Thyroid and Growth Hormones Interdependence: A “Reciprocal Potentiation”, the Synergy for Growth and Development at Childhood.

Aims: To investigate the effect of variations of plasma levels of thyroid and growth hormones on the physical growth indexes, the variation of these hormones with age, correlation of thyroid and growth hormones at varying concentrations of growth hormone in children aged 6-10 years. Study Designed: Department of Medical Laboratory Science, University of Calabar, conducted between March 2014 and February 2015. Methodology: Anthropometric uniqueness were described and Body Mass Index calculated for 180 (male 81 and female 99) participants of the study. Thyroid and growth hormones were determined by Enzyme-Linked Immunosorbent Assay (ELISA) specific for the various hormones, using STAT FAX 303 microtitre plate reader. Results: Nineteen percent (n=34) had GH values (≤0.6 ng/ml), lower than the expected normal, 64% (n=115) had values between (0.6-10.0ng/ml) while 17% (n=31) had values ≥10.0 ng/ml. Original Research Article Eworo et al.; BJMMR, 9(2): 1-9, 2015; