|
|
Growth Plate Changes of Catch-up Growth Following Caloric Restriction: Morphologic and Gene Expression Changes, Especially HIF1α« Back to Volume 24, Issue 2, November 2008 - Table of Contents To study the mechanism of catch-up growth, Even-Zohar et al examined growth plate morphology and gene expression in prepubertal male Sprague-Dawley rats subjected to 10 days of 40% caloric restriction. This degree of caloric reduction long-term had been shown to increase rat longevity, and was calculated based on the ad lib feeding of similar age and weight rats in a previous experiment (access to water remained unlimited). Rats were randomized into 3 groups: ad libitum (AL; unlimited food access throughout the experiment), food restricted (RES; 60% of the same chow throughout the experiment), and catch-up (CU; the 60% intake for 10 days followed by ad libitum feeding for the next 7 days). Growth parameters confirmed the experimental design, though the catch-up growth was partial. Weight gain was 6.5 gm/day in the AL group, and only 1.2 gm/day in the RES group. The CU period augmented weight gain to 15.1 gm on the first day of ad lib feeding, followed by an average daily weight gain rate of 8% their total body weight compared to 4.5% in the AL group. Nonetheless, the CU group failed to completely regain their weight deficit by the end of the week. Similarly, humeral length was significantly reduced in the RES group throughout, and significantly improved at day 7 of CU but still shorter than the AL group. The humeral epiphyseal growth plates (EGP) reflected the gross growth parameters. The EGP length (from the reserve zone to the ossification front of the metaphyseal bone) was constant in the AL group, shorter in the RES group, and showed progressive catch-up in the CU group. The average number of chondrocytes per column (proliferative zone through the last hypertrophic cell) was reduced in RES, and improved in the CU group from day 2 to 7 of ad lib feeding. The ratios of proliferative to hypertrophic zones were unaffected by the nutritional interventions. Towards a mechanistic understanding, the EGPs were microdissected and total RNA was pooled from at least 15 sections from each zone of each animal to be studied by Affymetrix microarray. Between the RES and AL groups 4144 probes differed significantly. Interestingly, the gene expression profile of the CU group differed from RES by the first day of liberated feeding, yet remained different from AL for the remainder of the week. The investigators focused on genes with a so-called “up-down-up” (UDU) expression profile (highly expressed in AL, reduced in RES and increased again in CU). At least 2-fold changes were shown in 714 genes, going down from AL to RES and up from RES to CU; of these, 550 were differentially expressed among all 3 groups on the first day of refeeding. In silico analyses of functional groups and promoter of cluster revealed the UDU genes to be enriched for synthetic (macromolecule metabolism, RNA processing and translation, protein transport, secretion and degradation) rather than proliferative functions. Of the 7 transcription factors whose downstream targets were enriched in the UDU gene list, hypoxia inducible factor (HIF)1α was selected by the investigators for further study. Messages of HIF1α and 3 of its target genes (one representing each function: glycolysis, proliferation/survival and chondrogenic/structural activity) were quantified by RT-PCR to validate the microarray findings. The UDU expression changes were specific to the growth plates, as hepatic expression of HIF1α did not differ among the 3 groups on the first day of liberated feeding. Immunohistochemistry of the proximal humeral EGPs showed HIF1α protein most intensely in the AL and CU proliferative zone cells, mainly in the nucleus (consistent with its function as a transcription factor); RES cells stained weakly for HIF1α. Microdissection and RT-PCR of tibial EGPs showed proliferative zone cells to exceed hypertrophic zone cells in HIF1α message levels as well; the 3-fold expression difference between the zones was constant across experimental conditions. In summary, the authors found that nutritional restriction/refeeding has a significant effect on expression of HIF1α and its targets in the prepubertal rat EGP, and proposed that HIF1α plays an important role in regulating chondrogenesis. They further speculated on the mediators of HIF1α regulation by nutritional restriction/refeeding, including oxygen tension (presumably decreased in CU growth plates due to the increased EGP dimension and/or increased oxygen consumption, both of which can be expected from the rapid growth) and circulating hormone levels (such as insulin-like growth factor [IGF]-I, which is known to induce HIF1α and is itself regulated by nutritional status). One can easily see how this paper has opened the door for many follow-up studies, not just of HIF1α but of the other identified genes as well. Editor’s CommentThis elegantly designed study marks an exciting new era of growth research. Historically, endocrinologists studied growth by focusing on changes in circulating hormone levels. Creation of the LID mouse (liver-specific IGF-I deficient via the cre-lox technique) shocked the traditional paradigm. The LID mice grew normally despite a 75% reduction in circulating levels of IGF-I, highlighting the surprising importance of autocrine/paracrine IGF-I for growth.1 Thus, “the action” is now understood to be local, in the growth plates, rather than the circulation. Growth plate studies have increasingly permeated the growth literature, some of which have been reported in previous issues of GGH.2,3 By applying technological advances, like the microarray and gene database analyses, to the newer growth-plate focus, this paper’s study design has the power to not only spotlight previously expected “players” but identify new ones as well that are important for mediating growth and its various perturbations. Such experimental approaches are likely to herald accelerated advances in the growth field, though confirmatory evidence through alternative models are still required to validate the biologically significant findings and to rule out the potential for inter-species differences. Adda Grimberg, MD References - (linked to
|
Volume 26 Number 1
September 2010
www.GGHjournal.com
ISSN 1932-9032
)