (affinity-purified rabbit antibodies against murine IGFBP-2, as described above, diluted 1:100 in PBS) at 4 C for 24 h. After a rinse in PBS (2 x 10 min), sections were incubated with peroxidase-labeled swine antirabbit Ig (DAKO Corp. Diagnostika, Hamburg, Germany; diluted 1:100 in PBS containing 5% normal mouse serum) for 1 h at room temperature. After another rinse in PBS (2 x 10 min), peroxidase activity was visualized with 3,3‘-diaminobenzidine tetrahydrochloride (Fluka Feinchemikalien, Neu-Ulm, Germany), 10 mg in 20 ml of PBS containing 0.01% hydrogen peroxide, 5–10 min at room temperature. Sections were finally counterstained with Mayer’s hematoxylin, rinsed in water, dehydrated in a graded series of ethanol, cleared with xylene, and mounted in mounting medium (Eukitt, Kindler, Freiburg, Germany). For negative controls, the primary antibody was substituted by PBS or normal rabbit serum diluted 1:100 in PBS.

Measurement of IGFBP-2, IGF-I, and IGF-II
IGFBP-2, IGF-I, and IGF-II serum levels were quantified by specific RIAs, as described previously (10). For all assays, dilution curves of mouse serum samples were linear, and they paralleled those of human standards. For the statistical analysis, Student’s unpaired t test was used.

Serum insulin and glucose levels
Blood glucose concentrations were determined in both overnight-fasted and 5-h-refed animals using the Precision QID system (Medi-sense, Taufkirchen, Germany). The corresponding serum insulin levels were measured using a commercial insulin RIA (Insulin-CT, CIS-Bio International, Gif-sur-Yvette, France), as described previously (10). For the statistical analysis, the unpaired Student’s t test was performed.

Analysis of body and organ growth
Mice were weighed, twice weekly, to the nearest 0.1 g. To estimate average growth of the individual groups, data were transformed to a weighing day of n x 3, by linear interpolation, as described previously (21). Then 5.5-month-old mice were ether anesthetized and killed by bleeding from the retroorbital sinus. Nose-rump length was measured, as the distance between nose and base of the tail, as described before (21). For the nose-rump length measurement, the animals were gently stretched (25 g). The weight of mesentery and fat tissue surrounding the genital organs and kidneys, which is correlated with total body fat content, was determined as the amount of intraabdominal fat tissue. For the analysis, organs were removed, blotted dry, and weighed to the nearest mg. Carcasses were weighed, after removal of the organs, without skin, head, and tail. Data for body weight were analyzed by the General Linear Models procedure (SAS Institute, Inc., Cary, NC). The statistical model included group (transgenic vs. control), sex, and age. Least-squares means for group x sex were calculated per weighing age and compared using the unpaired Student’s t test. Organ and carcass weights were analyzed by ANOVA, taking effects of group and sex into account.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CMV-IGFBP-2 transgenic mice overexpress biologically active IGFBP-2
Genomic integration of CMV-int-mIGFBP-2 vector copies was demonstrated by Southern blot analysis. In nontransgenic mice, hybridization signals of the endogenous IGFBP-2 gene were detected at 7 and 4 kb, after EcoRI digestion. In addition to these bands, in strain 1 and strain 2, a fragment of 3.2 kb (length of the expression vector) was detected, indicating the presence of tandem repeats in the genome. Furthermore, a band slightly above 6 kb and a second band below 2 kb were detected in strain 2 (Fig. 1Go). Absolute quantitation by TaqMan technology revealed two copies integrated in strain 1 and nine copies integrated in strain 2 (not shown).

