The Physiology and Mechanism of Growth

Introduction

In the present chapter, an international group of endocrinologists tried to select some of the most important articles dealing with the physiology of growth published in the period July 1, 2019 to June 2020. We specially looked for articles that shed more light on the already known mechanisms related to linear growth and those dealing with the interaction between nutrition and growth. We probably missed some important articles that we did not find or could not include in our chapter because of lack of space. In our comments, we tried to explain the reasons for selecting the specific articles and to highlight our own personal conclusion drawn from them. We encourage the readers to read the full article whenever possible.

The role of microRNAs in influencing body growth and development

Comments: There are three main epigenetic mechanisms: DNA methylation, histone modifications, and RNA-based mechanisms. In the present mini-review, Cirillo et al. reviewed the literature related to the role of the microRNAs (miRNAs) in linear growth, specifically they focused on the regulatory effects of miRNAs on growth hormone, insulinlike growth factors, and their related receptors. For many years, clinicians and researchers speculated that epigenetic mechanisms have a role in children’s linear growth even before we knew about miRNA and its mechanism, based on fast changes in the height of populations in different geographical areas around the globe in the last century that could not be explained by genetic differences only. Indeed, miRNAs were found to be influenced by internal and external environmental changes and stimulate or interfere with children’s and adolescents’ linear growth. It is important to stress the fact that in addition to the effect miRNAs have on the GH-IGF-1 axis and their receptors, miRNAs were also shown to directly mediate the effect of the nutritional changes on the growing epiphyseal growth plate [1].

Persistent SOX9 expression in hypertrophic chondrocytes suppresses transdifferentiation into osteoblasts

Comments: The growth of the long bones is dependent on the normal functioning endochondral ossification process, which is a complex process that involves many genes, growth factors, hormones, and environmental factors. In the growth plate, chondrogenesis is an organized process of chondrocyte proliferation, hypertrophy, and cartilage matrix secretion which create a new cartilage which then is remodeled into bone. In the past, we have all learned that in the hypertrophic zone the chondrocytes eventually undergo apoptosis and are replaced by osteoblasts derived directly from the bone marrow mesenchymal progenitor cells in the metaphysis. Recently, it has been shown that many osteoblasts near the growth plate originated from chondrogenic cells in the growth plate.

In the present study, the authors used a mouse model to study the role of SOX9 in the transdifferentiation of chondrocytes into osteoblasts at the cartilage-bone junction. The cause of linear growth abnormalities remains unelucidated in most cases during childhood and adolescence, and most children are therefore diagnosed as suffering
from idiopathic short stature. Many systemic syndromes involve growth retardation of unknown origin. The present study is important since it shed some more light on the role of the SOX family also in the endochondral ossification.

Cartilage ablation of SIRT1 causes inhibition of growth plate chondrogenesis by hyperactivation of mTORC1 signaling

Comments: Sirtuline proteins are involved in regulating cell survival, apoptosis, aging, and other physiological activities. Expression of sirtuline SIRT1 is significantly affected by food restriction [2]. Two articles describing the role of SIRT1 in endochondral ossification, an essential process for physiological skeletal growth were published, expanding the knowledge on mechanisms involved in the regulation of growth by nutritional factors. In the first study, the role of Sirt1 in modulating the response of the epiphyseal growth plate to nutritional manipulation was investigated. Ablation of Sirt1 in the proliferative zone of the growth plate mice resulted in mice being heavier, having shorter bones and less organized and mineralized growth plate. They were less responsive to the nutritional manipulation, and their catch-up growth was less efficient. These results corroborate the data that Sirt1 is important for normal regulation of the epiphyseal growth plate especially in relation to nutrition. In the second study, a causative link between SIRT1 and mTORC1 signaling in the growth plate was determined. Altogether, data from both studies not only add to the knowledge that nutrition has significant influence on skeletal growth, but also suggest SIRT1 as a possible novel therapeutic target in short stature.

Long-term growth in phenylketonuria: A systematic review and meta-analysis

Comments: Neonatal screening and early introduction of phenylalanine-restricted diet dramatically changes outcomes in children, adolescents, and adults with phenylketonuria. Dietary management of phenylketonuria represents a blueprint for treatment of metabolic diseases [3]. In the present meta-analysis, it was, however, determined that optimal growth outcomes are still not achieved in children and adolescents with phenylketonuria. Of importance, it was determined that linear growth is already compromised in the first years of life, where compliance to dietary restrictions is presumed to be more optimal than, e.g., in the adolescence. Further development of dietary treatments is therefore needed. On the other hand, these results advocate for the development of more etiologically based treatment modes as is, e.g., gene-therapy, making dietary treatment less or completely necessary altogether [4].

