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Departments of Internal Medicine (L.J.M.) and Physiology (J.V.S., Y.G., T.M., L.J.M.), University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3; and Department of Pediatrics, Baylor College of Medicine (A.S., S.K.D., D.R.P.), Houston, Texas 77030
Address all correspondence and requests for reprints to: L. J. Murphy, M.B., Ph.D., Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3. E-mail: ljmurph{at}cc.umanitoba.ca
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Abstract |
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A significant reduction in litter size was also observed in each of the acid-labile subunit transgenic mouse strains. This reduction in litter size was due to a maternal effect, as it was apparent when transgenic female mice were crossed with wild-type male mice, but not when male transgenic mice were crossed with female wild-type mice.
The transgenic mice were phenotypically normal at birth, but demonstrated a significant reduction in postnatal body weight gain, particularly during the first 3 wk of life. Over the first 3 months of life, average body weights were significantly reduced by 5.3 ± 0.6%, 4.2 ± 0.6%, 8.1 ± 0.9%, and 5.6 ± 0.8%, compared with those in wild-type mice, for male and female CMVALS-1 mice and male and female CMVALS-2 mice, respectively. Double transgenic mice, generated by crossing acid-labile subunit transgenic mice with transgenic mice that overexpress IGF-binding protein-3, demonstrated a significantly more marked reduction in body weight gain than acid-labile subunit transgenic mice.
These data demonstrate that overexpression of acid-labile subunit has significant effects on postnatal growth and reproduction. As there is little measurable alteration in the circulating components of the IGF system, these effects are most likely to be mediated via disturbances in tissue IGF availability.
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Introduction |
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Unlike binary complexes of IGFBP-3 and IGF-I or IGF-II, which can egress from the circulation across the endothelium, ternary complexes appear to be restricted to the circulation (8). The appearance of the ternary complex in the circulation shortly after birth is believed to serve as a relatively stable reservoir of circulating IGFs. However, it is not clear what physiological function this reservoir serves or how IGFs are liberated from the ternary complex to exert their biological actions in tissues.
Although the role of ALS in the formation of ternary IGF complex has been extensively investigated in vitro, there are as yet few reported data on the in vivo effects of ALS. To investigate the biological effects of ALS in vivo we generated acid-labile subunit transgenic (TG) mice that overexpress ALS. To maximize our chances of observing phenotypic effects of ALS overexpression, the ubiquitous cytomegalovirus (CMV) promoter was used. This promoter results in high level expression in most tissues from early embryonic life. Here we report significant growth retardation and reduction in female fecundity in ALS TG mice.
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Materials and Methods |
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TG mice were generated by pronucleus injection of the linearized 3.8-kb transgene fragment, devoid of plasmid sequences, into fertilized CD-1 zygotes. The microinjected embryos were transferred into CD-1 foster mice using standard techniques (11). The founders, CMVALS-1, and CMVALS-2 were bred to homozygosity with wild-type CD-1 mice. CD-1 mice from the same colony, bred in a similar fashion, provided wild-type (Wt), non-TG control mice of the same genetic background. The ALS TG mice were also bred with phosphoglycerate kinase-binding protein-3 (PGKBP-3) TG mice previously generated in this laboratory (12). PGKBP-3 TG mice overexpress a human IGFBP-3 cDNA using the phosphoglycerate kinase promoter. The double TG mice express both the IGFBP-3 and ALS transgenes. All experiments were performed in accordance with protocols approved by the animal care committee of the Faculty of Medicine, University of Manitoba.
Southern blot analysis
The presence of the transgene was detected by Southern blot
analysis of tail DNA. Filters were hybridized with the ALS fragment of
the transgene under stringent conditions. For determination of
transgene copy number, serial dilutions of tail DNA from homozygous TG
mice were analyzed by dot-blot hybridization and quantified
densitometrically. The data were compared to a standard curve of human
ALS cDNA generated in a similar fashion.
RIAs
ALS and total plasma IGF-I was measured using human ALS
ELISA and rat IGF-I RIA kits (Diagnostics Systems Laboratories, Inc., Webster, TX). GH was measured by RIA using a rat GH RIA
kit (Amersham Pharmacia Biotech, Baie d’Urfé,
Canada). Serum samples pooled from ALS TG mice were chromatographed on
a Sephacryl S300 column as previously described (12).
Fractions were collected and analyzed for ALS.