IGFBP-2 mRNA expression was investigated by Northern blot hybridization, which revealed a transgene-specific band (1.6 kb) in a number of organs (including heart, stomach, kidney, jejunum, spleen, skeletal muscle, colon, and lung; Fig. 2Go). In addition, transgenic IGFBP-2 mRNA was detected in brain, salivary glands, adipose tissue, and adrenal glands (data not shown). In contrast, no transgene-specific transcript could be detected in liver (Fig. 2Go). A transcript of the same length has been described previously in 293 cells transfected with the same expression vector (19). An endogenous signal was visible at 1.4 kb in spleen, colon, lung, and liver (Fig. 2Go). Increased IGFBP-2 protein levels were demonstrated in pancreas, heart, skeletal muscle, brain, and stomach in 2 independent transgenic strains, as shown by Western ligand blotting (Fig. 3Go). Increased IGFBP-2 protein levels were also detected in kidney, small intestine, spleen, salivary glands, lung, and adrenal glands (data not shown). In spite of endogenous IGFBP-2 mRNA expression in the liver, IGFBP-2 protein was undetectable in liver samples of both transgenic animals and controls (Fig. 3Go). In both transgenic strains, the transgene expression varied considerably between animals, consistently at elevated levels, compared with controls. Overall, the highest transgene expression was found in pancreas, followed by stomach, heart, colon, and adipose tissue (Fig. 4Go). By Western ligand blotting, a single band at 32 kDa was detected (Fig. 4AGo). Under reducing conditions, using IGFBP-2 specific peptide-induced antibodies, a single band was detected at 34 kDa (Fig. 4BGo). No signal was present in nontransgenic littermates demonstrating marked overexpression of IGFBP-2 and the specificity of the peptide-induced antiserum for IGFBP-2. To test whether the transgene was active during the first weeks of postnatal life, we determined serum and pancreatic IGFBP-2 levels by Western ligand blot analysis in 2-day- and in 2- and 4-week-old mice. Increased IGFBP-2 serum levels (Fig. 5AGo) and strong transgene expression in the pancreas (Fig. 5BGo) were found at all timepoints investigated. Endogenous IGFBP-2 serum levels were high 2 days after birth in control animals and declined thereafter.



View larger version (106K):
[in this window]
[in a new window]
  		Figure 2. IGFBP-2 mRNA expression in different organs, detected by Northern blot hybridization, in two nontransgenic littermates [controls (-)] and in two IGFBP-2 transgenic mice (+) of strain 1 (F2 animals). The bent arrow indicates endogenous (e) IGFBP-2 mRNA expression (1.4 kb) in spleen, colon, lung, and liver; whereas a signal around 1.6 kb of transgenic IGFBP-2 mRNA is detected in the organs displayed, with exception of the liver (linear arrow).

 


View larger version (97K):
[in this window]
[in a new window]
  		Figure 3. Comparison of transgene expression in two different IGFBP-2 transgenic strains in several organs, as shown by Western ligand blotting. Protein was extracted from different organs, as described in Materials and Methods. In both transgenic strains (F3 animals), increased activity of IGFBP-2 was detected in pancreas, heart, muscle, brain, and stomach. IGFBP-2 could not be detected in the liver.

 


View larger version (78K):
[in this window]
[in a new window]
  		Figure 4. IGFBP-2 levels in several organs from IGFBP-2 tg and co, as shown by Western ligand- (A) and Western immunoblotting (B). Protein was extracted from different organs, as described in Materials and Methods. The strongest signal was detected in the pancreas, followed by stomach, heart, colon, and adipose tissue. IGFBP-2 was visualized as a band at 32 kDa (by Western ligand blotting) and as a band at 34 kDa (by Western immunoblotting) under reducing conditions. In the immunoblot, no signal was detected in control animals demonstrating marked overexpression of IGFBP-2 in transgenic mice and the specificity of the peptide-induced antiserum.

 


View larger version (95K):
[in this window]
[in a new window]
  		Figure 5. IGFBP-2 levels in serum (A) and pancreas (B) of IGFBP-2 tg and co during the early postnatal period, detected by Western ligand blot. Samples were taken 2 days, 2 weeks, and 4 weeks after birth. Increased IGFBP-2 levels were found in serum and pancreas samples of transgenic mice at all developmental stages investigated.

