Longitudinal growth of the long bones in the postnatal period occurs in the epiphyseal growth plates (EGP), located in the proximal and distal parts of the long bones. Among the numerous growth factors, local and systemic, that regulate EGP growth are growth hormone (GH) and insulin-like growth factor 1, insulin, thyroid hormones, sex steroids, and others. However, additional, as yet uncharacterized growth factors may also exist because changes in the above-mentioned growth factors do not explain all of the growth abnormalities. Indeed, growth without GH has also been described and the compensating factor not identified, thus suggesting alternative regulatory systems that control linear growth (1).
The effect of nutrition on linear growth is well established. Growth stunting constitutes the most common effect of malnutrition, and numerous reports describe considerable height gain with food supplementation. However, because most studies were performed in severely malnourished children, it was impossible to dissect the effect of protein deficiency from deficiency of the other nutrients required in children's diet, namely phosphorus, calcium, zinc, potassium, and other micronutrients (2).
To this day an average of 33% of all children younger than 5 years of age in the developing countries have linear growth retardation or stunting due to chronic malnutrition, which is caused by food shortage as well as by infectious diseases. Malnutrition is also associated with developmental delay, including cognitive deficits, poorer school achievement, and lower IQ. In younger children it is associated with conduct, poorer attention, and poorer social skills at school (3). Most studies were performed on malnourished children in developing countries and only a few were performed in developed countries; however, no significant breakthrough was made in the last decades to understand the precise association between nutrition and growth.
Deciphering the mechanisms that translate the signals of energetic resources to a signal that allows growth may allow the development of novel therapeutic regimen for children with idiopathic growth abnormalities.
Catch-up (CU) growth is defined as “height velocity above the normal statistical limits for age and/or maturity during a defined period of time, following a transient period of growth inhibition.” Resolution of growth-inhibiting condition is followed by a period of spontaneous CU growth and, depending on the age of the child, may lead to correct growth. One excellent example for nutrition-induced CU growth is in childhood celiac disease. In these children, there is remarkable CU growth shortly after the onset of a gluten-free diet.
Although several hormones have been shown to be affected by nutritional manipulations, CU growth is probably an intrinsic capability of the EGP (4). In our attempts to study the mechanisms governing nutritional-induced CU growth in the EGP, we subjected prepubertal rodents (rats and mice) to 10 days of 40% food restriction, followed by a renewal of the regular food supply for up to 7 days. We found a dramatic difference in weight between the control and the restricted groups, as well as a significant difference in the length of the tibias. The EGP height was significantly lower in the restricted group, and a height change was observed in all cellular zones. Under these conditions, the changes were partially reversible because refeeding led to an instantaneous increase in body weight, which was accompanied by an increase in tibial and EGP length (5,6).
Among the hormones known to be affected by nutritional manipulation, we decided to focus on leptin, a hormone secreted from the adipocytes. It was found to be involved in the regulation of food intake and body weight as well as of bone density. Leptin leads to reduced food consumption and reduced weight gain in rodents. We have shown that under leptin treatment, leptin increased the length of the tibia, the overall size of the EGP, and stimulated proliferation activity in the chondrocytes of the EGP compared with pair-fed animals. The length of the tibia increased significantly in the leptin-treated animals compared with the untreated controls. Although it was previously described that leptin affects growth centrally by stimulating GH secretion through its effect on GH-releasing hormone, we have shown that the effect of leptin was independent of insulin-like growth factor 1 and that leptin has a local, direct, GH-independent stimulatory effect on the EGP through its receptor (7). We have shown that leptin's effect on the growth-plate chondrocytes is specifically mediated through ERK1/2 and STAT3 (8).
Leptin appears to be an important mediator between nutrition and growth and may play an important role in CU growth. However, further studies suggested that leptin alone is not enough to explain the major changes during nutrition-induced CU growth, and we are still searching for other possible mediators.
NUTRITION AND THE EPIPHYSEAL GROWTH PLATE
Examining the processes occurring in the EGP during CU growth would enable us to characterize the most important pathways for growth acceleration occurring during CU.
