An experimental model of partial insulin-like growth factor-1 deficiency in mice
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- Castilla-Cortazar, I., Guerra, L., Puche, J.E. et al. J Physiol Biochem (2014) 70: 129. doi:10.1007/s13105-013-0287-y
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Insulin-like growth factor-1 (IGF-1) is responsible for many systemic growth hormone (GH) functions although it has an extensive number of inherent activities (anabolic, cytoprotective, and anti-inflammatory). The potential options for IGF-1 therapy arise as a promising strategy in a wide list of human diseases. However, deeper studies are needed from a suitable animal model. All human conditions of IGF-1 deficiency consist in partially decreased IGF-1 levels since total absence of this hormone is hardly compatible with life. The aim of this work was to confirm that heterozygous Igf-1+/− mice (Hz) may be considered as an appropriate animal model to study conditions of IGF-1 deficiency, focusing on early ages. Heterozygous Igf-1+/− mice were compared to homozygous Igf-1+/+ by assessing gene expression by quantitative PCR, serum circulating levels by ELISA, and tissue staining. Compared to controls, Hz mice (25 days old) showed a partial but significant reduction of IGF-1 circulating levels, correlating with a reduced body weight and diminished serum IGFBP-3 levels. Hz mice presented a significant decrease of IGF-1 gene expression in related organs (liver, bone, testicles, and brain) while IGF-1 receptor showed a normal expression. However, gene expression of growth hormone receptor (GHR) was increased in the liver but reduced in the bone, testicles, and brain. In addition, a significant reduction of cortical bone thickness and histopathological alterations in the testicles were found in Hz mice when compared to controls. Finally, the lifelong evolution of IGF-1 serum levels showed significant differences throughout life until aging in mice. Results in this paper provide evidence for considering heterozygous mice as a suitable experimental model, from early stages, to get more insight into the mechanisms of the beneficial actions induced by IGF-1 replacement therapy.
KeywordsIGF-1IGF-1 receptorGH receptorLiver cirrhosisLaron syndromeGH/IGF-1 axisIGF-1 deficiencyIGFBP-3Gene expressionAging
Control group (wild-type mice)
Enzyme-linked immunosorbent assay
Glutamate–cysteine ligase catalytic subunit
Growth hormone receptor
Heat shock protein 1B
Insulin-like growth factor-1
Insulin-like growth factor binding protein
Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone produced mainly by the liver in response to the endocrine growth hormone (GH) stimulus, although it is also secreted by multiple tissues for autocrine/paracrine purposes  and it is tightly maintained in a close physiological range, mainly by IGFBP-3 (which binds ~90 % of circulating IGF-I) . IGF-1 is partly responsible for systemic GH activities although it possesses a wide number of its own properties (anabolic, antioxidant, anti-inflammatory, and cytoprotective activities) [5, 9, 17, 24, 39].
IGF-1 deficiency conditions (as it is the case with every other hormone) produce effects that culminate in recognizable syndromes with significant clinical consequences. Until now, the best known conditions of IGF-1 deficiency are  Laron syndrome in children ; advanced liver cirrhosis in adults [9, 11, 12, 20, 28, 30, 34, 36, 37]; aging, including cardiovascular and neurological diseases associated to aging [2, 6, 15, 16, 21, 39, 46, 48, 51]; and more recently, intrauterine growth restriction [1, 42, 49]. In these conditions, replacement therapy can logically induce beneficial actions.
In addition, many other diseases, or their complications, are being proposed as a consequence of either a global or local IGF-1 deficiency [18, 22, 29, 31, 38, 41, 45]. However, deeper studies are needed to properly characterize these possible new conditions of IGF-1 deficiency. Consequently, the potential options for IGF-1 therapy arise as a promising strategy in a wide list of diseases with evident translational relevance.
For the last decades, our team has been working on some IGF-1 deficiency states, such as chronic liver disease and aging, studying the beneficial effects of IGF-1 replacement therapy with low doses of IGF-1 on these conditions [8–12, 14, 20, 24, 28, 30, 34, 36, 39, 47]. However, many of the mechanisms underlying the described effects (neuro- and hepatoprotection, mitochondrial function recovery, antioxidant actions, glucose and lipid metabolism restoration, etc.) are not fully understood.