 
Immunohistochemistry revealed that IGFBP-2 expression in the pancreas was restricted to the islets of Langerhans (Fig. 6DGo). The immunohistochemical staining pattern within the islets suggests transgene expression in ß-cells. In contrast, no IGFBP-2 immunoreactivity was found in pancreatic tissue from control mice (Fig. 6BGo).



View larger version (115K):
[in this window]
[in a new window]
  		Figure 6. Immunohistochemical analysis of transgene expression in the pancreas. Photomicrographs of pancreatic tissue of an F2 control mouse (A and B) and of a F2 CMV-IGFBP-2 transgenic mouse (C and D). Each panel represents a section that is 120 µm in length. Pancreata were fixed in 4% formaldehyde and embedded in paraffin wax, and several serial paraffin sections were cut at a nominal thickness of 3 µm. Histological sections were stained with hematoxylin and eosin (A and C). An indirect immunoperoxidase technique was used for the immunohistochemical demonstration of IGFBP-2, as described in Materials and Methods. Peroxidase activity was visualized using 3,3‘-diaminobenzidine tetrahydrochloride. All immunostained sections (B and D) were counterstained with hematoxylin. Within the pancreata of transgenic mice, IGFBP-2 immunostaining was exclusively observed in the islets, with the majority of endocrine islet cells demonstrating intensive cytoplasmic staining (D). No immunostaining was seen in pancreatic tissue of control animals (B).

 
Serum concentrations of IGFBP-2, IGF-I, and IGF-II
Serum IGFBP-2 concentrations were about 3-fold increased in transgenic mice (females, 843 ± 282 ng/ml; males, 1199 ± 254 ng/ml), compared with their nontransgenic littermates (females, 298 ± 66 ng/ml; males, 387 ± 77 ng/ml; Fig. 7AGo). In contrast, total serum IGF-I (Fig. 7BGo) and IGF-II (Fig. 7CGo) levels were not affected by IGFBP-2 overexpression in transgenic mice.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 7. Serum levels of IGFBP-2 (A), IGF-I (B), and IGF-II (C), measured by RIAs. Significantly (P < 0.05) increased IGFBP-2 serum levels were measured in 5.5-month-old male (n = 4) and female (n = 3) F2 transgenic animals. Error bars indicate SE. Serum levels for IGF-I and IGF-II were unaffected by the genotype. C, Control; T, transgenic.

 
Effects on other serum IGFBPs
Western ligand blot analysis of serum samples from transgenic mice and controls confirmed increased IGFBP-2 levels, which were detected by a specific RIA. However, serum levels of IGFBP-3 and IGFBP-4 were not affected by IGFBP-2 overexpression in transgenic mice (Fig. 8Go).



View larger version (71K):
[in this window]
[in a new window]
  		Figure 8. Western ligand blot analysis of IGFBPs in serum from 4-week-old F1 offspring. Serum samples were subjected to SDS-PAGE (Fig. 8AGo), as described in Materials and Methods, and the binding proteins were detected using [125I]-IGF-II as a tracer. The relative mobility of molecular weight markers is indicated. The bands were quantified, as described in Materials and Methods (Fig. 8BGo). Significant differences (P < 0.05) were detected for IGFBP-2 using the unpaired Student’s t test (AU, arbitrary units).

 
Effects on glucose homeostasis
Fasted serum glucose and insulin values were reduced in CMV-IGFBP-2 transgenic mice, compared with nontransgenic littermates. For fasted glucose serum levels, borderline significance (P = 0.051) was found. After refeeding, serum glucose and insulin levels of transgenic mice were not different from those of controls (Table 1Go).