RNA extraction and ribonuclease protection assays (RPAs)
Total RNA was isolated from mouse liver using TRIzol reagent
(Life Technologies, Inc., Burlington, Canada). RT-PCR was
used to generate a mouse ALS cDNA fragment. First strand cDNA was
synthesized from total RNA with AMV reverse transcriptase
(Promega Corp., Madison, WI). PCR was used for the
amplification of human ALS cDNA fragment. Amplification was performed
in a thermocycler (Perkin-Elmer Corp., Norwalk, CT) under
the following conditions: 95 C for 45 sec, 60 C for 30 sec, and 72 C
for 90 sec for 30 cycles. The primers used for mouse ALS cDNA were
5'-CTGCTACTTACTGGCCTGGT-3' (sense) and 5'-TGCCCACCCTATTTAGGCC-3'
(antisense). For human ALS cDNA the primers used were 5'-CAG CCC GCC
CGA GGT CGT G-3' (sense) and 5'-ACC CCA TCA GGC CCT TGC GTC-3'
(antisense). Fragments of the anticipated sizes (176 and 195 bp for
mouse and human ALS cDNAs, respectively) were obtained.
The PCR products of mouse and human ALS were extracted from 1% agarose gel with the QIAquick gel extraction kit (QIAGEN, Mississauga, Canada) and cloned into the pCR II vector using reagent supplied by the manufacturer (Invitrogen, San Diego, CA). Bacterial colonies were selected with X-galactosidase plates. The sequence and orientation of the cDNA fragments were confirmed by DNA sequencing. The plasmids containing the mouse and human ALS cDNA fragments were linearized with XbaI and BamHI, respectively, for use as a template for RNA probe synthesis.
The in vitro transcription and labeling reaction was carried out using 80 µCi [32P]UTP (NEN Life Science Products, Boston, MA) and the Maxiscript SP6/T7 polymerase kit (Ambion, Inc., Austin, TX). A riboprobe for mouse cyclophilin (Ambion, Inc.) was used as the internal standard, and century RNA markers (Ambion, Inc.) were used as the molecular size indicators.
Total RNA was isolated with TRIzol reagent from 17-d-old fetal mice and from various tissues of 8-wk-old mice. A ribonuclease protection assay (RPA) kit (Ambion, Inc.) was used to quantify mouse ALS or human ALS mRNA abundance. Total RNA (20 µg) from mouse tissues was hybridized in hybridization buffer containing approximately 3 x 105 cpm 32P-labeled human or mouse ALS cRNA and cyclophilin cRNA by incubation overnight at 45 C. After hybridization, single stranded RNA was digested with ribonuclease A/T at 37 C for 30 min. The remaining RNA duplexes were separated on 5% polyacrylaminde/8 M urea gel. The hybridization signal was detected by autoradiography. The protected sizes for human ALS, mouse ALS, and mouse cyclophilin fragments were 195, 176, and 103 bp, respectively.
Western and ligand blotting
For immunodetection of human ALS, mouse embryos were homogenized
in buffer [10 mm Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0),
0.15 M NaCl, 1% Nonidet P-40, 0.5% SDS, 1 µg/ml
aprotinin, and 1 mM phenylmethylsulfonylfluoride]. The
homogenate was clarified by centrifugation at 15,000 x
g for 15 min at 4 C and separated by discontinuous 8%
SDS-PAGE. The separated proteins were transferred onto nitrocellulose
membrane (MSI, Westborough, MA) at 200 mA for 2 h. Serum samples
(2 µl) from TG and Wt mice were analyzed on a 10% SDS-PAGE gel and
transferred to nitrocellulose membrane. The blots were incubated
overnight at 4 C with a 1:7,500 dilution of goat anti-ALS antibody
(DSL-R00941, Diagnostics Systems Laboratories, Inc.).
After washing with buffer [5 mM Tris-HCl (pH
7.4), 136 mM NaCl, and 0.05% Tween-20], the
blots were incubated for 2 h at room temperature with horseradish
peroxidase-conjugated donkey antigoat IgG (Promega Corp.)
at a dilution of 1:5,000. Detection of immune complexes was achieved
using an ECL Western blotting kit (Amersham Pharmacia Biotech).
For ligand blotting, the membrane was incubated with [125I]IGF-I, (500,000 cpm, NEN Life Science Products) at 4 C overnight. The membrane was subsequently washed four times with Tris-buffered saline, pH 7.6, and 0.1% Tween 20 and exposed to Kodak XAR film (Rochester, NY) at -70 C for 24–72 h.