Humanin prevents undesired apoptosis of chondrocytes without interfering with the anti-inflammatory effect of dexamethasone in collagen-induced arthritis

Comments: In addition to the chronic autoimmune disease itself, glucocorticosteroid treatment has an important negative effect on growth. Glucocorticosteroids remain a cornerstone treatment for many autoimmune diseases in children; therefore, growth-protecting treatment approaches are needed. Glucocorticosteroids affect linear growth by increasing apoptosis and suppressing proliferation in growth plate chondrocytes. Recently, it was shown that endogenous antiapoptotic protein humanin rescues growth plate chondrocytes from glucocorticosteroid-induced apoptosis by regulating Hedgehog signaling [5]. Hedgehog signaling pathway has a central regulatory role in growth plate development [6]. It was therefore proposed that humanin possibly prevents impaired growth in relation to the glucocorticosteroid treatment. To this effect, a synthetic analogue of humanin was developed. Indeed, it prevented glucocorticosteroid-induced toxicity in growth plate chondrocytes of mice, without interfering with the desired anti-inflammatory effect.

Further studies regarding safety and efficacy of humanin analogs in humans are needed; however, it seems a feasible treatment approach to prevent glucocorticosteroidinduced impairment of growth in children with autoimmune disease.

Non-coding RNAs in cartilage development: An updated review

Comments: Long bone growth occurs as a result of complex processes as a result of endochondral ossification by which cartilage is replaced by bone. Whilst it is known that numerous environmental factors like nutrition, disease factors, and host factors (genetics) play a role in these complex processes, the role of epigenetics combines the influences of genetic and environmental factors. This review provides a comprehensive summary of the published role of non-coding RNA and the role of epigenetic regulation of cartilage development. Such studies are only preliminary, and further research is needed to carefully delineate the role of these non-coding RNA and how factors like nutrition, lifestyle factors, disease factors can influence their role in physiology and pathophysiological states.

The majority of transcribed microRNA (mRNA) (non-coding RNA) are not transcribed into proteins and are known as non-protein-coding RNA (ncRNA). ncRNA are further categorized into small RNAs (<200 bp); the most well recognized are mRNA or long RNAs (>200 bp). It is now known that mRNA play an important role in epigenetic regulation of numerous steps of cartilage and bone growth development. mRNA have been shown to play a role in cartilage development and homeostasis, growth plate chondrocyte differentiation. This opens up potential for future epigenetic-based biomarkers of growth and bone disorders, including those as a result of nutritional and insult by drugs/disease. Research into non-coding RNA-based therapies for growth and bone disorders is anticipated.

Duodenal microbiota in stunted undernourished children with enteropathy

Comments: Environmental enteric dysfunction (EED) is a disorder of the small intestine with characteristic histopathological changes which include reduction in the number and height of the intestinal villi. Associated changes also include loss of absorptive surface area, impairment of the epithelial barrier, and chronic inflammatory infiltrate. Evidence suggests that EED is associated with stunting. Previous studies investigating the association between stunting and EED have relied on fecal or plasma biomarkers. The researchers in this present study investigated the levels of bacterial strain from duodenal aspirates in 80 children with biopsy-confirmed EED in Bangladesh. A larger group of children aged 12–18 months received a 3-month nutritional intervention. The participants were divided into those who were stunted (defined as length Z-score <–2.0) and those at risk of stunting (defined as length Z-score between –1.0 and –2.0). Non-responders were defined as those who remained stunted or at risk of stunting after 3 months of nutritional therapy. These non-responders were investigated with esophagogastroduodenoscopy following the 3 months nutritional intervention and also had plasma sample collected. The investigators quantified the levels of 4,077 plasma proteins and 2,619 proteins from these duodenal biopsies. The four plasma proteins most correlated with length Z-score in a positive fashion were insulin growth factor 1, insulin growth factor acid labile subunit, insulin-binding protein 3, and procollagen C endopeptidase enhancer 2. Insulin growth factor-binding protein 2 was negatively associated with length Zscore. As previously reported, there was lack of correlation between length Z-score and histopathological scores from biopsies. Bacterial strains from duodenal biopsies in 14
taxa was negatively correlated with length Z-score. These 14 duodenal taxa could represent biomarkers of EED and provide insight into the mechanism of stunting in EED.