View this table:
[in this window]
[in a new window]
  		Table 1. Fasted and postprandial glucose and insulin serum levels in IGFBP-2 transgenic (tg) and nontransgenic littermates (co)

 
Effects on body and organ weights
Transgenic mice and their nontransgenic littermates were weighed twice a week to investigate potential effects of transgene expression on body weight. At days later than postnatal day 23, the least-squares means for body weight in the first transgenic strain were significantly reduced (P < 0.05; 9%, on average, in males; 11% in females), compared with control littermates (Fig. 9Go). Body weights in the second transgenic strain were similarly reduced (data not shown). Body weight of CMV-IGFBP-2 transgenic mice was still reduced in the period between days 150 and 240 and did not seem to be compensated with time (data not shown).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 9. Growth of IGFBP-2 tg (hemizygous mice from the F1 and F2 generations) and nontransgenic littermates (co). Mice were weighed twice a week, and body weight data were transformed by linear interpolation, as described in Materials and Methods. Least-squares means were calculated and plotted with their respective SE. Significant differences (P < 0.05) were measured at days later than 23 in both sexes (n = 9).

 
Organ weights were studied in 5.5-month-old mice. A significant (P < 0.01) reduction in weight was seen only for the spleen in male CMV-IGFBP-2 transgenic mice (Fig. 10AGo). However, in male and female transgenic mice, 10/14 and 8/14 parameters investigated, respectively, displayed slightly reduced values, compared with controls. In total, the organ weights analyzed were reduced by only about 3% in both sexes in transgenic mice, compared with their nontransgenic littermates. When relative organ weights were calculated, significant (P < 0.01) increases were found for kidneys, lung, stomach, and colon of female transgenic mice (Fig. 10BGo). Significantly decreased (P < 0.05) absolute carcass weights (Fig. 11Go) were measured in IGFBP-2 transgenic mice (10.6 ± 1.1 g), compared with nontransgenic controls (12.1 ± 1.2 g), corresponding to approximately 13% reduction. Importantly, a significant (P < 0.05) reduction of the relative carcass weights was present in transgenic mice (40.0 ± 3.5%), compared with controls (45.4 ± 3.8%), which represents a similar reduction of about 12% (Fig. 11Go). During the dissection of the mice, no obvious pathological alterations were found.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 10. Absolute (A) and relative (B) organ weights and dimensions of 5.5-month-old IGFBP-2 transgenic F2 mice, compared with nontransgenic littermates (males, n = 5; females, n = 4). Organs were removed after cervical dislocation, as described in Materials and Methods. Data are presented as percent of controls; error bars indicate SE.

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 11. Absolute and relative carcass weights in adult IGFBP-2 transgenic mice (T) and nontransgenic controls (C). The empty carcasses were analyzed as described in Materials and Methods (n = 6). Error bars represent SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have established IGFBP-2-overexpressing transgenic mice to investigate the functional role of IGFBP-2 in vivo. Genetic ablation of IGFBP-2 (13) suggested functional redundancy of the IGFBPs and failed to reveal clear functions for IGFBP-2 in vivo. IGFBP-2 expression is elevated in a number of pathological alterations (6, 7, 9), whereas the role of the induction is unclear. In a recent study on mice selected for body weight, we screened for effects on components of the IGF-system (15) and thereby found increased IGFBP-2 expression associated with reduced body weight.

Transgene expression
Transgenic mice displayed transgene expression on mRNA and protein level in nearly every organ tested, with exception of the liver. The spectrum of tissues expressing the transgene was almost identical to that of a high-expressing transgenic strain of mice harboring a CMV-chloramphenicol expression vector (22). The only exception was the pancreas. In our model, the pancreas represented the organ with highest transgene expression of the organs investigated, whereas the pancreatic expression level in the CMV-chloramphenicol transgenic model was intermediate. In addition, expression was restricted to the islets and probably to the ß-cells. The intron used in the expression vector was derived from the rat insulin II gene. It is tempting to speculate, and worth further investigation, whether the intron used is responsible for the strong transgene expression within the ß-cells of the pancreas observed in our transgenic mouse strains. Transgene expression was characterized by a relatively high variation between individual animals in both transgenic strains. However, the overall transgenic IGFBP-2 secretion seemed similar in the different individuals, resulting in a reproducible 3-fold increase of IGFBP-2 serum levels and a consistent phenotype of reduced body weight gain. No expression of transgene-specific mRNA was found in the liver, and no IGFBP-2 protein was detected in protein extracts from the liver. The liver is known to express and secrete endogenous IGFBP-2. It is most likely that endogenous IGFBP-2 was below the detection limit because of IGFBP-2 secretion by the liver. Nevertheless, the liver (besides pancreas, spleen, salivary glands, and brain) was among those organs displaying more pronounced weight reductions, indicating endocrine vs. auto- or paracrine mechanisms of IGFBP-2 action in the liver.