Statistical analysis
Data are expressed as the mean ± SEM.
Statistical differences between the growth curves for each group of
mice were determined from an analysis of covariance with the repeated
measure model using the SPSS for Windows software package (SPSS, Inc., Chicago, IL). A t test was used for single
comparisons between TG and Wt mice. For determining statistical
differences between multiple groups, an ANOVA, followed by Dunnett’s
t test, were used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the
restriction endonuclease pattern obtained when DNA from each strain of
TG mice was digested with SphI and BamHI
endonucleases. The predominant band, 3.8 kb in size, resulted from
multiple head to tail copies of the transgene. Less prominent bands of
different sizes were apparent in the DNA from the two TG mouse strains,
indicating different transgene insertion sites.
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). This reduction in litter size was a
maternal, rather than fetal, effect, because a significant reduction in
litter size was apparent when female TG mice were crossed with male Wt
mice, but not when female Wt mice were crossed with male TG mice.
Furthermore, the reduction in litter size was of the same magnitude in
litters from female TG mice mated with Wt or TG mice.
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). Using a human ELISA, most serum
samples from Wt mice gave very low or undetectable levels of ALS,
indicating minimal cross-reaction between mouse ALS and the antibody
used in the ELISA assay. The levels of human ALS in the sera of TG mice
ranged from 16–23 µg/ml. There were no significant differences in
serum ALS levels between the two TG strains or between TG mice of
different sexes. Sera from ALS TG mice were fractionated by gel
chromatography. Two distinct immunoreactive peaks were identified (Fig. 4). The most prominent peak eluted at a
position that corresponded to the elution position of alcohol
dehydrogenase (mol wt, 150,000). A less prominent, more rapidly eluting
peak was also observed and may represent an ALS dimer or ALS bound to
other serum proteins.
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). Immunoreactive
human ALS was detected in the TG mice. When the autoradiographs were
analyzed by densitometry, there was no significant difference in the
abundance of human ALS as detected by Western blotting between CMVALS-1
and CMVALS-2 mice.
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). Three bands were
identified in each of the serum samples. The largest and most abundant
of these was presumed to represent IGFBP-3. Additional bands with
apparent sizes of 30 and 20 kDa were also recognized. These were
presumptively identified as IGFBP-2 and IGFBP-4. When quantified by
densitometry there were no significant differences between
CMVALS-1 and CMVALS-2 TG mice or between TG mice and Wt mice in the
abundance of any of these IGFBPs.
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A human ALS mRNA-specific RPA was used to assess the expression of the
transgene products. Using this assay, transgene expression was easily
detected in RNA extracted from 17-d-old embryos (data not shown). A
careful analysis of tissue transgene expression was undertaken in
2-month-old mice (Fig. 7A). Using
a human ALS mRNA-specific RPA, expression of the transgene was detected
in the liver, kidney, lungs, heart, brain, and spleen. The highest
level was detectable in the heart, and the lowest level was present in
the spleen. Using a mouse ALS mRNA-specific RPA, expression of the
mouse ALS gene was detectable only in the liver (Fig. 7B). There was no
significant difference in the level of endogenous mouse ALS
expression in TG and Wt mice (Fig. 7C).
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). A modest reduction in postnatal body
weight gain was observed in both CMVALS-1 and CMVALS-2 mice. Using
analysis of covariance with repeated measures, there was a significant
difference in the growth curves for the ALS TG mice compared with Wt
mice (P = 0.045, P = 0.001,
P = 0.05, and P = 0.001 for CMVALS-1
males, CMVALS-2 males, CMVALS-1 females, and CMVALS-2 female mice,
respectively). Over the first 3 months of life, the average body weight
of TG mice was significantly reduced by 4.2–8.1% compared with that
of Wt mice. The difference between ALS TG mice and Wt mice was
particularly apparent during the first 3 wk of life, when body weight
was reduced by 9–17% of that of Wt mice (Fig. 8). CMVALS-2 TG mice
demonstrated a more marked reduction in body weight than CMVALS-1 TG
mice.
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). The relative weight of the testes was
significantly increased in CMVASL-2 TG mice compared with Wt mice,
but this increase in relative weight was not observed in CMVALS-1 TG
mice.
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). PGKBP-3/CMVALS-1 and PGKBP-3/CMVALS-2
double TG mice weighed significantly less than CMVALS-1 and CMVALS-2 TG
mice, respectively. PGKBP-3 TG mice weighed significantly less than
CMVALS-2 and CMVALS-2 TG mice. However, the PGKBP-3/CMVALS-1 double TG
mice were significantly lighter than the PGKBP-3 mice. A similar trend
was observed for PGKBP-3/CMVALS-2 double transgenic mice; however, the
difference between these double TG animals and PGKBP-3 TG mice did not
achieve significance. Similar data were obtained when the mice were
analyzed at 3 months of age (data not shown).
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Discussion |
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Although some ALS is present at birth in the rodent and is functionally capable of interacting with the IGFBP-3/IGF binary complex, the concentration appears to be insufficient to lead to the generation of ternary complex (13). The enhanced expression of ALS, with an eventual 2- to 3-fold molar excess over IGFBP-3 (13, 14), results in the gradual appearance of the ternary complex in the circulation in the first few weeks of postnatal life. This change in the compartmentalization of IGF in the circulation is thought to limit the mitogenic and hypoglycemic actions of IGF-I and IGF-II in the first few weeks of life (1, 17). In the rodent, IGF-II declines in importance during this period because of developmental down-regulation of expression (18), and IGF-I becomes the most abundant IGF in adult life. The appearance of the ternary complex during the early postnatal period may serve as a circulating reservoir of IGF-I. The functional significance of this reservoir has been called into question in recent times with the demonstration that normal postnatal growth can occur in mice with conditional nullification of hepatic IGF-I expression (19, 20). These mice have low circulating total IGF-I levels, although free IGF-I levels may be normal.
To date there are no reported data on the in vivo effects of ALS on growth or other parameters. To investigate the biological effects of chronic elevated ALS levels in vivo, we generated TG mice that overexpress ALS. Here we report the characterization of transgenic mice in which ALS is expressed at high levels from very early in embryonic life. The CMV promoter is known to be transcriptionally active in mouse embryos (10, 21), and human ALS mRNA was easily demonstrable in 17-d-old embryos. High levels of human ALS were observed in the circulation of both TG strains. In addition, transgene-derived ALS mRNA expression was abundant in most tissues examined in TG mice, indicating that the potential exists for the formation of ternary complex in the extravascular space.
Although the actual serum levels of endogenous ALS in the mouse circulation are not known, the level in the adult rat is approximately 40 µg/ml (14, 15). In ALS TG mice, the measured level of human ALS was about 20 µg/ml. As the transgene was transcriptionally active early in embryogenesis, similar circulating human ALS levels were probably present throughout fetal life, when endogenous ALS levels are low in humans and rodents (1, 15). Despite this increase in circulating ALS and the expression during early embryonic life, the ALS TG mice were phenotypically normal at birth, and only a modest effect on postnatal growth was observed.
Estimation of the serum levels of IGFBP-3 from ligand blots indicated that the circulating IGFBP-3 concentration was not increased in TG mice compared with Wt mice. Similarly, serum IGF-I levels were not elevated in ALS TG mice. Thus, it would appear that enhanced expression of ALS does not result in a compensatory increase in IGFBP-3 and IGF-I levels or increased amounts of ternary complex. The absence of an effect on IGFBP-3 and IGF-I levels is not attributable to the inability of human transgene-derived ALS to form a ternary complex with mouse IGFBP-3, as chromatography and SDS-PAGE demonstrated that the majority of the human ALS was present as a 150-kDa complex. Although ALS expression and secretion are tightly regulated by GH, with cortisol and insulin also having an effect (15, 16), the actual levels of ALS surprisingly do not appear to be rate limiting in the formation of ternary complexes in perinatal life (13) or GH deficiency (22). Rather, ternary complex formation appears to be limited by the availability of IGF-I (22). Thus, excess ALS in the circulation of TG mice would be unlikely to have any major effect on ternary complex formation.
The failure to perturb the circulating IGF system may account for the relatively modest growth retardation observed in the ALS TG mice. A small, but significant, reduction in body weight gain was observed in both strains of ALS TG mice. This was most apparent in the first 4 wk, when growth velocity, as measured by weight gain, is at its maximum.
Under normal circumstances, the expression of ALS is tightly restricted to the liver (15), with smaller amounts of expression detected by in situ hybridization in the kidney cortex, bone, and ovarian tissue (23, 24). In ALS TG mice, tissue expression of the transgene was ubiquitous, raising the possibility that the local tissue IGF system could be perturbed in these mice. Recent reports of normal growth after selective disruption of hepatic IGF-I expression has suggested that paracrine/autocrine IGF-I may be more important than circulating IGF-I in normal growth (19, 20). In contrast, Ueki et al. (17) recently reported modest postnatal growth retardation in ALS null mutant mice. These ALS knockout mice had reduced circulating levels of IGFBP-3 and IGF-I (17), suggesting that the circulating ternary complex may indeed have some role in postnatal growth. Interestingly, as in the overexpressing ALS TG mice reported here, birth weight was normal in the ALS knockout mice.