The strength of this study is the use of duodenal aspirates instead of plasma or fecal biomarkers which were used in previous studies. Limitations include the fact that
there were no control subjects (i.e., infants from the same population without EED) with biopsy samples, but this is of course a very challenging and perhaps not ethical research plan. In addition, biopsies in the cases with EED were performed after a 3-month nutritional intervention, and therefore the impact of the nutritional intervention is unknown. Nevertheless, this elegant study provides detailed information which could lead to future development of therapeutic targets.

Early fortification of human milk versus late fortification to promote growth in preterm infant

Comments: Preterm infants are at very high risk of postnatal growth restriction due to numerous factors which include a catabolic state secondary to illnesses like ongoing sepsis, necrotizing enterocolitis, chronic lung disease, need for respiratory support or use of
postnatal steroids. There is no doubt that human breast milk is the best enteral nutrition for preterm infants. Unfortified human milk provides 67 kcal and 1.1 g protein per 100 mL. On the other hand, human milk with human milk fortifier provides approximately 80 kcal and 2 g protein per 100 mL. Generally, current neonatal clinical practice recommends that fortification of human milk should occur when feed volume has reached approximately 100 mL/kg/day. Fortification is often delayed because of clinical concerns about the risk of feed intolerance and necrotizing enterocolitis. However, some clinical reports have introduced fortification from the first feed. This Cochrane systematic review/meta-analysis aims to assess the impact of early fortification of human milk compared with late fortification on growth and safety parameters. Randomized controlled trials of preterm infants (<37 weeks gestation) were included in this review. Early fortification was defined as commencing at <100 mL/kg/day or at postnatal age <7 days. Late fortification was defined as commencing at 100 mL/kg/day or more or at postnatal age 7 days or later. Primary outcome measures were time to regain birth weight, rate of weight gain, rate of gain in length, and increase in head circumference during hospital stay and incidence of necrotizing enterocolitis. Full text review of nine articles was performed, of which only 2 were included in the final qualitative and quantitative analysis. The two RCTs included 237 infants. Shah et al. [7] included infants with birth weight <1,500 g, whereas Sullivan et al. [8] included infants with birth weight of 500–1,250 g.

Meta-analysis of the primary growth outcomes did not show any differences between the 2 groups. There was also no difference between frequency of necrotizing enterocolitis between early and late fortification. Other secondary outcome measures of efficacy and safety were not different between the groups. The authors conclude that there is insufficient evidence to support or refute early fortification. The authors also recommend that future trials should include an adequately powered number of subjects at high risk including those with low birth weight. The issue of more precise estimates on important outcome measures in future clinical trials was raised. Another important factor is also the consideration of the main outcome measures that such future studies should be powered on. Perhaps engagement with patient group, i.e. parents of infants who were previously born preterm is also important so that clinical trials can be powered on outcomes which are of importance to the family. Wider engagement with experts in nutrition, neonatology, and clinical academics with interest in infant growth is also important.

Early life malnutrition causes gastrointestinal dysmotility that is sexually implicit

Comments: Slow gastrointestinal transit time is reported in moderate to severe malnutrition. This dysmotility may further exacerbate poor weight gain due to coexisting gastrointestinal symptoms. The underlying mechanisms of the dysmotility in malnutrition is poorly understood and needs to be teased out using animal models. The investigators of this research used mice models of malnutrition in this study to evaluate gut dysmotility. Neonatal mice underwent timed maternal separation or were fed low-protein, low-fat diet. Adult mice fed low-protein low-fat diet were also used. Carmine red gavage, colonic motility, and fluorescein isothiocyanate-conjugated dextran were used for assessing gastric emptying and small bowel motility. All models of malnutrition exhibited growth failure and had abnormal stomach grossly. In addition, gastrointestinal dysmotility was observed, in particular delayed gastric emptying and poorer small bowel motility. Malnutrition also impaired mucosal structure, intestinal contractility, and gut microbiota. These findings are observed in the clinical conditions like small for gestational age, children with severe malnutrition, and adults with anorexia nervosa.

A major and striking finding of the study is the effect of sexual dimorphism on gastrointestinal dysmotility outcomes. Delayed gastric emptying was observed in the females, whereas there was increased contractile activity in the males but also atrophic gut mucosa and increased gut barrier permeability. Future studies should address the contribution of sex and endocrine factors on dysmotility outcomes to further shed light on the contribution of sex and nutritional status to these gastrointestinal complications.