Glucose homeostasis
The levels of IGFBP-2 within the pancreatic islets of CMV-IGFBP-2 transgenic mice were extremely high. This finding is particularly interesting in the context of potential effects on glucose homeostasis in IGFBP-2 transgenic mice. Interestingly, glucose levels were, as a tendency, reduced in IGFBP-2 transgenic mice. However, fasted glucose serum level differences did show only borderline significance (P = 0.051). In addition, fasted insulin serum levels were slightly (but not significantly) reduced in transgenic mice, compared with controls (P = 0.111). After refeeding, serum levels of both insulin and glucose were in the same range in both genetic groups. A role of insulin in the regulation of IGFBP-2 has been suggested from several studies in which diabetic children displayed increased IGFBP-2 levels (7, 23). In contrast, insulin therapy tended to reduce IGFBP-2 levels (24). Moreover, nutritional regulation of IGFBP-2 expression has been well documented (10, 25, 26, 27). In a sharp contrast to IGFBP-2 transgenic mice, an increase of relative pancreas weight, as well as of serum glucose and insulin levels, have been observed in IGFBP-1 transgenic mice (28, 29). Because, in IGFBP-2 transgenic mice, neither hyperglycemia nor hyperinsulinemia have been observed, and the pancreas was among the organs of the more severe weight reductions, distinct roles in the control of growth and metabolism for IGFBP-1 and IGFBP-2 are likely to suppose. Further studies are required to study a potential involvement of IGFBP-2 in glucose homeostasis.

Expression of other components of the IGF system
IGFBP-2 serum levels were markedly (about 3-fold) increased in transgenic mice. Interestingly, no effect on IGF-I or -II serum levels was demonstrated in transgenic mice. IGFBP-2 is supposed to have higher affinity for IGF-II than for IGF-I (2). In a goat model, iv injection of IGFBP-2 resulted in increased plasma clearance of IGF-I and -II (30). It was concluded that IGFBP-2 targets the IGFs to distinct tissues. Such effects were not seen in CMV-IGFBP-2 transgenic mice, which may be attributable to differences in the levels of serum IGFBP-2 reached or to unknown species-specific factors. Because serum IGF-I levels (which monitor GH serum levels and, therefore, GH growth control) were unaffected by IGFBP-2 overexpression, reduced weight gain of CMV-IGFBP-2 transgenic mice is most likely attributable to local inhibition of IGF actions at the tissue level by IGFBP-2.

Body and organ weights
Body weight of IGFBP-2-overexpressing mice was significantly reduced at days later than postnatal day 23, a fact which is surprising, because transgene expression was active from postnatal day 2. However, we found very high endogenous IGFBP-2 serum levels in the early postnatal period. Therefore, differences in IGFBP-2 serum levels between transgenic and control mice were relatively small, which might be a reason for the absence of a clear phenotype during the first weeks of life. Similarly, the impairment of body weight gain in two different transgenic mouse models overexpressing IGFBP-1 (29, 31) occurred mainly between 3 and 8 weeks of age. Accordingly, it is possible that increased IGFBP-2 expression could substantially decrease the GH-induced IGF-I action during pubertal growth spurt.

