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. 2009 Jun 4;150(9):4395–4403. doi: 10.1210/en.2009-0272

Elevated Levels of Insulin-Like Growth Factor (IGF)-I in Serum Rescue the Severe Growth Retardation of IGF-I Null Mice

YingJie Wu 1, Hui Sun 1, Shoshana Yakar 1, Derek LeRoith 1
PMCID: PMC2819739  PMID: 19497975

Abstract

IGF-I plays a vital role in growth and development and acts in an endocrine and an autocrine/paracrine fashion. The purpose of the current study was to clarify whether elevated levels of IGF-I in serum can rescue the severe growth retardation and organ development and function of igf-I null mice. To address that, we overexpressed a rat igf-I transgene specifically in the liver of igf-I null mice. We found that in the total absence of tissue IGF-I, elevated levels of IGF-I in serum can support normal body size at puberty and after puberty but are insufficient to fully support the female reproductive system (evident by irregular estrous cycle, impaired development of ovarian corpus luteum, reduced number of uterine glands and endometrial hypoplasia, all leading to decreased number of pregnancies and litter size). We conclude that most autocrine/paracrine actions of IGF-I that determine organ growth and function can be compensated by elevated levels of endocrine IGF-I. However, in mice, full compensatory responses are evident later in development, suggesting that autocrine/paracrine IGF-I is critical for neonatal development. Furthermore, we show that tissue IGF-I is necessary for the development of the female reproductive system and cannot be compensated by elevated levels of serum IGF-I.

Tissue IGF-1 is important for neonatal and early postnatal growth; however, superphysiological levels of IGF-1 in serum which can restore body size in both males and females in the absence of tissue IGF-1 expression, are insufficient for female reproductive function.

The GH/IGF-I axis plays a critical role in postnatal growth and development. IGF-I is produced by virtually all tissues and acts in an endocrine and autocrine/paracrine fashion. Endocrine IGF-I is secreted primarily by the liver and is tightly regulated by pituitary GH, whereas in nonhepatic tissues IGF-I is regulated by GH and tissue-specific factors. Nonetheless, the biological role of serum IGF-I has been a matter of debate for many years, and many studies have focused on addressing endocrine vs. autocrine/paracrine IGF-I functions in growth and development and various critical functions of different tissues (1) including cancer growth and metastases (2).

Mouse models with genetic ablation/mutations of different components of the GH/IGF axis, such as IGF-I (3,4,5), IGF-II (3), IGF-I receptor (4), GHRH (6), GH receptor (7,8), insulin receptor substrate-1/2 (9,10), and various IGF binding proteins (IGFBPs) (11) revealed significant alterations in skeletal development, metabolism, and reproduction. For example, IGF-I null mice exhibit severe growth retardation and smaller bones, are infertile, and die early (3,4,5). Another model of IGF-I deficiency is the GH receptor null mouse, which has blunted GH action and exhibits 90% reductions in serum IGF-I levels (8). These mice are also growth retarded and have increased body adiposity. Whereas studies of these mouse models provide significant insights into the GH/IGF system, interpretations of the observations are challenging; IGF-I and IGF-I receptor (IGF-IR) null mice exhibit increased lethality and therefore assessment of growth and metabolism in the adult age is unfeasible. Additionally, IGF-I acts in a dual mechanistic mode, and these models cannot unequivocally distinguish endocrine vs. autocrine/paracrine IGF-I actions. Addressing the latter question is critical with respect to the advent of recombinant human IGF-I therapy for multiple conditions including diabetes (12) and short stature (13,14).

We used the tissue-specific IGF-I null (KO) approach and generated a liver IGF-I-deficient (LID) mouse model using the Cre-loxP system (15). Liver-specific igf1 gene deletion abrogated expression of IGF-I in the liver and caused dramatic reductions (75%) in circulating IGF-I levels with no change in IGF-I mRNA expression in nonhepatic tissues. Serum GH levels were elevated about 4-fold in LID mice due to inadequate serum IGF-I levels to provide feedback inhibition. Surprisingly, however, the overall growth of LID mice, as determined by body weight, was not significantly different from that of control littermates. In a subsequent study, we showed that linear growth of LID mice was only mildly affected (6%); however, bone mineral density was markedly reduced compared with controls. The LID mouse model confirmed that liver is indeed the major source of circulating IGF-I and provided direct evidence for the importance of endocrine IGF-I in the development of peak bone mineral density. Nevertheless, in the LID model, there is no complete ablation of serum IGF-I, and the possibility of direct effect of (the elevated levels of) GH could not be excluded. In a consequent study, we generated double-gene deletion of the acid labile subunit (ALS; which stabilizes IGF-I in serum) together with LID (LID/ALSKO mice). The LID/ALSKO mice demonstrated a further decrease in circulating IGF-I levels and a definite reduction (30%) in body weight (16). Yet the ALSKO mice exhibited mild growth retardation (16,17), and the possibility of IGF-I-dependent or independent effects of ALS alone in extrahepatic tissues was not excluded. Using another approach to reduce serum IGF-I levels, Ning et al. (18) generated a triple knockout of IGFBP-3, -4, and -5, the main IGFBPs in serum. In that study, despite 65% decreases in serum IGF-I levels, only 22% reductions in body weight were reported. We recently generated mice, which carry liver IGF-I gene deletion (LID) and null alleles of both als and igfbp-3 genes (LAB mice) (19). LAB mice show undetectable levels of IGF-I in serum and exhibit about 30% decrease in body weight. Lastly, a study by Stratikopoulos et al. (20) also showed that endocrine IGF-I contributes approximately 30% of the adult body size. Together, these models led to the conclusion that circulating IGF-I is not a major determinant of body growth and that tissue IGF-I expression plays an important role in growth and development.

It is still unclear, however, whether in the absence of tissue IGF-I action, serum IGF-I can compensate for growth or organ development and function. To address that, we developed a mouse model, in which only the endocrine mode of IGF-I is active (i.e. mice that have a null IGF-I gene in all tissues but overexpress a rat IGF-I transgene specifically in the liver). Using this model, we show that in the total absence of tissue IGF-I, elevated levels of serum IGF-I can support normal body size at puberty and postpubertal ages. We further show that in the absence of tissue IGF-I, increases in serum levels of IGF-I are insufficient to fully support female reproductive system.

Materials and Methods

Animals

Hepatic IGF-I transgenic mice were generated on FVB/N genetic background. These mice were crossed with heterozygous IGF-I+/− mice that were backcrossed 13 generations to FVB/N genetic background. Mice were housed four per cage in a clean mouse facility and fed a standard mouse chow (Purina Laboratory Chow 5001; Purina Mills, St. Louis, MO) and water ad libitum and kept on a 12-h light, 12-h dark cycle. Female mice that were used for determination of the different states of estrous cycle were housed separately in distance from male cages on a ventilated rack. Animal care and maintenance were provided through the National Institutes of Health and Mount Sinai School of Medicine Association Assessment and Accreditation of Laboratory Animal Care-accredited animal facility. All procedures were approved by the Animal Care and Use Committee of Mount Sinai School of Medicine and the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD. The crossing strategy is presented in Fig. 1.

Figure 1.

Figure 1

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Generation of the KO-HIT mice. A, Schematic representation of the transgenic construct used to drive liver expression of the rat transgene under the TTR promoter. R-IGFI, Rat IGF-I. B, The crossing strategy used to generate the KO-HIT mice. Briefly, male and female heterozygous mice for the endogenous gene harboring the rat IGF-I transgene (+/−HIT) were crossed and gave birth to control (+/+), HIT (+/+HIT), IGF-I null (−/−), and KO-HIT (−/−HIT) mice. C, The rat IGF-I transgene expression restricted to liver. RNA was extracted from different tissues, transcribed into cDNA, and amplified by PCR. The rat IGF-I transgene (RnIGF-I) is evident in livers of HIT and KO-HIT mice only, whereas the endogenous mouse IGF-I (MmIGF-I) is present in all tissues of all mice except the KO-HIT mice. D, Hepatic expression of als, igfbp-3, ghr, and insulin receptor genes, assessed by RT-PCR (n = 3/group). E, Real-time PCR analyses (n = 5–6/group). BP3, IGFBP3. *, Significance was determined at P < 0.05.

Determination of serum hormones

Mice were bled through the mandibular vein and serum samples were collected at the indicated ages. Serum IGF-I (with sensitivity of 1 ng/ml), GH (with sensitivity of 0.07 ng/ml), and insulin (with sensitivity of 0.02 ng/ml) levels were determined using commercial RIAs as previously described (19). IGFBP-3 levels were determined using ELISAs, developed at the University of California, Los Angeles, (Los Angeles, CA) using recombinant mouse proteins from R&D Systems (Minneapolis, MN) and monoclonal antibodies as previously described (11).

Determination of serum levels of ALS

Sera were diluted 1:4 with saline and subsequently separated on 4–12% acrylamide gel and transferred to nitrocellulose. The membrane was blocked with block solution (LI-COR, Lincoln, NE) and incubated overnight at 4 C with a goat anti-ALS antibody (R&D Systems). After washing, the membrane was incubated with IRDye800CW-donkey antigoat antibody (LI-COR). Bound antibodies were detected by Odyssey infrared imaging system (LI-COR).

Determination of body composition

Body adiposity was measured using a Bruker minispec nuclear magnetic resonance analyzer mq 10 in nonanesthetized mice (Bruker Optics, The Woodlands, TX).

Determination of bone and body lengths

Body length was measured from nose to anus of anesthetized mice. Femur length was assessed by microcomputed tomography images as described elsewhere (19).

Glucose tolerance test (GTT)

Intraperitoneal GTT was performed after overnight fasting by administering 20% glucose (2 g/kg) to mice. Blood glucose was measured using a Glucometer Elite (Bayer, Elkhart, IN) at the indicated time points.

Gene expression studies

Total RNA from tissues was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA). RNA integrity was verified using a bioanalyzer (2100 Bioanalyzer-Bio Sizing, version A.02.12 SI292; Agilent Technologies, Palo Alto, CA). One microgram of RNA was reverse transcribed to cDNA using oligo(dT) primers with a RT-PCR kit according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA). Real-time PCR was performed with the QuantiTect SYBR green PCR kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions in ABI PRISM 7900HT sequence detection systems (Applied Biosystems, Foster City, CA). Each transcript in each sample was assayed three times, and the fold change ratios between experimental and control samples for each gene used in the analysis were calculated using β-actin or glucose-3-phosphate dehydrogenase (G3DPH) levels as a reference.

Sequences of primers used for real-time PCR are available on request

Vaginal smear assay

Estrous cycle was evaluated in 6- to 8-wk-old virgin females according to Champlin et al. (21) housed together three to five per cage. Vaginal smears were performed daily over 10 d during the morning. Specimens were collected in 50 μl saline, smeared on glass slides, stained with Giemsa solution, and viewed under the microscope.

Superovulation and oocyte retrieval

Six-week-old virgins female mice were injected ip with 5 IU pregnant mare serum gonadotropin (PMSG; Sigma Corp. of America, St. Louis, MO) and 5 IU of human chorionic gonadotropin (hCG; Sigma) 48 h later. Oviducts were dissected 14 h after hCG injection and placed in petri dishes containing M2 medium (Sigma) supplemented with 5% (vol/vol) heat-inactivated fetal bovine serum (Invitrogen). Mouse oocytes were collected and counted.

Statistical analysis

All differences in mean serum hormones, organ weights, body composition, and growth among the different groups were assessed by ANOVA. Values are presented as mean ± se. P < 0.05 was considered statistically significant.

