Differential inhibition of postnatal brain, spinal cord and body growth by a growth hormone antagonist
© McIlwain et al; licensee BioMed Central Ltd. 2004
Received: 05 December 2003
Accepted: 23 February 2004
Published: 23 February 2004
Growth hormone (GH) plays an incompletely understood role in the development of the central nervous system (CNS). In this study, we use transgenic mice expressing a growth hormone antagonist (GHA) to explore the role of GH in regulating postnatal brain, spinal cord and body growth into adulthood. The GHA transgene encodes a protein that inhibits the binding of GH to its receptor, specifically antagonizing the trophic effects of endogenous GH.
Before 50 days of postnatal age, GHA reduces spinal cord weight more than brain weight, but less than body weight. Thereafter, GHA ceases to inhibit the increase in body weight, which approaches control levels by day 150. In contrast, GHA continues to act on the CNS after day 50, reducing spinal cord growth to a greater extent and for a longer duration than brain growth.
Judging from its inhibition by GHA, GH differentially affects the magnitude, velocity and duration of postnatal growth of the brain, spinal cord and body. GH promotes body enlargement more than CNS growth early in postnatal life. Later, its CNS effects are most obvious in the spinal cord, which continues to exhibit GH dependence well into adulthood. As normal CNS growth slows, so does its inhibition by GHA, suggesting that reduced trophic effects of GH contribute to the postnatal slowing of CNS growth. GHA is a highly useful tool for studying the role of endogenous GH on organ-specific growth during aging.
The mammalian CNS acquires most of its mature size after birth. Human brain weight increases 3–4 fold between birth and maturity , while the spinal cord enlarges about 10 times postnatally . After maturity is reached, the human brain declines in size by approximately 2% each decade , while spinal cord size declines approximately 3% per decade .
Growth hormone helps to drive postnatal CNS growth. Evidence for its role comes from several sources, including reduced brain size in human GH deficiency disorders and in experimental animals [4, 5]. Transgenic mice have been especially useful in demonstrating the role of GH in CNS growth [6–9]. Strong evidence for a trophic role of GH in brain growth has come from transgenic mice whose somatotropes were selectively destroyed after birth by the expression of a GH promoter-driven diphtheria toxin transgene [6, 10, 11]. Additional evidence for trophic effects of GH on the CNS was obtained from transgenic mice that overexpress GH. Our laboratory previously found a 12% increase in mean brain weight in adult mice expressing excess GH (GH+) , consistent with other studies showing slight brain enlargement in such animals [8, 9].
There is much less information about the effects of GH on the spinal cord than on the brain. We previously reported a mean increase of 35% in spinal cord weight in GH+ mice, indicating that the spinal cord has a much greater capacity for GH-dependent, supranormal growth than does the brain . There is no information about the effects of reduced endogenous GH action on spinal cord size in transgenic mice. To examine further the role of GH in postnatal spinal cord, brain and body growth, we have utilized transgenic mice expressing GHA, an antagonist of human GH that inhibits the binding of endogenous GH to its receptor . We find evidence for differential trophic effects of GH on the magnitude, rate and duration of postnatal growth of the body, brain and spinal cord in GHA transgenic mice, providing strong evidence for organ-specific dependence on GH during early postnatal life and adulthood.
Postnatal body weight increase in WT(wild-type) mice
GHA strongly inhibits early postnatal body weight
At the time of weaning, GHA mice were already 30% smaller (p < 0.01) than gender-matched, WT mice (Fig. 1, day 21). At each successive age analyzed, differences in body weight between GHA and control mice remained significant (p < 0.01), except for 150 day-old males. Body weight increased rapidly in GHA mice between weaning and day 50, as it did in WT mice. GHA strongly inhibited the rate of increase in body weight during this period of early, rapid growth, with body weight increasing 0.23 ± 0.02 g/day in GHA males and 0.18 ± 0.2 g/day in GHA females, about one-half as fast as in WT mice. At day 50, GHA males weighed 56% less than WT males (Fig. 1), and GHA females weighed 62% less than WT females. By day 50, male and female transgenic mice had reached only 58.0% and 50.0%, respectively, of the body weight they were to achieve by day 150 (Fig. 1). By 150 days of age, GHA and females had attained 3.5 and 3.1 times their wean weight, respectively.
GHA does not inhibit body weight increases after day 50
GHA inhibits the magnitude and velocity of postnatal brain growth
GHA inhibits the magnitude and velocity of postnatal spinal cord growth
GHA has more potent and prolonged inhibitory effects on spinal cord than brain growth
GHA differentially inhibits the postnatal growth of the body, brain and spinal cord. Because GHA antagonizes the action of GH at the GH receptor, we infer that endogenous GH also must differentially influence their growth. Much of the trophic action of GH on the CNS is probably mediated by IGF-1, which increases brain and spinal cord weight largely by increasing white matter [6, 7, 11, 13]. Spinal motor neuron size is also increased postnatally by GH, but may not depend upon IGF-1 .