High levels of tissue expression of ALS could potentially perturb paracrine IGF-I action if indeed IGF-I present in the ternary complex is functionally inactive. However, to our knowledge there are no published data that directly addresses this question. Indeed, IGF-I complexed to IGFBP-3 appears to be more potent than free IGF-I under many conditions (25, 26, 27, 28). ALS may simply limit the biological activity of circulating IGF-I by confining it to the intravascular space rather than inhibiting IGF-I interaction with its receptor. Careful allometry failed to demonstrate any consistent significant differences in organ weight between ALS TG and Wt mice. Of note is the fact that the brain, a tissue particularly sensitive to IGF-I levels (29, 30), was of normal size in ALS TG mice. ALS is not normally expressed in the brain (31). The normal brain size in ALS TG mice would suggest that tissue ALS overexpression does not impede the paracrine actions of IGF-I, at least in this organ. A possible explanation for this lack of effect of ALS may be the fact that IGFBP-3 and -5, the two binding proteins that can interact with ALS, are expressed in the brain at a relatively late stage in embryogenesis (32, 33).
A consistent abnormality in ALS TG was a significant reduction in litter size. IGF-I has a crucial role in ovarian follicular development. Expression of IGF-I increases in the growing follicle in the mouse (34). Similarly, IGFBP-5, which is capable of binding to ALS, is important in the survival of slow-growing and immature preantral follicles (34). There are no data for ALS expression in mouse ovarian tissue. However, in the porcine ovary ALS is present in follicular fluid and is expressed in both thecal and granulosa cells in a similar pattern as IGFBP-3 during follicular development (24). A significant reduction in litter size was observed in both ALS TG mouse strains. This effect was a maternal effect, rather than an effect of ALS expression in the developing embryo, as it was observed both in litters where all pups were TG and in mixed litters derived from mating TG females with Wt males. In contrast, no reduction in litter size was apparent when TG male mice were mated with Wt females. A similar reduction in litter size has been reported in TG mice that overexpress IGFBP-1 in the ovary, and in this case the reduction in litter size was attributable to a decrease in the number of ovulatory follicles (35). Decreased litter size was also observed in IGFBP-3 TG mice (12). Thus, it would appear that litter size might be a very sensitive marker of perturbations in the ovarian IGF system.
Although overexpression of ALS had only a modest growth effect, the combination of both ALS and IGFBP-3 overexpression resulted in a more marked reduction in body weight than overexpression of either IGFBP-3 or ALS alone. Usually there is a molar excess of ALS to IGFBP-3 in serum (1). However, in IGFBP-3 TG animals there may be insufficient endogenous ALS to allow for the full growth-attenuating effect of IGFBP-3 overexpression to be observed. In the double TG mice the increased levels of ALS together with overexpressed IGFBP-3 may allow for a larger pool of ternary complex and for the full manifestation of the effect of excess IGFBP-3.
In summary, overexpression of ALS in transgenic mice has no significant effect on the levels of major components of the circulating endocrine IGF system, but has a modest effect on growth and reproduction. Furthermore, overexpression of ALS appears to modulate the effects of concomitantly overexpressed IGFBP-3.
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Acknowledgments |
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Footnotes |
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1 Present address: Animal Health Discovery Research, Pharmacia,
Kalamazoo, Michigan 49007-4940. 
2 Recipient of a Medical Research Council Senior Scientist award and
an endowed Research Professorship in Metabolic Diseases.
Abbreviations: ALS, Acid-labile subunit; CMV, cytomegalovirus; IGFBP, IGF-binding protein; PGK, phosphoglycerate kinase; PGKBP, phosphoglycerate kinase-binding protein; RPA, ribonuclease protection assay; TG, transgenic; Wt, wild-type.
Received May 14, 2001.
Accepted for publication June 18, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3% of
the mean) have been deleted for clarity. The statistical differences
between the entire growth curves for TG and Wt mice were determined by
analysis of covariance with repeated measures, followed by Dunnett’s
t test. The inset shows the mean percent
reduction in body weight of TG mice compared with Wt mice.