Results

Generation of mice with elevated serum IGF-I levels and total ablation of tissue IGF-I

Hepatic IGF-I transgenic (HIT) mice express the rat IGF-I transgene under the transthyretin (TTR) promoter, specifically in liver (Fig. 1A). HIT mice were crossed with IGF-I+/− heterozygous mice to generate IGF-I +/− mice harboring the hepatic rat IGF-I transgene (Fig. 1B, +/−HIT). The latter mice were crossed to generate IGF-I null (KO) mice expressing the hepatic rat IGF-I transgene (Fig. 1B, −/−HIT), namely KO-HIT mice. This crossing strategy gave rise to four groups of mice with similar genetic backgrounds: control mice that express normal tissue and circulating IGF-I; HIT mice that express normal tissue IGF-I but have increased serum IGF-I levels; IGF-I null mice; and KO-HIT mice. KO-HIT mice have no tissue IGF-I but exhibit increased serum IGF-I levels. Note that whereas IGF-I null mice were lethal at birth and were not born at the expected ratios, the KO-HIT mice (that resulted from the crossing of IGF-I +/− mice harboring the hepatic rat IGF-I transgene, i.e. +/−HIT) were born at the expected ratios (∼18%) (data not shown), suggesting no prenatal lethality.

Elevated levels of serum IGF-I do not alter liver expression of igfbp-3 or als genes

Gene expression of endogenous and transgenic IGF-I was examined in liver, muscle, fat, spleen, heart, kidney, brain, ovary, and uterus. As shown in Fig. 1C, endogenous IGF-I gene expression was detected in all tissues of control and HIT mice but was not observed in IGF-I null or KO-HIT mice. In contrast, the rat IGF-I transgene was expressed in livers of HIT and KO-HIT mice only. The TTR promoter, which drives the rat IGF-I transgene, is turned on early during embryogenesis (22,23,24). We studied liver expression of the TTR-IGF-I transgene as early as postnatal d 1 and were able to detect the rat-IGF-I mRNA (data not shown). Expression of the rat IGF-I transgene did not alter hepatic histomorphology, as depicted by hematoxylin and eosin (H&E) staining (supplemental Fig. S1).

Upon IGF-I secretion by hepatocytes, IGFBP-3 and the ALS form ternary or binary complexes to stabilize IGF-I in circulation (11,25,26). To study the effect of hepatic IGF-I transgene on the expression of the two major binding proteins of circulating IGF-I, namely IGFBP-3 and ALS, we analyzed hepatic gene expression of these genes (Fig. 1, D and E) using RT-PCR and real-time PCR. Interestingly, there was no difference in als or igfbp-3 gene expression in livers of 16-wk-old mice among HIT, KO-HIT, and control mice. Serum levels of IGFBP-3 (Fig. 2A) were similar in all groups from 8 wk on. Serum levels of ALS (Fig. 2B) did not differ significantly between the groups. Additionally, liver expression of the insulin receptor did not differ between the groups. Unexpectedly, liver (Fig. 1, D and E) and kidney (data not shown) expression of the GH receptor (GHR) increased about 2-fold in HIT and KO-HIT mice. Note that expression data in tissues of IGF-I null (KO) mice were not feasible due to early lethality of these mice.

Figure 2.

Figure 2

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Serum IGF-I levels are increased in HIT and KO-HIT mice. A, IGFBP-3 levels were measured as described in Materials and Methods at the indicated ages (n = 8 mice/group per age). B, Western immunoblotting was used to determine serum ALS levels in 16-wk-old male and female mice (n = 6 mice/genotype) serum from ALSKO served as negative control. Serum IGF-I (n = 10 mice/group) (C) and GH levels (D) were measured as described in Materials and Methods at 8 wk of age. E, Pituitary gene expression (in 4 wk old male mice) of GH (GenBank no. NM_008117), prolactin (Prl, NM_011164), thyroid stimulating hormone (Tshb, NM_009432), ACTH (Acth/Pomc, NM_008895), FSH (Fshb, NM_008045), LH (Lhb, NM-008497) and G3DPH (G3dph, NM_008084), which served as reference control (n = 6 mice/group). *, Significance was determined at P < 0.05.