Whereas IGFBP-2 transgenic mice displayed a significant reduction of body weight, organ weights were only partly and slightly reduced, with the spleen of male transgenic mice being the only organ significantly reduced in weight. However, although not significant, those organs (spleen, pancreas, brain, kidney, and liver) that were most markedly increased in IGF-I-overexpressing transgenic mice (32) showed the clearest tendency of reduced weight in IGFBP-2-overexpressing mice. This inverse phenotype of IGF-I- and IGFBP-2-overexpressing mice suggests an inhibitory effect of IGFBP-2 on IGF-I. It is known that IGF-I represents a postnatal regulator of growth in mice; in contrast, IGF-II is important for fetal growth (33, 34), whereas its expression is almost completely shut down in adult tissues.

Although there were slight reductions in absolute organ weights of CMV-IGFBP-2 transgenic mice, the difference in the sum of organ weights (accounting for about 20% of total body weight), between transgenic and control mice, corresponded to only about 3% and could thus not explain the overall 10% difference in total body weight. If calculated relative to body weight, we found significant increases in the weight of kidneys, lung, stomach, and colon in female transgenic mice. We conclude that the organs contributed to the overall body weight reduction only to a limited extent. Therefore, we determined the carcass weight, which accounts for 40–45% of total body weight, in six CMV-IGFBP-2 transgenic and in six control mice. The mean carcass weight was significantly (P < 0.05) smaller in transgenic (10.6 ± 1.1 g) than in control mice (12.1 ± 1.2 g), resulting in a difference of about 13%. A similar reduction (~12%) was also evident when relative carcass weights were calculated, a fact which strongly suggests the carcass as a main target of direct or indirect IGFBP-2 actions in vivo. Various cell types comprising the carcass, including osteoblasts, chondroblasts, and myoblasts, are target tissues of IGF-I (35, 36) and are therefore likely to be sensitive to IGFBP-2-mediated inhibition of IGF-I. Conversely, carcass weight was increased by 20% in transgenic mice overexpressing IGF-I (32).

Our data suggest that IGFBP-2 is capable of inhibiting the biological actions of IGF-I in vivo via endocrine or paracrine mechanisms, resulting in reduced postnatal weight gain.


   Acknowledgments
 
We appreciate the kind gift of the cDNA for mIGFBP-2 by Dr. S. Drop, Rotterdam, The Netherlands. We thank Dr. Ingrid Renner-Müller for veterinary management; Petra Renner for expert animal care; and Petra Demleitner, Norman Rieger, Tamara Holy, and Karin Weber for excellent technical assistance.