Using an Affymetrix expression microarray, we analyzed changes in gene expression during food restriction and CU growth (5). The results showed changes in expression in hundreds of genes, from which we focused only on those showing a decrease in expression in the food-restricted group and a concomitant increase in expression in the CU group, compared with the control group. We identified among these genes the transcription factor hypoxia-inducible factor 1α and several of its downstream targets, suggesting that hypoxia-inducible factor 1α is a possible mediator between nutrition and growth and may play an important role in CU growth.
Further studies suggested the involvement of additional regulatory mechanisms, such as microRNAs (miRNAs) and epigenetic regulation. The first are small nonprotein-coding RNAs, measuring approximately 21 to 23 nucleotides in length, that negatively regulate the expression of a large portion of protein-encoding and nonprotein-encoding genes at the posttranscriptional level. Each miRNA can regulate 1 to several mRNA transcripts, and conversely, a single mRNA may be regulated by 1 to several miRNA sequences. The central role of miRNAs in skeletal development was demonstrated in mice devoid of the cytoplasmic RNAse III Dicer enzyme, an essential enzyme in the metabolism of miRNAs, in their cartilage. These animals showed that Dicer is required for the formation of normal mouse limbs (9). In addition, several miRNAs were shown to be involved in metabolism; thus, it is reasonable to suggest a regulatory role for miRNAs in nutritional-induced growth regulation of the EGP.
Epigenetic mechanism, defined as DNA methylation patterns and associated posttranscriptional modifications of histones, is thought to influence the programming of gene expression profiles. In the growth plate, histone deacetylase (HDAC) 4 was recently shown to be essential for the hypertrophy process. Furthermore, it was shown that the cartilage-specific miR-140 regulates HDAC4 in growth plate, thus suggesting a complex mode of epigenetic regulation. Another class of HDACs, the sirtuins, are highly conserved enzymes that use nicotinamide adenine dinucleotide (NAD+) to deacetylate a number of histone and nonhistone substrates. Recently, it was shown that both SIRT1 and SIRT6 are increased in response to long-term energy restriction in several organs, suggesting that similar effect can occur in the EGP (10).
Longitudinal bone growth at the growth plate is governed by a complex network of signals; however, to this day, the exact mechanism by which nutrition affects growth has not been elucidated. CU growth is a fascinating capability of the growth plate that is associated with systemic as well as local growth factors, microRNAs, and epigenetic mechanisms. By using innovative experimental approaches we aim to decipher the regulatory signals that mediate the effect of nutrition on CU growth. Understanding these processes may lead to the development of novel therapeutic regimines and diagnostic approaches to treat children with idiopathic growth abnormalities, especially when the response to GH treatment is not satisfactory.
1. Phillip M, Moran O, Lazar L. Growth without growth hormone. J Pediatr Endocrinol Metab 2002; 15(Suppl 5):1267–1272.
2. Rosado JL. Separate and joint effects of micronutrient deficiencies on linear growth. J Nutr 1999; 129(2S Suppl):531S–533S.
3. Walker SP, Wachs TD, Gardner JM, et al
. Child development: risk factors for adverse outcomes in developing countries. Lancet 2007; 369:145–157.
4. Gafni RI, Weise M, Robrecht DT, et al
. Catch-up growth is associated with delayed senescence of the growth plate in rabbits. Pediatr Res 2001; 50:618–623.
5. Even-Zohar N, Jacob J, Amariglio N, et al
. Nutrition-induced catch-up growth increases hypoxia inducible factor 1 alpha RNA levels in the growth plate. Bone 2008; 42:505–515.
6. Gat-Yablonski G, Shtaif B, Abraham E, et al
. Nutrition-induced catch-up growth at the growth plate. J Pediatr Endocrinol Metab 2008; 21:879–893.
7. Gat-Yablonski G, Ben-Ari T, Shtaif B, et al
. Leptin reverses the inhibitory effect of caloric restriction on longitudinal growth. Endocrinology 2004; 145:343–350.
8. Ben-Eliezer M, Phillip M, Gat-Yablonski G. Leptin regulates chondrogenic differentiation in ATDC5 cell-line through JAK/STAT and MAPK pathways. Endocrine 2007; 32:235–244.
9. Harfe BD, McManus MT, Mansfield JH, et al
. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb. Proc Natl Acad Sci U S A 2005; 102:10898–11103.
10. Cohen HY, Miller C, Bitterman KJ, et al
. Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science 2004; 305:390–392.