Elevated serum IGF-I levels do not change GH gene expression in the pituitary

Expression of the hepatic IGF-I transgene in HIT and KO-HIT mice is driven by the TTR promoter and therefore is not regulated by pituitary GH secretion. As revealed in Fig. 2C, circulating IGF-I levels were increased about 2.5-fold in HIT and KO-HIT mice compared with controls in both male and female. Surprisingly, the elevated IGF-I levels did not inhibit GH secretion such that serum GH levels did not differ significantly between control, HIT, and KO-HIT male mice between 4 and 16 wk of age (Fig. 2D). Of importance is that GH is secreted in a pulsatile manner, and therefore, modest differences would be hard to detect. Gene expression studies in pituitary extracts from the various groups (at 4 wk of age) showed no difference in mRNA levels of GH, prolactin (Prl), thyroid-stimulating hormone (Tshb), ACTH hormone (Acth/Pomc), FSH (Fshb), LH (Lhb), and G3DPH (G3dph), which served as reference control (Fig. 2E).

Elevated levels of serum IGF-I support normal body size in the absence of tissue IGF-I

IGF-I null mice exhibit severe growth retardation and commonly die soon after birth (3,4,5). Importantly, elevated concentrations of endocrine IGF-I was able to rescue the severe growth-retarded phenotype observed in the IGF-I null mice. Both male and female HIT mice showed an increase in body weight starting at 2 wk of age (Fig. 3, A and B) that persisted throughout 16 wk (Fig. 3, C and D). In contrast, despite the 3-fold increase in serum IGF-I levels, the body weight of KO-HIT mice was similar to that of control mice from 2 to 16 wk of age (Fig. 3, C and D). Female KO-HIT mice were born smaller and normalized their body weight at 2 wk of age (Fig. 3, A and B). This suggests that IGF-I-stimulated increased growth at early postnatal age requires both endocrine and autocrine/paracrine IGF-I functions (Fig. 3, A and B).

Figure 3.

Figure 3

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Body weight during growth and development. A and B, Body weight of male and female mice from 1 to 3 wk of age. C and D, Body weight of male and female mice from 3 to 16 wk of age. Data on IGF-I null mice were derived from our previous study (48). *, Significance was determined at P < 0.05.

Body length (from nose to anus) was used to assess linear growth in both males and females (Fig. 4, A and B). Female HIT mice exhibit increased body length at 4 wk of age. In contrast, the body length of KO-HIT mice (both genders) was reduced at 4 wk but normalized later on during growth, suggesting that tissue IGF-I plays a role pre- and early postnatally. At 16 wk of age, femur length of HIT and KO-HIT male and female mice did not differ significantly from controls, despite the 2.5-fold increase in serum IGF-I levels (Fig. 4, C and D).

Figure 4.

Figure 4

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Body length during growth. A and B, Body length of male and female mice from 4 to 16 wk of age (measured from nose to anus of anesthetized mice). C and D, Femoral lengths of male and female mice at 16 wk of age measured from microcomputed tomography images of femurs. *, Significance was determined at P < 0.05.

Similar to the previous IGF-I transgenic models (27,28), which reported organomegaly, HIT mice show a 25–35% increase in relative weights of liver, kidney, and spleen (supplemental Table S1). Spleen weight was increased in HIT and KO-HIT mice, most likely due to the increased circulating IGF-I levels. Liver weight increase in HIT mice could have been caused by the increase in the expression of GHR.

Elevated levels of serum IGF-I do not alter insulin sensitivity

We first examined the consequences of increased serum IGF-I on body composition. Using nuclear magnetic resonance, we evaluated lean and fat mass in control, HIT, and KO-HIT mice at 4, 8, and 16 wk of age. As shown in Fig. 5, A and B, percent body adiposity was decreased in male mice harboring the hepatic IGF-I transgene (HIT and KO-HIT mice) at 4 and 8 wk but reached control levels at 16 wk of age. Similarly, gonadal fat pad mass was significantly decreased in HIT and KO-HIT male mice compared with controls (supplemental Table S1). In females we observed a similar decrease in body adiposity at 4 and 8 wk in the HIT mice. However, KO-HIT females displayed decreased body adiposity at 4 wk but not at 8 wk of age (Fig. 5B). Serum insulin levels in the fed state in HIT and KO-HIT mice tended to reduce compared with controls but remained within normal range (Fig. 5C). GTT, performed in 16-wk-old mice of both genders, showed normal fasting glucose (at 0 time point) and normal glucose clearance (Fig. 5D).