In this context, an animal model of IGF-1 null mice (Igf-1−/−) has been used to get more insight into the physiological properties of IGF-1 [5, 25]. However, since all human conditions of IGF-1 deficiency consist in partially decreased IGF-1 levels due to the fact that total or severe absence of this hormone is barely compatible with life and development [25, 33], we suggested the hypothesis that heterozygous Igf-1+/− mice could be considered a more suitable animal model to mimic recognizable syndromes associated to human conditions of IGF-1 deficiency. For this reason, the aim of the present work was to confirm this hypothesis in early animal ages by measuring key parameters related to IGF-1 system and function, both in control wild-type mice Igf-1+/+ (CO) and Igf-1+/− (Hz) mice, such as (1) body and organ weights; (2) circulating levels of IGF-1 and IGFBP-3; (3) gene expression of IGF-1, IGFBP-1, IGFBP-3, and GH receptor (GHR) in the liver (the main source of this hormone); (4) gene expression of IGF-1, IGF-1 receptor, and GH receptor in the main target tissues (bone, brain, and testicle); (5) histopathological analysis of the bone and testicle; and (6) expression of genes recently reported as involved in oxidative stress response during IGF-1 deficiency in the liver: heat shock protein 1B (Hspa1b), catalase (Cat), and glutamate–cysteine ligase catalytic subunit (Gclc) [3, 44]. Finally, a study about the lifelong evolution of IGF-1 circulating levels and animal weight (from both CO and Hz mice) was performed to justify the use of this model as a proper tool for future studies about IGF-1 deficiency conditions.
Material and methods
Animals and experimental design
Heterozygous MF1 Igf1+/− mice (Mus musculus) were kindly provided by Dr. Argiris Efstratiadis. They were obtained by gene disruption as previously reported . Briefly, a vector was employed to target exon 4 of the Igf1 gene. Transfected countercurrent centrifugal elutriated embryonic stem cells were selected in vitro (by Neomicine) for Igf1 disrupted gene and then injected to blastocoels from naturally mated MF1 females. Finally, groups of 6–12 blastocysts were transferred into pseudopregnant females.
For genotyping of mice by reverse transcription PCR (RT-PCR) analysis, DNA was extracted from a piece of tail as previously described  and specific primers were used to identify both Igf1 and Neo genes (Neo sense: 5′-CCAGCTCTTCAGCA-ATATCACGGG-3′and antisense: 5′-CCTGTCCGGTGCCCTGAATGAACT-3′; Igf1 sense: 5′-GACTCGATTTCACCCACTCGATCG-3′and antisense: 5′-GTCTAACACCAGCCCATTCTGATT-3′).
Heterozygous MF1 Igf1+/− mice were mated with 129sv wild-type mice for colony establishment. Then, the resulting Hz mice were crossed to obtain a proper number of heterozygous Igf1+/− mice for further experiments: 54.8 % wild-type, 43.7 % heterozygous, and 1.5 % knockout.
Both food (Teklad Global 18 % Protein Rodent Diet, Harlan Laboratories, Spain) and water were given ad libitum. Mice were housed in cages placed in a room with a 12-h light/12-h dark cycle, and constant humidity (50–55 %) and temperature (20–22 °C). All experimental procedures were performed in compliance with The Guiding Principles for Research Involving Animals  and approved by the Bioethical Committee from the University CEU San Pablo (Madrid, Spain).
Two groups of 25-day-old male mice were included in the experimental protocol: control group of wild-type animals Igf1+/+ and Igf1+/− animals with heterozygous IGF-1 expression, n = 10–15 each group (depending on the assay). Initially, an additional group of knockout (KO) animals Igf−/− (n = 5, same age) was used to compare IGF-1 circulating levels and body weights vs the CO and Hz groups. Additional series of both control (Igf1+/+) and Hz (Igf1+/−) mice were used to determine IGF-1 circulating levels and body weight at different ages (0.5, 1, 2, 3, 6, 10, 16, 19, and 23 months old; a minimum of five animals for each point).
After animal weighing, blood was obtained from the submandibular vein with capillary tubes (70 mm; Laboroptik, Germany), centrifuged (2,500×g, 15 min), aliquoted, and stored at −20 °C until used. The animals were then sacrificed by cervical dislocation. The liver, testicle, bone (femur and tibia), and brain were dissected, weighted, divided, and stored separately at −80 °C after immersion in liquid nitrogen for protein determinations and at −20 °C in RNA later (Ambion Inc., UK) for genetic analyses; and liver, testicle, and femur specimens were immersed in 4 % paraformaldehyde at room temperature for immunohistochemistry purposes.
Analytical methods in serum
Serum levels of IGF-1 and IGFBP-3 were assessed by ELISA in a Varioskan spectrophotometer (Thermo Scientific, Finland), using specific commercial assay systems following protocol instructions (Chiron Corporation, USA). Transaminases (alanine aminotransferase and aspartate aminotransferase), alkaline phosphatase, total bilirubin, albumin, and total proteins were determined in serum by routine laboratory methods using an autoanalyzer (Hitachi-Cobas Integra 400 plus, Roche Diagnostics, Spain).
Liver, testicle, and femur specimens were fixed in 4 % paraformaldehyde (and then transferred to 70 % ethanol) and embedded in paraffin. Tissue sections (4-μm-thick) were stained with hematoxylin–eosin and observed by optic microscopy (Leica DFC425, Switzerland). Changes in bone cortical thickness and testicular structure were evaluated. A histopathological score was assessed in the testicles by a simple semiquantitative index of damage attending to loss of germinal line (0 to 4 points), germinal epithelium degradation (0 to 4 points), and presence of aberrant cells inside the lumen of the seminiferous tubules (0 to 2 points), by measuring ten tubules per field in the whole preparation.
Total RNA extraction, RT-PCR, and qPCR
Cryopreserved tissues were homogenized in TRIzol reagent (Invitrogen, UK), and RNA was extracted and further purified using the Qiagen RNeasy Mini Kit including digestion with RNase-free DNase I following the manufacturer's instructions . RNA quality was checked using the A260:A280 ratio and with Bioanalyzer 2100 (Agilent Technologies Inc., USA). Purified RNA was then converted to cDNA by using the RNA-to-DNA EcoDryTM Premix (Clonetech Labs, USA) for quantitative real time PCR (qPCR) assays.
qPCR assays were performed in a 3100 Avant Genetic Analyzer (Applied Biosystems Hispania, Spain) based on the manufacturer's instructions with specific Taqman® probes for IGF-1, IGF-1 receptor, GH receptor, and other genes recently related to oxidative response in IGF-1 deficiency, such as Hsp1b, Cat, and Gclc [3, 44]. The thermal profile consisted in an initial 5-min melting step at 95 °C followed by 40 cycles at 95 °C for 10 s and 60 °C for 60 s. The relative mRNA levels of the genes of interest were normalized to 18S expression using the simplified comparative threshold cycle delta, cycle threshold (CT) method (2−(ΔCT gene of interest − ΔCT actin)).
Unless otherwise stated, all data represent mean ± SEM. Statistical analysis was performed on SPSS 17 (Statistical Package for Social Sciences, USA). Significance was estimated using Student's t test or, when appropriate, by analysis of variance (ANOVA). Differences were considered significant at a level of p < 0.05.
IGF-1 serum levels, body and IGF-1 key organs weights, and lifelong evolution of IGF-1 circulating levels and body weights
Weights of the bone (femur), liver, testicle, and brain from control wild-type and heterozygous mice
(n = 15)
(n = 15)
Femur weight (mg)
33.00 ± 2.00
25.50 ± 2.00*
Femur (g/100 g bw)
0.28 ± 0.02
0.29 ± 0.02
Liver weight (mg)
645.00 ± 41.00
523.00 ± 43.00*
Liver (g/100 g bw)
5.45 ± 0.04
6.10 ± 0.16*
Testicle weight (mg)
10.50 ± 0.50
8.00 ± 0.30*
Testicle (g/100 g bw)
0.09 ± 0.05
0.09 ± 0.07
Brain weight (mg)
435.50 ± 21.00
359.00 ± 24.00*
Brain (g/100 g bw)
3.66 ± 0.35
4.22 ± 0.36
On the other hand, IGF-1-deficient mice showed a decrease of IGFBP-3 levels, the main regulator of circulating IGF-1 bioavailability (CO 3,495.64 ± 342.77 ng/mL vs Hz 1,480.37 ± 226.07 ng/mL, p < 0.05). A significant correlation between IGF-1 and IGFBP-3 serum levels was also found (Fig. 1d, r = 0.866, p < 0.01).