The contribution of GH to body, spinal cord and brain growth
An intriguing finding from our study was that GHA mice exhibited catch-up growth after day 50 by increasing body growth rates to levels higher than in WT mice. During this period, body length showed a constant inhibition of 16.2 ± 0.03% in all GHA mice. Part of the catch-up growth may be due to greater increases in adipose tissue mass in GHA mice, as we have reported previously . It is also possible that high levels of GHA and inhibition of GH action in the period of rapid growth between day 25 and 50 elicit compensatory increases in GH responsiveness either at the level of GH receptor expression or subsequent intracellular signal transduction systems.
Does GH play a role in the slowing of postnatal CNS growth?
Relative to its weight at birth, the spinal cord grows more postnatally than does the brain [1, 2], and GH stimulates the enlargement of the spinal cord more than the brain . Normal postnatal brain growth slows earlier than spinal cord growth (Figs. 3, 4), at the same time as GHA inhibition of their growth diminishes (Fig. 5). Taken together, these data suggest that cessation of GH action plays a role in the age-related slowing of brain and spinal cord enlargement. Three possible mechanisms by which GH might influence the slowing of CNS growth are: 1) a decrease in the concentration of circulating GH with age; 2) a decrease in GH responsiveness in the CNS at the level of the GH receptor or downstream events; or 3), hypothetically, an age-dependent increase in a growth antagonist that competes with the trophic action of GH on the CNS. GH responsiveness includes the GH-dependent synthesis and action of IGF-I, which has powerful trophic effects on postnatal growth of the brain [6, 11, 13] and spinal cord . There is an inverse correlation between serum GHA and IGF-I concentrations among GHA mice .
An age-dependent decrease in the concentration of GH accessible to all parts of the CNS cannot account for the observation that GHA continued to inhibit spinal cord growth well after its action on brain growth was no longer apparent (Fig. 6). For the same reason, if either an age-related decrease in GH responsiveness or the action of some growth antagonist decreased the rate of brain growth, then such changes must have occurred more slowly in the spinal cord than in the brain. A decrease in GH responsiveness is consistent with reports of age-dependent decreases in GH binding in human brain  and in rat brain and spinal cord . However, Zhai et al.  reported that GH binding began to decline earlier in the rat spinal cord than in the brain.
Does GH help to maintain CNS size in adult mice?
That GH is responsible for some of the postnatal growth of the brain and spinal cord suggests that GH could help to maintain CNS size with age. The dramatic decrease in circulating GH in older human beings  might contribute to the age-dependent atrophy observed in the spinal cord  and brain . In order to investigate this possibility in experimental animals, one would ideally want to allow the CNS to reach its mature size and then to alter the action of endogenous GH. Transgenic mice expressing an inducible GHA gene could be used to investigate this possibility. Such transgenic mice would be useful in studying not only the effects of GH in the adult mouse CNS, but also a wide variety of other actions GH may have throughout the body.
GH plays a significant, but not an exclusive nor uniform role in postnatal CNS and body growth. Based upon the actions of GHA, we conclude that GH differentially affects the magnitude, velocity and duration of postnatal growth of the brain, spinal cord and body. Early in postnatal life, GH promotes body enlargement more than CNS growth. Later, its CNS effects are most obvious in the spinal cord, which continues to exhibit GH dependence well into adulthood. Studies using GHA transgenic mice could help to determine whether the somatotrophic system contributes to the age-related slowing of CNS growth and whether it helps to maintain CNS size in adulthood.
GHA mouse line
Mice hemizygous for the GHA transgene were obtained from the laboratory of Dr. John J. Kopchick, Edison Biotechnology Institute, Ohio University. We used line hGH G120R, which bears a human GH transgene that has an arginine substituted for the normal glycine at position 120, converting its product to a GH antagonist [12, 18].
At 25-day intervals between ages 25 and 150 days, mice were injected i.p. with a mixture of xylazine (4 mg/kg) and ketamine (400 mg/kg), and when fully anesthetized, the animals were decapitated. The brain of each animal, including the cerebellum, but not the brainstem, was dissected and weighed on a Mettler semi-micro (P1210) or micro (H20) balance. The spinal cord was dissected under a stereomicroscope from the mid-cervical through the sacral segments, the cauda equina and all other spinal roots were removed, and the spinal weight was recorded.
Means ± s.e.m are presented in the text wherever possible, while percentages only are used when expressing the percent difference between two unpaired means. Tests of significance were performed using Student's paired or unpaired t-test. Where absolute weights are presented, values for WT and GHA mice are derived from all litters, including those containing only WT or GHA mice.
This study was supported by grants from the UNC University Research Council to DLM and by NIH grants DK40247 and AG09973 to PKL. JJK is supported in part by the state of Ohio's Eminent Scholars Program that includes a gift from Milton and Lawrence Goll and by DiAthegen LLC. Mouse facilities of the UNC Center for Gastrointestinal Biology and Disease aided this work.
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