Figure 5.

Figure 5

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Increased serum IGF-I levels do not affect insulin sensitivity. Body adiposity was assessed in both males (A) and females (B) using magnetic resonance imaging (n = at least 10 per each time point per each genotype). BW, Body weight. C, Serum insulin levels (n = 8/group per age). D, Intraperitoneal GTT. *, Significance was determined at P < 0.05.

Tissue IGF-I is necessary to support normal female reproductive system

Mating KO-HIT males with control females yielded 100% pregnancies with normal litter size (supplemental Table S2). Histological examination of the male reproductive organs revealed no differences from control mice (data not shown). In sharp contrast, mating the KO-HIT female mice was largely unsuccessful. KO-HIT females crossed with control males did not yield litters and crosses of KO-HIT females with KO-HIT males led to 20% KO-HIT pregnancies (supplemental Table S2), leading to a litter size of about one third of control mice. The difference between the two crosses may result from the differences in the number of mating pairs. Detailed examination of the female reproductive system showed irregular estrous cycle in the KO-HIT mice. Daily vaginal smears were taken from females housed three to five per cage; a total of eight mice per genotype was analyzed. Our data revealed that in control and HIT mice, diestrus and proestrus states last 1–2 d and estrus 2–3 d. In contrast, KO-HIT mice show diestrus and proestrus of 3–7 d and estrus 0–1 d. These data are consistent with reduced number of pregnancies in KO-HIT females (even when housed with males, avoiding pheromonal influences).

To test whether ovaries of KO-HIT mice respond to exogenous hormone stimulation, we injected PMSG and hCG followed by oocyte washings from the oviducts. We found that young HIT and KO-HIT mice (4 wk old) responded normally to exogenous hormone stimulation, and the number of eggs washed from the oviducts was significantly higher than that of control mice (Fig. 6A). This suggests that ovarian IGF-I is not obligatory for follicular development, a morphological observation previously made in the IGF-I null females (29). It is important to note that the number of retrieved follicles is not reflective of follicular function. Histological examination of adult KO-HIT mice revealed ovarian hypoplasia (Fig. 6B, 1–3) with hypoplastic corpora lutea and very few mature graafian follicles. Furthermore, the uterus of KO-HIT female mice exhibits endometrial hypoplasia, little glandular development, and immature epithelium (Fig. 6B, 4–6). Despite increased endocrine IGF-I levels, KO-HIT mice display marked myometrial hypoplasia in myometrial layers. IGF-I null females exhibit underdeveloped mammary glands with little branching and very few terminal end buds compared with controls (30). Whole-mount staining of mammary glands from HIT and KO-HIT mice indicated increased ductal branching (Fig. 6B, 7–9). Cluster sites of branch initiations can be seen toward the terminal end buds in the KO-HIT mice, suggesting that elevated levels of serum IGF-I are sufficient to induce mammary ductal formation. Taken together, our results suggest that the main reason for reduced fertility in the absence of ovarian IGF-I is estrogen insufficiency, which leads to impaired estrous cycle, hypoplastic ovaries and uterus.

Figure 6.

Figure 6

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Endocrine IGF-I was not sufficient to rescue infertility of KO-HIT females. A, Number of eggs washed from the oviducts 48 h after ip hormone stimulation with 5 IU PMSG and 5 IU hCG. B, H&E staining of ovaries (one to three) dissected from 16-wk-old virgin control, HIT, and KO-HIT mice. Corpora lutea (CL) and graafian follicles (GF) are marked with arrows. H&E staining of uterus (four to six) dissected from 16-wk-old virgin control, HIT, and KO-HIT mice. Uterine glands are marked with arrows; myometrium layer is marked with I. Whole-mount staining of mammary glands (seven to nine) isolated from 16-wk-old control, HIT, and KO-HIT mice. Clustered sites of branch initiations toward the terminal end of ducts in the KO-HIT are marked with arrows. Scale bar, 100 μm. *, Significance was determined at P < 0.05.

Discussion

In the present study, we evaluated body growth and organ function in mice with abrogated tissue IGF-I gene expression but elevated levels of serum IGF-I. We created a transgenic mouse model on an IGF-I null background that exclusively expresses IGF-I in the liver and thereby delivers IGF-I by the endocrine route only (namely KO-HIT mice). Unlike IGF-I null mice, which exhibit severe growth retardation and metabolic abnormalities, adult KO-HIT mice exhibited normal body weight and carbohydrate metabolism. We found that tissue IGF-I is fundamental for early postnatal growth but a catch-up growth is evident in KO-HIT mice at pubertal age. Furthermore, in the absence of tissue IGF-I, elevated levels of serum IGF-I were insufficient to sustain normal function of female reproductive organs, evident by irregular estrous cycle, impaired development of ovarian corpus luteum, reduced number of uterine gland, and development of endometrial hypoplasia, all leading to decreased number of pregnancies and litter size.

Surprisingly, we found that serum levels of GH in both HIT and KO-HIT mice did not differ significantly from control levels, despite approximately 2.5-fold elevations in serum IGF-I. These data are in line with the fat-specific IGF-IR knockout mice, in which serum IGF-I levels were increased approximately 2-fold, with no change in serum GH levels (31). Moreover, the normal levels of GH in serum of HIT and KO-HIT mice are consistent with normal igfbp-3 and als gene expression in liver. These findings possibly point to local IGF-I action that may be important for the regulation of GH secretion.

Human and animal studies show unequivocally that both tissue and serum IGF-I are necessary to support normal growth and organ function. Children who are born with GH resistance (Laron’s dwarfs) show both serum and tissue IGF-I insufficiency, resulting in short stature, increased body adiposity, poor muscle mass, and delayed bone age (32). High concentrations of recombinant human IGF-I are effective in promoting growth of those children (33,34,35,36,37); however, their growth response is less than that of severely GH-deficient children treated with GH, most likely due to stimulation of both serum and tissue IGF-I production (38).

To understand the contribution of endocrine IGF-I to body size, Stratikopoulos et al. (20) took a genetic approach and clearly showed that activation of the IGF-I gene exclusively in liver of IGF-I null mice restored 44% of serum IGF-I levels. Accordingly, body weight was increased to approximately 50% of wild-type controls, suggesting a linear relationship between serum IGF-I and body size. In the present study, we took a different approach, whereby we overexpressed the IGF-I transgene specifically in livers of IGF-I null mice (the KO-HIT mice). Characterization of the KO-HIT mice revealed that despite approximately 2.5-fold increase in serum IGF-I levels, KO-HIT mice were born smaller than controls. However, in contrast to the model presented by Stratikopoulos, both KO-HIT genders exhibited a catch up growth, and at 16 wk of age, their body length and body weight were indistinguishable from controls. Our data imply that tissue IGF-I production is important for early neonatal growth and that postnatally, increased serum IGF-I concentrations can compensate linear growth. Yet KO-HIT mice show increases in liver expression of GHR, and therefore, we cannot exclude the possibility of increased GHR in other tissues and potential direct effects of GH on growth in those mice. It is important to note that a similar approach of overexpressing IGF-II under the phosphoenolpyruvate carboxykinase promoter, leading to increased serum IGF-II levels, on an IGF-I null background failed to rescue the dwarfism of IGF-I null mice (39).