Received March 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Drop SL, Schuller AG, Lindenbergh-Kortleve DJ, Groffen C, Brinkman A, Zwarthoff EC 1992 Structural aspects of the IGFBP family. Growth Regul 2:69–79[Medline]
  2. Oh Y, Muller HL, Lee DY, Fielder PJ, Rosenfeld RG 1993 Characterization of the affinities of insulin-like growth factor (IGF)-binding proteins 1–4 for IGF-I, IGF-II, IGF-I/insulin hybrid, and IGF-I analogs. Endocrinology 132:1337–1344[Abstract]
  3. Landwehr J, Kaupmann K, Heinrich G, Schwander J 1993 Cloning and characterization of the gene encoding murine insulin-like growth factor-binding protein-2, mIGFBP-2. Gene 124:281–286[CrossRef][Medline]
  4. Schuller AG, Zwarthoff EC, Drop SL 1993 Gene expression of the six insulin-like growth factor binding proteins in the mouse conceptus during mid- and late gestation. Endocrinology 132:2544–2550[Abstract]
  5. Schuller AGP, Groffen C, vanNeck JW, Zwarthoff EC, Drop SLS 1994 cDNA cloning and mRNA expression of the six mouse insulin-like growth factor binding proteins. Mol Cell Endocrinol 104:57–66[CrossRef][Medline]
  6. Blum WF, Horn N, Kratzsch J, Jorgensen JO, Juul A, Teale D, Mohnike K, Ranke MB 1993 Clinical studies of IGFBP-2 by radioimmunoassay. Growth Regul 3:100–104[Medline]
  7. Strasser-Vogel B, Blum WF, Past R, Kessler U, Hoeflich A, Meiler B, Kiess W 1995 Insulin-like growth factor (IGF)-I and -II and IGF-binding proteins-1, -2, and -3 in children and adolescents with diabetes mellitus: correlation with metabolic control and height attainment. J Clin Endocrinol Metab 80:1207–1213[Abstract]
  8. Young SC, Underwood LE, Celniker A, Clemmons DR 1992 Effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone on serum IGF-binding proteins in calorically restricted adults. J Clin Endocrinol Metab 75:603–608[Abstract]
  9. Zapf J, Kiefer M, Merryweather J, Musiarz F, Bauer D, Born W, Fischer JA, Froesch ER 1990 Isolation from adult human serum of four insulin-like growth factor (IGF) binding proteins and molecular cloning of one of them that is increased by IGF I administration and in extrapancreatic tumor hypoglycemia. J Biol Chem 265:14892–14898[Abstract/Free Full Text]
  10. Wolf E, Kramer R, Blum WF, Föll J, Brem G 1994 Consequences of postnatally elevated insulin-like growth factor-II in transgenic mice: endocrine changes and effects on body and organ growth. Endocrinology 135:1877–1886[Abstract]
  11. Rogler CE, Yang D, Rossetti L, Donohoe J, Alt E, Chang CJ, Rosenfeld R, Neely K, Hintz R 1994 Altered body composition and increased frequency of diverse malignancies in insulin-like growth factor-II transgenic mice. J Biol Chem 269:13779–13784[Abstract/Free Full Text]
  12. Wood TL, Rogler L, Streck RD, Cerro J, Green B, Grewal A, Pintar JE 1993 Targeted disruption of IGFBP-2 gene. Growth Regul 3:5–8[Medline]
  13. Pintar JE, Schuller A, Cerro JA, Czick M, Grewal A, Green B 1995 Genetic ablation of IGFBP-2 suggests functional redundancy in the IGFBP family. Prog Growth Factor Res 6:437–445[CrossRef][Medline]
  14. Wolf E, Jehle PM, Weber MM, Sauerwein H, Daxenberger A, Breier BH, Besenfelder U, Frenyo L, Brem G 1997 Human insulin-like growth factor I (IGF-I) produced in the mammary glands of transgenic rabbits: yield, receptor binding, mitogenic activity, and effects on IGF-binding proteins. Endocrinology 138:307–313[Abstract/Free Full Text]
  15. Hoeflich A, Schmidt P, Föll J, Rottmann O, Weber MM, Pirchner F, Wolf E 1998 Altered growth of mice divergently selected for body weight is associated with complex changes in the growth hormone/insulin-like growth factor system. Growth Horm IGF Res 8:113–123[CrossRef][Medline]
  16. Tapanainen PJ, Bang P, Wilson K, Unterman TG, Vreman HJ, Rosenfeld RG 1994 Maternal hypoxia as a model for intrauterine growth retardation: effects on insulin-like growth factors and their binding proteins. Pediatr Res 36:152–158[Medline]
  17. Price WA, Stiles AD, Moats-Staats BM, D’Ercole AJ 1992 Gene expression of insulin-like growth factors (IGFs), the type 1 IGF receptor, and IGF-binding proteins in dexamethasone-induced fetal growth retardation. Endocrinology 130:1424–1432[Abstract]