An additional study to determine the lifelong evolution of IGF-1 circulating levels was performed to elucidate if the initial IGF-1 deficiency in Hz mice (25 days old) was either restored or maintained throughout the different ages (see Fig. 2a). A significant and persistent decrease of IGF-1 serum levels was observed in Hz mice until aging (23 months old) compared to controls.
In parallel, body weights from the same animals were assessed to establish a time-dependent curve. Figure 2b shows how from early stages, the previously described IGF-1 deficiency correlates with lower body weights (Hz group) when compared to CO mice, with the only exception of the older mice (23 weeks old).
Analytical serum parameters
Analytical parameters of liver function, cholestasis, and cytolysis from control wild-type and heterozygous mice
(n = 10)
(n = 10)
Alanine aminotransferase (ALT) (U/L)
18.60 ± 3.95
16.10 ± 2.97
Aspartate aminotransferase (AST) (U/L)
43.76 ± 15.67
57.99 ± 11.89
Alkaline phosphatase (U/L)
32.86 ± 8.25
30.25 ± 5.04
Total bilirubin (mg/dL)
0.11 ± 0.01
0.09 ± 0.01
195.03 ± 14.52
208.57 ± 8.75
3.80 ± 0.13
3.45 ± 0.10
Total proteins (g/dL)
5.49 ± 0.18
5.34 ± 0.26
IGF-1 and IGF-1-related gene (IGFBP-3, IGFBP-1, IGF-1R, and GHR) expression in the liver
Accordingly with well-known data, IGF-1R was not detectable in the liver (data not shown). Interestingly, an overexpression of GHR gene was observed (Fig. 3d), a result that may suggest a compensating response to IGF-1 deficiency.
IGF-1 and IGF-1 receptor gene expression in target organs
GH receptor gene expression in target organs
Histopathological changes associated to partial IGF-1 deficiency in the bone and testicles
Additional study of gene expression in IGF-1-deficient mice
Liver gene expression of transcripts related to IGF-1 deficiency, group Hz
Fold change (vs CO)
(n = 10)
Heat shock protein 1B (Hsp1b)
−1.85 ± 0.12*
−1.50 ± 0.07*
Glutamate–cysteine ligase (Gclc)
−1.99 ± 0.05*
In the last decades, the availability of an animal IGF-1 gene null model has provided a wide knowledge about the physiological roles of IGF-1 [4, 25–27, 33]. However, all described human conditions of IGF-1 deficiency consist in decreased IGF-1 circulating levels, but only partially, since total or severe absence of this hormone is almost incompatible with survival [25, 33].
IGF-1 deficiency produces a multitude of effects related with recognizable syndromes with significant clinical consequences. At this moment, the best characterized conditions of IGF-1 deficiency are Laron syndrome in children , advanced liver cirrhosis in adults [9, 11, 12, 20, 28, 30, 34, 36, 37], and aging [2, 6, 15, 16, 21, 39, 46, 48, 51]. Recently, intrauterine growth restriction has also been suggested [1, 42, 49]. In all these conditions, replacement therapy can potentially be considered as an effective therapeutic approach. On the other hand, the multiple physiological properties of IGF-1 may generate excessive prospects when exogenous administration of IGF-1 is intended to exploit its anti-inflammatory, hematopoietic, antioxidant, metabolic, or anabolic properties. However, despite the limited results of these strategies, it may entail obvious risks.
In our experience, low doses of IGF-1 seem to be able to restore circulating levels of this hormone, which promotes beneficial actions without secondary effects (including hypoglycemia) [9, 21]. However, both physiopathological mechanisms related to IGF-1 deficiency and many of the effective actions of IGF-1 replacement therapy are not fully understood [13, 18, 22, 29, 31, 41, 45, 47]. In this context, the usefulness of this model in confirming the role of IGF-1 during liver fibrosis development and resolution, metabolic and neurodegenerative diseases, as well as its implication in life expectancy and oxidative damage in latter states of life, may be a tempting approach, with translational perspectives, especially in clarifying the awkward concern about the controversial relationship between IGF-1 and cancer. All these potentialities and risks claim for an experimental model of partial IGF-1 deficiency that could be able to mimic human conditions of IGF-1 deficiency as an appropriate tool for further translational strategies.
Consistent with previous reports , the present work provides evidence that, from early ages, heterozygous mice (Igf1+/−) show a partial, but significant, reduction of IGF-1 circulating levels. In addition, these differences are maintained throughout life until, at least, 23 months old. Since serum IGF-1 levels are persistently diminished in Hz mice compared to CO animals, in the present study, 25-day-old mice were chosen to start characterizing, from early stages, this model of partial IGF-1 deficiency. This age is previous to the maximum IGF-1 peak that in human occurs by 18 years old  while in mice, by 1 month old (Fig. 2).
In these young mice, the significant reduction of circulating levels of IGF-1 was associated to a significant decrease of IGF-1 gene expression in those studied organs: the liver (the main source of circulating IGF-1), bone, testicles, and brain (relevant target organs of this hormone); meanwhile IGF-1 receptor shows a normal expression, with the only exception of the liver, accordingly with well-known data that demonstrate the absence of IGF-1R in a normal liver .
In parallel, insufficient IGF-1 feedback could cause an increase of circulating GH values and, consequently, a decrease in GH receptor gene expression in target organs. As it has already been mentioned, in this study it was not possible to assess serum GH levels due to the high blood volumes needed to avoid disturbances due to endogenous pulsatile GH peaks along the day. However, reduced gene expression of GH receptor in relevant target organs seems to suggest a compensatory physiological mechanism to counteract presumable high levels of GH (due to the unbalanced suppression of GH by IGF-1).
Of interest, IGF-1-deficient mice showed an overexpression of GHR in the liver, probably stating an attempt to upregulate IGF-1 production (since the liver is the main source of circulating IGF-1) . In this sense, only the relative liver weight was higher in Hz animals as compared to controls.
On the other hand, at this early animal age, no differences were found regarding serum parameters of liver function, cholestasis, or cytolysis. Keeping in mind that, although liver is the main source of IGF-1, it does not usually benefit from its effects in normal conditions [33, 43, 50], this finding is congruent with the fact that our model consisted of young and uninjured mice. Further studies are required to uncover how mere IGF-1 deficiency may influence the development of liver diseases (at later ages) or its damage susceptibility.
Data in this paper are relevant because conditions of partial IGF-1 deficiency occur also from the early stage of life, such as Laron syndrome and intrauterine growth retardation [1, 24, 42, 49]. Accordingly, absolute weights of the bone, liver, testicle, and brain were significantly reduced in IGF-1-deficient mice as compared to control mice. Interestingly, partial IGF-1 deficiency was associated to a significant reduction of bone cortical thickness and testicular alterations from early stages, consistent with data in some conditions of IGF-1 deficiency, such as Laron dwarfism and cirrhosis [1, 24, 42, 49].
Furthermore, in the present work, several genes involved in IGF-1 physiology, such as Igfbp1 and Igfbp3 and more recently described genes related to oxidative response associated to IGF-1 deficiency (Hsp1b, Cat, and Gclc), were found to be reduced in the liver from animals with partial IGF-1 deficiency [3, 44]. These data reinforce that this animal model of partial IGF-1 deficiency may be a suitable tool to assess the underlying mechanisms of IGF-1 activities.
In conclusion, results in this paper may provide evidence for considering heterozygous Igf1+/− mice as a suitable experimental model in order to get more insight into the pathological mechanisms related to IGF-1 deficiency. Further studies, currently in progress, will be needed to confirm the suitability of this model in advanced ages, where it may be used to unravel potential conditions of IGF-1 deficiency, from which patients would benefit from an adequate replacement therapy.
The authors specially thank Dr. Argiris Efstratiadis for kindly providing the transgenic IGF-1 mice and for his helpful advice. We are also grateful to Ms. Amalia Calderón, Ms. Raquel Romero, and Ms. Susana Arahuetes for their expert secretarial and technical assistance. Special thanks to Dr. Maria Cruz Sádaba and Ms. Elena Ávila for their generous help. This work was supported by the Spanish “I + D Program” SAF 2009–08319.