The development and maturation of both male and female reproductive systems are regulated by gonadotropins, sex steroids, and members of the GH/IGF axis. IGFs and the IGF-IR are widely expressed in ovary, uterus, mammary, and testis (40,41,42). In rodents, ovarian IGF-IR is up-regulated at puberty (∼3–4 wk of age, as determined by vaginal opening), at which time FSH peaks in serum. During follicular development, granulose cells and mature oocytes express the IGF-IR gene. Morphological characterization of follicular development in the IGF-I null mice revealed that IGF-I is not necessary for that process (29). Nonetheless, detailed studies show that the ovarian follicles of the IGF-I null mice are arrested at an early stage of development and that that IGF-I is essential for normal basal and estrogen-induced granulosa cell proliferation and follicular growth (43). Restoring the liver IGF-I gene expression in livers of the IGF-I null mice reestablish only 44% of serum IGF-I with normal fertility of both male and female mice (20). However, in this study only three females were evaluated and the female reproduction system was not addressed in detail. In our study, histological examinations of ovaries from KO-HIT mice show a decrease in mature follicles, which may result from gonadotropin insufficiency, decreased ovarian responsiveness to gonadotropins, or estrogen insufficiency. However, pituitary expression of gonadotropins in KO-HIT mice (at 4 wk of age) was similar to controls. Furthermore, ovarian response to exogenous hormones was even greater than controls, implying that in the KO-HIT mice, the absence of ovarian IGF-I is the major cause for KO-HIT female subfertility. In uterus, the IGF-IR is expressed in both myometrial layers and is up-regulated during proestrus in rodents (44,45,46). Estrogens stimulate uterine expression of IGF-I and IGF-IR (47). Uterus of KO-HIT mice exhibits severe hypoplasia with marked decrease in uteral glands. These findings strongly suggest that uterus expression of IGF-I is necessary for glandular development and cannot be compensated by elevated levels of serum IGF-I.

Finally, examination of the metabolic consequences of increased serum IGF-I levels in the presence (HIT) or absence (KO-HIT) of tissue IGF-I revealed normal response to glucose load, normal levels of fasted and fed glucose, and normal levels of serum insulin. Interestingly, body adiposity of both HIT and KO-HIT mice decreased dramatically between 4 and 8 wk of age and normalized thereafter.

In summary, using the KO-HIT model, we demonstrate that most autocrine/paracrine actions of IGF-I that determine organ growth and function can be compensated by elevated levels of endocrine IGF-I. However, in mice, full compensatory responses are evident later in development, suggesting that autocrine/paracrine IGF-I is critical for neonatal development. Furthermore, we show that tissue IGF-I is necessary for the development of female reproductive system and cannot be compensated by elevated levels of serum IGF-I.

Supplementary Material

[Supplemental Data]
en.2009-0272_index.html (1KB, html)

Acknowledgments

We thank Dr. Terry Van Dyke (Department of Genetics and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC) for the TTR1 promoter. We also thank Dr. Clifford Rosen (Maine Medical Center, Research Institute, Scarborough, ME) for measurements of serum IGF-I, Dr. Pinchas Cohen (Division of Pediatric Endocrinology, Mattel Hospital for Children, David Geffen School of Medicine, Los Angeles, CA) for measuring serum IGFBP-3 levels, and Dr. John J. Kopchick (School of Human and Consumer Sciences, Ohio University, Athens, Ohio) for measuring serum GH levels.

Footnotes

This work was supported by the National Institutes of Health Grants AR054919 (to S.Y.), AR055141 (to S.Y.), and 1R01CA128799 (to D.L.).

Disclosure Summary: The authors have nothing to declare.

First Published Online June 4, 2009

Abbreviations: ALS, Acid labile subunit; G3DPH, glucose-3-phosphate dehydrogenase; GHR, GH receptor; GTT, glucose tolerance test; hCG, human chorionic gonadotropin; H&E, hematoxylin and eosin; HIT, hepatic IGF-I transgenic; IGFBP, IGF binding protein; IGF-IR, IGF-I receptor; KO, IGF-I null; LID, liver IGF-I-deficient; LID/ALSKO, ALS together with LID; PMSG, pregnant mare serum gonadotropin; TTR, transthyretin.

References

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