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Endocrinology, doi:10.1210/en.2003-0236
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Endocrinology Vol. 144, No. 8 3505-3513
Copyright © 2003 by The Endocrine Society

PERK eIF2{alpha} Kinase Regulates Neonatal Growth by Controlling the Expression of Circulating Insulin-Like Growth Factor-I Derived from the Liver

Yulin Li, Kaori Iida, Jeff O’Neil, Peichuan Zhang, Sheng’ai Li, Ami Frank, Aryn Gabai, Frank Zambito, Shun-Hsin Liang, Clifford J. Rosen and Douglas R. Cavener

Department of Biology (Y.L., K.I., J.O., P.Z., S.L., A.F., A.G., F.Z., S.-H.L., D.R.C.), The Pennsylvania State University, University Park, Pennsylvania 16802; and The Jackson Laboratory (C.J.R.), Bar Harbor, Maine 046049

Address all correspondence and requests for reprints to: Douglas R. Cavener, Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802. E-mail: drc9{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Humans afflicted with the Wolcott-Rallison syndrome and mice deficient for PERK (pancreatic endoplasmic reticulum eIF2{alpha} kinase) show severe postnatal growth retardation. In mice, growth retardation in Perk-/- mutants is manifested within the first few days of neonatal development. Growth parameters of Perk-/- mice, including comparison of body weight to length and organ weights, are consistent with proportional dwarfism. Tibia growth plates exhibited a reduction in proliferative and hypertrophic chondrocytes underlying the longitudinal growth retardation. Neonatal Perk-/- deficient mice show a 75% reduction in liver IGF-I mRNA and serum IGF-I within the first week, whereas the expression of IGF-I mRNA in most other tissues is normal. Injections of IGF-I partially reversed the growth retardation of the Perk-/- mice, whereas GH had no effect. Transgenic rescue of PERK activity in the insulin- secreting ß-cells of the Perk-/- mice reversed the juvenile but not the neonatal growth retardation. We provide evidence that circulating IGF-I is derived from neonatal liver but is independent of GH at this stage. We propose that PERK is required to regulate the expression of IGF-I in the liver during the neonatal period, when IGF-I expression is GH-independent, and that the lack of this regulation results in severe neonatal growth retardation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POSTNATAL GROWTH IN humans and other mammals is primarily regulated by the concerted action of IGF-I and GH. Genetic experiments have confirmed the importance of IGF-I and GH in postnatal growth in mice and have delineated the developmental periods in which these factors are individually and jointly required for normal growth (1). Igf1-/- knockout mice are 30–40% smaller than normal at birth and continue to lag in postnatal growth (2, 3). In contrast, mice that are deficient for GH, the GH release factor, or GH receptor (GHR) grow normally for the first 2 wk before subsiding to postnatal growth retardation (1, 4, 5, 6). Thus, early neonatal growth in mice appears to be independent of GH. Later in postnatal development, a primary function of GH is to induce the expression of IGF-I mRNA in the liver via the transmembrane GHR and a signaling cascade that culminates in the transcriptional activation of the Igf-1 gene. IGF-I, synthesized in the liver, is secreted into the circulatory system, and most evidence supports the hypothesis that the liver is the predominant if not sole source of circulating IGF-I (4, 7, 8). IGF-I is also expressed in many other tissues, including the skeletal system, where it may have autocrine and/or paracrine functions. Unlike the liver, however, IGF-I expression is completely or partially independent of GH/GHR in most other tissues (4). Because liver-derived IGF-I has been argued to be strictly dependent upon GH (4, 9), it has been assumed that circulating levels of IGF-I are very low and functionally inconsequential during the first 2 wk of neonatal growth when longitudinal growth is not dependent on GH/GHR. Inasmuch as neonatal growth is clearly dependent upon IGF-I, this would imply that neonatal growth is exclusively dependent on the paracrine/autocrine action of IGF-I. The importance of circulating IGF-I to longitudinal growth was also questioned by experiments showing that mice with a liver-specific knockout of IGF-I (LID) did not show postnatal growth retardation, although circulating IGF-I was reduced by approximately 75% at the adult stage (7, 8). However, this interpretation, which arises from studies of the LID mice, is contradicted by investigation of the Ghr-/- knockout mice, which exhibit severe postnatal growth retardation and are completely deficient for circulating IGF-I, but have normal levels of IGF-I expressed in most other tissues. To explain the unexpected normal growth in the LID mice, it has been suggested that these mice may have substantial expression of circulating IGF-I during the early postnatal development before the Cre-induced deletion of the Igf-1 gene in the liver has occurred in most of the hepatocytes (4). The level of circulating IGF-I in the LID or Ghr-/- knockout mice during the neonatal period is unknown. Moreover, the expression and functional activity of IGF-I during the neonatal period has not been investigated in either humans or mice.

Genetic analysis of various mouse strains has revealed that polygenic variation for IGF-I expression maps to a small number of loci with major effects on skeletal growth and development (10). Such loci may correspond to genes that regulate IGF-I. PERK (pancreatic endoplasmic reticulum eIF2{alpha} kinase), a type I transmembrane protein kinase spanning the endoplasmic reticulum membrane, maps within a large multigene region on chromosome 6 of mice that bears one of these loci. Recently, we have generated a knockout mutation of the mouse Perk gene (11). Loss-of-function mutations in the mouse Perk gene result in three major defects: postnatal growth retardation, multiple skeletal dysplasias and osteopenia, and loss of endocrine and exocrine functions in the pancreas. The same array of defects is seen in the Wolcott-Rallison syndrome (WRS), a human genetic disease associated with mutations in the Perk gene (12). Perk-/- mice are normal at birth but exhibit an immediate lag in growth rate during the neonatal period. Inasmuch as this growth retardation precedes the development of diabetes and exocrine pancreatic insufficiency by more than 3 wk, we speculate that the early growth retardation is not caused by general metabolic defects. Because the early neonatal growth retardation in Perk-/- mice is similar than that seen in mice deficient for IGF-I, we investigated the expression of IGF-I in Perk-/- mice. We show herein that liver IGF-I mRNAs and serum IGF-I are dramatically reduced in Perk-/- mice during the neonatal period, whereas the IGF-I receptor and GH are elevated. Injection of IGF-I, but not GH, reversed the severe growth retardation of the Perk-/- mice, thus supporting the hypothesis that PERK is a major regulator of IGF-I-dependent neonatal growth. During the course of investigating the growth retardation of Perk-/- mice, we discovered that IGF-I is actively transcribed in the liver during early neonatal development, resulting in significant levels of circulating IGF-I before the period when liver IGF-I expression becomes GH-dependent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Growth plate analysis
All animal experiments were performed in accordance with the rules and regulations of the Institutional and Animal Care Use Committee. Tibias from prediabetic mice were isolated and fixed in 10% formalin overnight, decalcified using Cal-Ex (Fisher Scientific, Pittsburgh, PA) for 3–5 d, embedded in paraffin, cut longitudinally into 5-µm midsagittal sections, and stained with hematoxylin and eosin. Morphometry was performed on light microscopy photographs of the growth plates captured at x100 magnification. The total height of the epiphyseal growth plate was calculated as the distance between the proximal end of the chondrocyte resting zone to the distal base of the hypertrophic zone. Proliferative chondrocytes typically appear as flattened discs such that their height is much smaller than their width. The width of the hypertrophic chondrocytes is similar to proliferative chondrocytes, but their height is proportionally much greater. The boundary between the proliferative zone and hypertrophic zone was defined as the point where the ratio of cell width to cell height is approximately 2.0, less than 2.0 defined as hypertrophic chondrocytes, and more than 2.0 defined as proliferative chondrocytes (13). All zone height and cell height measurements were calculated in centimeters on photographs printed at x30 and x60 magnification, respectively. Averages from five Perk-/- mice and three Perk+/+ mice were analyzed, with multiple sections of each sample calculated. For cell number calculations, at least four columns per section around the midline were counted by at least three different coauthors. All measurements were performed on coded photographs to mask the genotype information from each observer.

RNA extraction and IGF-I mRNA analysis
Long bones (femur and tibia) were isolated from mice after removal of skin and skeletal muscles. Bone marrow was removed by flushing the bone cavities with PBS. The bones were cut into small pieces in TRIzol reagent (Sigma, St. Louis, MO) and homogenized with a Polytron homogenizer. Liver and pancreas were homogenized in TRIzol reagent with a sonicator, and the RNA was extracted. DNA contaminating the RNA sample was digested with RNase-free DNase (Ambion, Inc., Austin, TX) at 37 C for 60 min. An equal quantity of RNA was primed with random hexamer primers and was reverse transcribed into cDNA using M-MLV (Moloney murine leukemia virus) reverse transcriptase (Promega Corp., Madison, WI). PCR primers for IGF-I amplification are exon 1 forward 5'-GAT GGG GAA AAT CAG CAG CC-3', exon 2 forward 5'-TGC TGT GTA AAC GAC CCG-3', and common reverse primer 5'-CAA CAC TCA TCC ACA ATG CC-3'. Primers for tubulin are forward 5'-TCC TTC AAC ACC TTC TTC AGT GAG A-3' and reverse 5'-GAG GAT GGA ATT GTA GGG CTC AAC-3'. IGF-I and tubulin cDNA was quantified using real-time PCR. Real-time PCRs were carried out in 1x SYBR Green PCR buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.8% glycerol, 0.01% Tween 20, 0.25x SYBR Green (Molecular Probes, Inc., Eugene, OR), and 60 nM ROX I (Synthegen, Houston, TX). Real-time PCR was performed using the GeneAmp 5700 DNA amplification system (PE Applied Biosystems, Foster City, CA). The reaction program included a 94 C denaturation step for 5 min followed by 35 cycles of 94 C denaturation for 60 sec, 60 C annealing for 60 sec, and 72 C extension for 60 sec. Detection of fluorescent product was carried out at the end of the 72 C extension period. PCR products were subjected to a melting curve analysis and agarose gel electrophoresis. Data were analyzed and quantified with the GeneAmp 5700 SDS software. At a specific threshold in the linear amplification stage, the cycle differences between amplified tubulin and IGF-I cDNA were used to determine the relative quantity of IGF-I mRNA.

Calvarial osteoblasts isolation and culture
Calvarial osteoblasts were isolated from mice using a protocol modified from Ecarot-Charrier et al. (14). Calvaria were removed aseptically and stripped of the periosteum in culture media (low-glucose DMEM media with 100 U/ml penicillin and 100 µg/ml streptomycin). The calvaria were cut into small pieces and incubated in the same DMEM complete media with 10% fetal calf serum at 37 C, 5% CO2 for 1 wk, and then the bones were removed. Cells were grown for another week until reaching confluence. Half of the medium was changed with fresh media every 3 d. For IGF-I secretion, cells were first grown to confluence in the complete media and then washed and placed in conditioned media in which the 10% fetal calf serum was substituted with 0.1% BSA. After 24 h, conditioned media and cells were collected separately. Secreted IGF-I was normalized to total protein in the cell lysates.

IGF-I detection
Serum IGF-I was measured in mice using either enzyme immunoassay or RIA-based systems. IGF-I from the serum of neonatal mice and IGF-I secreted into the conditioned culture media were measured with an ultrasensitive RIA kit (022-IGF-R21, ALPCO, Windham, NH) with an assay sensitivity equal to 0.02 ng of IGF-I/ml. This IGF-I assay includes removal of IGF binding proteins by acid extraction and reducing cross-reactivity of IGF-II through the addition of a saturating concentration of exogenous IGF-II. Cross-reactivity to IGF-II is small (0.05%), and the intraassay coefficient of variation was 0.42%. For adult and juvenile mice, a rat IGF-I enzyme immunoassay kit (DSL-10-2900, Diagnostic Systems Laboratories, Webster, TX) was used to detect IGF-I after acid-ethanol extraction to remove IGF binding proteins. The intraassay and interassay coefficients of variation were 10 and 12%, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Perk-/- knockout mice exhibit postnatal growth retardation and a reduction in hypertrophic chondrocytes in the tibia growth plate
The body weight and size of Perk-/- mice is indistinguishable from wild-type littermates at birth, but within a few days they begin to noticeably lag in growth. By the end of the neonatal period [postnatal d 21 (P21)], male and female Perk-/- mice are on average 50% smaller than normal as a consequence of a 4-fold reduction in daily growth rate (Fig. 1Go). The growth rates of Perk-/- males are somewhat more negatively impacted than females, but this varies among litters. Examination of the tibia growth plate chondrocytes (Fig. 2Go) revealed that Perk-/- mice show on average a 22% reduction in the width of the growth plate, which is largely due to a 26 and 19% reduction in the width of the hypertrophic and proliferative zones, respectively (Table 1Go). The width of the resting chondrocyte zone of the Perk-/- growth plate is normal, however. The reduction in the hypertrophic zone is due to a 28% reduction of the number of cells in the longitudinal axis (Table 1Go), consistent with a deficiency in a primary growth factor. We also noted an abrupt transition from the proliferative to the hypertrophic zone in the Perk-/- growth plates, as indicated by a deficiency in chondrocytes exhibiting intermediate width/height ratios.



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  		FIG. 1. Growth curves of Perk-/- and wild-type mice. Total body weights of wild-type (WT; open circles and squares), Perk-/- (filled circles and squares with solid line), and ßPERK; Perk-/- (filled circles and squares with dashed lines) were recorded from birth until P40. Circles represent female mice and squares represent male mice. Average sample size: WT males, 33; WT females, 25; Perk-/- males, 7; Perk-/- females, 5; ßPERK; Perk-/- males, 6; and ßPERK; Perk-/- females, 2.

 


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  		FIG. 2. Tibia growth plates of Perk-/- and wild-type (WT) mice. Representative longitudinal sections of WT and Perk-/- proximal tibia growth plates showing similar resting zones (RZ) and decreased proliferative zone (PZ) and hypertrophic zone (HZ) in the Perk-/- mice. Micrographs are shown at x100 magnification.

 

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  		TABLE 1. Analysis of tibia growth plate chondrocyte zones in Perk-/- and wild-type (WT) mice

 
Comparison of body length (rump to nose) to total body weight of Perk-/- mice was assessed using the allometric scaling equation y = ax b, where y = body length, x = body weight, and b is the slope of the regression. A regression slope near 0.33 is typically found in normal mice as well as growth-deficient mice that retain allometric proportions (4, 15). The allometric scaling slope of body weight and length for the Perk-/- mice was determined to be 0.32, very close to previous estimates of wild-type mice as well as Ghr-/- and Igf1-/- deficient mice. With the exception of the brain, major organs of Perk-/- mice also showed a proportional reduction in size relative to body weight (Table 2Go). The weight of the brain was only marginally reduced in Perk-/- mice and constituted 5.36% of the total body weight, compared with 2.75% for wild-type mice. Others have noted that the brain does not decrease proportionally to total body weight in Igf-1- and Ghr-deficient mice and speculated that because the brain grows more rapidly in fetal development than other organs (16), it is less impacted by postnatal growth factor deficiencies (4). The spleen in Perk-/- mice is somewhat proportionally smaller than expected and shows considerable variation independent of total body size.


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  		TABLE 2. Comparison of organ weights between wild-type (WT) and Perk-/- mice

 
Transgenic rescue of PERK expression in the insulin-secreting ß-cell defect in Perk-/- mice rescues juvenile, but not neonatal, growth retardation
Perk-/- knockout mice continue to display a 3- to 4-fold reduction in growth rate during the juvenile period (3–6 wk). Between P22 and P25, these mice develop severe hyperglycemia (11), which may negatively impact their growth. To determine whether the onset of diabetes at the juvenile stage contributes to the continued growth retardation in Perk-/- mice, a Perk transgene targeted to the insulin-secreting ß-cells of the islet of Langerhans (denoted ßPERK) was introduced into the genome of the Perk-/- knockout strain. The resultant mice (ßPERK; Perk-/-) are deficient in PERK in all tissues except for the insulin-secreting ß-cells of the endocrine pancreas. The expression of PERK in the insulin-secreting ß-cells of these mice reverses the loss of ß-cell mass and the development of hyperglycemia (Zhang, P., and D. R. Cavener, unpublished data). The ßPERK; Perk-/- mice still exhibit a 4-fold reduction in growth rates during the neonatal period, indistinguishable from the Perk-/- global knockout strain (Fig. 1Go). However, at the neonatal-juvenile transition (P21) these mice exhibit accelerated growth compared with Perk-/- knockout mice. Although ßPERK; Perk-/- mice do not catch up to the wild-type mice in total size, their growth rates between P20 and P40 are indistinguishable from wild-type mice.

Perk-/- mice are deficient in serum IGF-I and liver IGF-I mRNA
Genetic analysis of Perk-/- mice showed that PERK is essential for normal growth, particularly during neonatal development. Comparison of the growth rates of Perk-/- mice with various mutations affecting GH, IGF-I, and their receptors suggested that the Perk-/- mice might be deficient in IGF-I because of the early neonatal manifestation of growth retardation. Analysis of serum IGF-I revealed a dramatic reduction in Perk-/- mice during neonatal development (Fig. 3Go, top). The severest reduction (25% of normal) was apparent during the early neonatal period in which IGF-I is clearly the dominant, if not exclusive, growth factor. In contrast, GH serum levels in juvenile mice were somewhat elevated (Perk-/-, 7.07 ± 2.64 ng/ml, vs. wild-type, 4.04 ± 0.43 ng/ml) as expected due to the reduction in the normal negative feedback regulation of IGF-I on GH (17, 18, 19). In late neonatal development, the ßPERK; Perk-/- rescued mice exhibit a deficiency in circulating IGF-I, which is indistinguishable from the nonrescued Perk-/- knockout mice (Fig. 3Go, bottom). However, IGF-I levels in the ßPERK; Perk-/- mice recover to normal levels during the second postnatal month, whereas in contrast, IGF-I levels in Perk-/- knockout mice remain relatively low.



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  		FIG. 3. Serum IGF-I levels in neonatal, juvenile, and adult mice. Top, Serum IGF-I levels of neonatal mice from WT (white bars) and Perk-/- (black bars). For each time point, n = 3–7 for WT, and n = 2–4 for Perk-/-. *, P < 0.05 based upon a one-tailed t test. Bottom, Serum IGF-I levels of WT, Perk-/-, and ßPERK; Perk-/- (hatched bars) mice (n = 3–4). Error bars represent SE values of the mean. *, P < 0.05. **, P < 0.01 based on a two-tailed t test comparing Perk-/- and ßPERK; Perk-/- mice to wild-type.

 
To ascertain the molecular and cellular basis for the reduction in IGF-I in the Perk-/- mice, the level of the two major IGF-I mRNA isoforms was determined in liver, because it is the major source of circulating IGF-I (Fig. 4Go). Both IGF-I mRNA isoforms 1 and 2 were substantially reduced in liver during neonatal development at P5, P7, P10, and P14. The relative abundance of liver IGF-I mRNA is highly correlated with serum IGF-I levels, suggesting that the primary defect in the IGF-I deficiency in Perk-/- mice is caused by misregulation of the steady state levels of liver IGF-I mRNA. The expression of IGF-I mRNA was also similarly reduced in the pancreas of neonatal Perk-/- mice (Table 3Go). However, in contrast, normal levels of IGF-I mRNA were found in long bone, kidney, lung, and skeletal muscle of neonatal Perk-/- mice (Table 3Go).



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  		FIG. 4. Relative abundance of IGF-I transcripts in livers of neonatal Perk-/- and wild-type (WT) mice. WT (white bars) and Perk-/- (black bars) levels of IGF-I transcripts initiated from exon 1 and exon 2. For each group, n = 3–4. Error bars represent SE values of the mean.

 

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  		TABLE 3. Igf-1 mRNA expression in major organs and tissues of neonatal wild-type (WT) and Perk-/- mice

 
Although the level of IGF-I mRNA in long-bone tissue is normal, we found that the level of the IGF-I receptor in bone tissue was elevated (Fig. 5Go). An increased level of the IGF-I receptor has been shown in other cases of IGF-I deficiency in humans (20) and is purported to be due to a relaxation of the normal negative feedback regulation of IGF-IR by circulating IGF-I. To further assess potential defects in IGF-I expression in bone tissue, IGF-I expression and secretion were assayed in primary cultures of calvarial osteoblasts isolated from Perk-/- mice. Calvarial osteoblasts of Perk-/- mice exhibit normal levels of IGF-I mRNAs and secrete normal amounts of mature IGF-I (Fig. 6Go). Together, these results suggest that the neonatal growth retardation is not due to an autocrine/paracrine IGF-I deficiency in bone or other tissues, but rather due to a reduction of circulating IGF-I secreted from the liver. However, we have not been able to quantitatively assess the level of IGF-I expression specifically within the growth plate chondrocytes, and therefore we cannot rule out a possible reduction in locally expressed IGF-I in the growth plate that may negatively impact longitudinal growth of long bones.



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  		FIG. 5. Expression of IGF-I receptor-{alpha} in bone tissue. IGF-IR ({alpha} subunit) level of WT (+/+) and Perk-/- tibia and thoracic vertebrae. Ten micrograms of total protein were loaded in each lane. ß-Actin was used for a loading control.

 


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  		FIG. 6. Expression of IGF-I mRNA and secretion of IGF-I in primary cultured osteoblasts. Top, Relative abundance of IGF-I mRNA in cultured osteoblasts from P7 and P17 WT (white bars) and Perk-/- mice (black bars). Bottom, Levels of IGF-I secreted into culture media. For WT, n = 7; for Perk-/-, n = 5. Error bars represent SE values of the mean.

 
Beyond the neonatal period, the expression of IGF-I mRNA is largely controlled by GH as mediated by the GHR (2, 4). To ascertain whether the deficiency of liver IGF-I in the Perk-/- mice was caused by a deficiency of GHR, as seen in the Laron syndrome (22), GHR mRNA was quantitatively measured in the liver by real-time RT-PCR and normalized to tubulin mRNA. GHR mRNA was detected in the livers of P14 Perk-/- mice and was not substantially different from that seen in wild-type littermates [GHR mRNA Perk-/-, 0.17 ± 0.008 (n = 3); wild-type, 0.26 ± 0.026 (n = 3)].

Injection of IGF-I reverses the severe growth retardation of the Perk-/- mice.
We reasoned that if the deficiency in circulating IGF-I in the Perk-/- mice is the cause of the neonatal growth retardation, injection of IGF-I would lead to a reversal of the growth retardation. Perk-/- neonatal mice were injected sc twice daily with 125 ng IGF-I/g total body weight during the entire neonatal period (P2–P21). Within a few days after the onset of IGF-I injection, the injected Perk-/- mice began to exhibit an accelerated growth rate compared with noninjected Perk-/- mice (Fig. 7Go). Averaged over the neonatal developmental period, the IGF-I injected Perk-/- mice exhibited a 2-fold higher daily growth rate than noninjected mutant mice. In contrast, twice-daily injection of GH (3 µg/g body weight) during the late neonatal and juvenile state (P15–P42) had no effect on the growth rate of the Perk-/- mice (data not shown).



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  		FIG. 7. Growth curves of Perk-/- and WT mice injected with IGF-I. Body weights were recorded from mice with and without IGF-I injection (125 ng/g body weight, sc injections, twice daily) from P2 to P21. On average, n = 22 for noninjected WT, n = 3 for noninjected Perk-/-, n = 6 for IGF-I-injected WT, and n = 3 for IGF-I-injected Perk-/-. Error bars represent SE values of the mean.

 
Serum titers of IGF-I and body weight are highly correlated during neonatal but not juvenile development
IGF-I levels are modulated by a number of environmental and genetic factors, and normal variation of IGF-I may impact postnatal growth. Considerable variation in IGF-I levels was seen for Perk mutant and wild-type mice. During neonatal and juvenile development, body weight generally reflects relative growth rates. We determined the correlation of body weight and IGF-I titers at the neonatal (0–3 wk) and juvenile (3–6 wk) stages for Perk-/- and wild-type mice (Fig. 8Go). During the neonatal period, a highly significant correlation (r = 0.93) was observed between body weight and IGF-I titers; this strong positive correlation was observed for both wild-type (r = 0.94) and Perk-/- (r = 0.80) mice when analyzed separately as well. In contrast, no significant correlation was observed during the juvenile stage for either wild-type mice or Perk-/- mice. These findings suggest that IGF-I levels present in both normal and Perk-/- mice are limiting for neonatal growth but permissive for juvenile growth.



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  		FIG. 8. Correlation of body weight to serum IGF-I in Perk-/- and WT mice. Top, Correlation of body weight to serum IGF-I in Perk-/- (black squares) and WT (white circles) neonatal mice ranging from age P5–P14. Correlation coefficient (r) for Perk-/- mice is 0.80, whereas the correlation coefficient for WT mice is 0.94. Bottom, Correlation of body weight to serum IGF-I in Perk-/- and WT juvenile mice ranging in age from P23–P42.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
According to the somatomedin hypothesis of postnatal growth, GH mediates skeletal growth by activating the expression of IGF-I in the liver and secretion into the circulation (17, 18). GH has been shown to regulate the transcription of IGF-I in the liver via signaling through the GHR and downstream effectors such as STAT5b (signal transducer and activator of transcription 5b) and hepatocyte nuclear factor (HNF)1{alpha}. During the first 2 wk of neonatal development, however, GHR appears to have no effect on growth (4). Knockout mutations of Igf1 in mice, however, show that the lack of IGF-I leads to perinatal growth retardation (2, 3, 23), and by P14 Igf1-/- mice are only half the size of normal. An unresolved issue is the source and regulation of IGF-I during the neonatal period when IGF-I expression is independent of GH. In addition to the liver, IGF-I has been shown to be expressed in a variety of tissues, and in many of these other tissues IGF-I expression is apparently independent of GH (4, 9). Thus, others have speculated that neonatal growth may be under paracrine and/or autocrine control of IGF-I independently of GH (7, 8). The potential contribution of liver-derived serum IGF-I to early neonatal growth has been largely ignored due to the prevalent view that IGF-I expression in the liver is strictly GH-dependent. However, the notion that IGF-I expression in the liver is strictly GH- dependent is based on studies of juvenile and mature adult mice. Genetic ablation of IGF-I in the mouse liver as achieved in the LID mice using the Cre/loxP system reduced serum IGF-I by 75%, but surprisingly had no impact on postnatal growth (7, 8). However, the ablation of IGF-I in these experiments either was not induced or likely did not occur in the liver of the LID mice until after the early neonatal period and therefore would not have significantly reduced the expression of IGF-I during neonatal development. We show herein that a significant level of IGF-I mRNA is expressed in the liver of 5- to 7-d-old mice as well as a significant level of IGF-I in the serum. Moreover, Rotwein and colleagues (24, 25) have reported significant IGF-I mRNA in the liver in neonatal rats (p7), which they argue is GH independent. In the absence of a functional PERK eIF2{alpha} kinase gene, both liver IGF-I mRNA and serum IGF-I are dramatically reduced in neonatal mice, whereas the IGF-I mRNA is normally expressed in most other tissues including bone. However, the tibia growth plates in neonatal Perk-/- mice showed a reduction in the proliferative and hypertrophic zone consistent with the longitudinal growth defect. Injection of IGF-I, but not GH, partially reverses the neonatal growth retardation of the Perk-/- mice. Together, these data support the hypothesis that liver-derived IGF-I, whose expression is PERK dependent but independent of GH, plays a major role in neonatal longitudinal growth. Our data do not exclude the possibility that IGF-I also plays a paracrine/autocrine role of IGF-I during neonatal growth; however, our finding that IGF-I is normally expressed in major organs and tissues in Perk-/- mice, with the exception of the liver and pancreas, suggests that the endocrine role of IGF-I dominates longitudinal growth during the neonatal period.

We showed that although a 2-fold reduction in circulating IGF-I during the neonatal period negatively impacts growth, it does not retard growth during the juvenile stage. This finding is consistent with genetic ablation experiments of the Igf-1 gene, which show that global and early ablation of IGF-I dramatically impacts growth, whereas a reduction of circulating IGF-I during the juvenile period has no significant impact on growth (2, 3, 7, 8, 23). In addition, we showed that serum IGF-I and body weights are highly correlated during neonatal development, but showed no correlation during juvenile development. Variation in circulating IGF-I among human infants has also been shown to be significantly correlated with growth and the age at which the childhood growth stage is reached (26). The correlation between IGF-I and growth is apparent in infants before the stage at which the effect of GH on linear growth becomes prominent. Similar to mice, IGF-I continues to rise in humans beyond the neonatal/childhood stage, but growth rates are no longer correlated with normal variation in IGF-I levels. We propose that circulating IGF-I is the major limiting factor for neonatal growth but may only limit juvenile growth if its levels are exceedingly low (<10% of normal). Thus, IGF-I is permissive for juvenile growth over a broad range.

Yakar et al. (27) have drawn a similar conclusion regarding the importance of a threshold of circulating IGF-I during the juvenile and adult stages. They showed that mice lacking liver-specific expression of IGF-I (LID) and mice globally deficient for the ALS (acid labile subunit) each exhibit a substantial reduction in circulating IGF-I (25 and 35% normal, respectively), but neither exhibited a substantial reduction in postnatal growth rates. However, the double mutant combination LID/ALSKO (LID/acid labile subunit knockout) exhibits a further reduction of circulating IGF-I (10–15% normal) and showed a substantial reduction in postnatal growth rates. Quantitative variation in circulating IGF-I above a minimal threshold at juvenile and adult stages thus appears to have a negligible impact on longitudinal growth; however, variation in IGF-I associated with different mouse strains has been shown to strongly influence bone density and skeletal morphology (10, 28, 29). Moreover, the LID mouse exhibits diminished bone density suggesting that the level of circulating IGF-I derived from the liver has a pronounced effect on bone architecture, although longitudinal growth is normal in these mice. Thus, we conclude that the endocrine function of IGF-I is rate limiting for longitudinal growth during the neonatal period, whereas it is only limiting for bone density and bone modeling during the juvenile and adult stages.

Humans afflicted with the WRS, caused by a loss-of-function mutation in the Perk gene, typically develop diabetes within the first 2 months, similar to Perk-/- mice. IGF-I deficiency has been reported in one WRS patient (30), but apparently has not been examined in most WRS patients. Because humans do not exhibit as rapid longitudinal growth during the first postnatal month as seen in mice, it has not been possible to determine whether the growth retardation apparent in the first 2 yr in WRS is due to a primary defect in IGF-I or due to an indirect effect of the severe hyperglycemia. For Perk-/- mice, however, we show that a dramatic reduction in neonatal growth precedes the development of hyperglycemia, which does not occur until the early juvenile stage (11). Thus, the neonatal growth retardation is not caused by diabetes. We speculate that growth retardation manifested in the WRS during the neonatal period is also caused by a deficiency of IGF-I and may therefore respond to neonatal IGF-I therapy.

In contrast, we have shown that rescuing PERK expression in the insulin-secreting ß-cells of the Perk-/- mice reverses the juvenile growth retardation thus implicating hyperglycemia as the cause of growth retardation in the Perk-/- knockout mice during this later postnatal stage. Hyperglycemia is known to precipitate a reduction in circulating IGF-I, and the longitudinal growth of some individuals suffering from other forms of early onset juvenile diabetes may be delayed (31, 32). The ß-cell rescued Perk-/- mice (ßPERK; Perk-/-) recover normal titers of IGF-I during the juvenile period, suggesting that hyperglycemia may be repressing the expression of IGF-I in the Perk-/- mice once hyperglycemia is manifested in the early juvenile state (P22–P24). Although the increase in IGF-I titers in the ßPERK; Perk-/- rescued mice is correlated with an increase in growth rates compared with the global Perk-/- mice, we suggest the increase in IGF-I may not be responsible for increasing growth rates based upon studies of conditional knockouts of IGF-I in the liver (LID). The LID mice exhibit IGF-I levels that are comparable to the Perk-/- mice in 1-month-old mice, yet do not exhibit growth retardation, suggesting that the reduced level of IGF-I does not limit growth at this stage. Hyperglycemia affects a number of other metabolic processes that directly impact growth, including repression of the GLUT4 transporter and the IGF-I receptor in humeral growth plates (33). We postulate that the growth retardation seen in the Perk-/- mutants, specifically during the juvenile period, may be caused by such hyperglycemic effects.

Liver dysfunctions are known to reduce the expression of IGF-I resulting in postnatal growth retardation (34, 35). However, Perk-/- mice appear to express normal levels of liver-enriched genes including albumin, apolipoprotein E, apolipoprotein CI, and serum AST (aspartate aminotransferase) levels (data not shown). In addition, histological examination of the liver of Perk-/- mice appears normal. We conclude that the down-regulation of IGF-I in the liver of Perk-/- mice has a specific effect on the transcription and/or stability of the IGF-I mRNAs and is not due to a general liver dysfunction. The mechanism underlying the regulation of IGF-I in the liver during the neonatal period by PERK is unknown. We have examined the expression of the key transcriptional regulatory factors that are purported to regulate IGF-I expression in the liver, including HNF1{alpha}, HNF3ß, HNF4{alpha}, CAAT enhancer binding protein (C/EBP){alpha}, and C/EBPß, but we did not detect any significant differences in their expression in the liver of Perk-/- mice (Li, Y., and D. R. Cavener, unpublished data). However, these factors may only be required for GH-dependent regulation of IGF-I later in development. Regulatory factors that specifically control GH-independent expression of liver IGF-I during the neonatal period have been speculated to exist in rats (24, 25) but have not yet been identified.

The translation initiation factor eIF2{alpha} is the prime, if not exclusive, substrate of PERK. Phosphorylation of eIF2{alpha} (Ser51) may have two diametrically opposed effects on translation initiation, repression or activation, depending on the specific mRNA and the degree to which the eIF2{alpha} is phosphorylated. Hyperphosphorylation, particularly under stress conditions, may lead to the repression of global protein synthesis, whereas more modest levels of eIF2{alpha} phosphorylation under normal developmental and physiological states can have a very different effect, namely the activation of translation of specific genes, particularly those encoding regulatory functions (36, 37). We speculate that PERK may be required in the liver to regulate the translation initiation of one or more regulatory factors that control neonatal expression of IGF-I mRNA. Although some of these factors that control the neonatal expression of IGF-I in the liver may be identical to those required for juvenile and adult regulation of IGF-I, we propose that one or more neonatal-specific factors are required for IGF-I expression and are under the control of PERK.


   Acknowledgments
 
We thank Julie Burgess for excellent technical assistance.


   Footnotes
 
This work was supported by the Pennsylvania State University and NIH Grants GM 56957 (to D.R.C.) and AR 45433 (to C.J.R).

Abbreviations: GHR, GH receptor; HNF, hepatocyte nuclear factor; LID, liver-specific knockout of IGF-I; PERK, pancreatic endoplasmic reticulum eIF2{alpha} kinase; P21, postnatal d 21; WRS, Wolcott-Rallison syndrome.

Received February 21, 2003.

Accepted for publication April 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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