  18. Kampman KA, Ramsay TG, White ME 1993 Developmental changes in hepatic IGF-2 and IGFBP-2 mRNA levels in intrauterine growth-retarded and control swine. Comp Biochem Physiol [B] 104:415–421[CrossRef][Medline]
  19. Höflich A, Lahm H, Blum W, Kolb H, Wolf E 1998 Insulin-like growth factor-binding protein-2 inhibits proliferation of human embryonic kidney fibroblasts and of IGF-responsive colon carcinoma cell lines. FEBS Lett 434:329–334[CrossRef][Medline]
  20. Nakane PK, Pierce Jr GB 1967 Enzyme-labeled antibodies: preparation, and application for the localization of antigens. J Histochem Cytochem 14: 929–931
  21. Wolf E, Wanke R, Hermanns W, Brem G, Pirchner F, von Butler-Wemken I 1991 Growth characteristics of metallothionin-human growth hormone transgenic mice as compared to mice selected for high eight-week body weight and unselected controls. I. Body weight gain and external body dimensions. Growth, Development & Aging 55:225–235
  22. Furth PA, Hennighausen L, Baker C, Beatty B, Woychick R 1991 The variability in activity of the universally expressed human cytomegalovirus immediate early gene 1 enhancer/promoter in transgenic mice. Nucleic Acids Res 19:6205–6208[Abstract/Free Full Text]
  23. Knip M, Tapanainen P, Pekonen F, Blum WF 1995 Insulin-like growth factor binding proteins in prepubertal children with insulin-dependent diabetes mellitus. Eur J Endocrinol 133:440–444[Abstract/Free Full Text]
  24. Bereket A, Lang CH, Blethen SL, Wilson TA 1995 Insulin-like growth factor-binding protein-2 and insulin: studies in children with type 1 diabetes mellitus and maturity-onset diabetes of the young. J Clin Endocrinol Metab 80:3647–3652[Abstract]
  25. Tseng LY, Ooi GT, Brown AL, Straus DS, Rechler MM 1992 Transcription of the insulin-like growth factor-binding protein-2 gene is increased in neonatal and fasted adult rat liver. Mol Endocrinol 6:1195–1201[Abstract]
  26. Ketelslegers JM, Maiter D, Maes M, Underwood LE, Thissen JP 1996 Nutritional regulation of the growth hormone and insulin-like growth factor-binding proteins. Horm Res 45:252–257[Medline]
  27. Ogawa E, Breier BH, Bauer MK, Gallaher BW, Grant PA, Walton PE, Owens JA, Gluckman PD 1996 Pretreatment with bovine growth hormone is as effective as treatment during metabolic stress to reduce catabolism in fasted lambs. Endocrinology 137:1242–1248[Abstract]
  28. Rajkumar K, Krsek M, Dheen ST, Murphy LJ 1996 Impaired glucose homeostasis in insulin-like growth factor binding protein-1 transgenic mice. J Clin Invest 98:1818–1825[Medline]
  29. Rajkumar K, Barron D, Lewitt MS, Murphy LJ 1995 Growth retardation and hyperglycemia in insulin-like growth factor binding protein-1 transgenic mice. Endocrinology 136:4029–4034[Abstract]
  30. Prosser CG, Schwander J 1996 Influence of insulin-like growth factor-binding protein-2 on plasma clearance and transfer of insulin-like growth factors-I and -II from plasma into mammary-derived lymph and milk of goats. J Endocrinol 150:121–127[Abstract/Free Full Text]
  31. Gay E, Seurin D, Babajko S, Doublier S, Cazillis M, Binoux M 1997 Liver-specific expression of human insulin-like growth factor binding protein-1 in transgenic mice: repercussions on reproduction, ante- and perinatal mortality and postnatal growth. Endocrinology 138:2937–2947[Abstract/Free Full Text]
  32. Mathews LS, Hammer RE, Behringer RR, D’Ercole AJ, Bell GI, Brinster RL, Palmiter RD 1988 Growth enhancement of transgenic mice expressing human insulin-like growth factor I. Endocrinology 123:2827–2833[Abstract]
  33. Baker J, Liu JP, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82[CrossRef][Medline]
  34. Liu JP, 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]
  35. Salmon WD, Daughaday WH 1957 A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825–836[Medline]
  36. Froesch ER, Schmid C, Schwander J, Zapf J 1985 Actions of insulin-like growth factors. Annu Rev Physiol 47:443–467[CrossRef][Medline]




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 Google Scholar
Google Scholar
Right arrow Articles by Hoeflich, A.
Right arrow Articles by Wolf, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoeflich, A.
Right arrow Articles by Wolf, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals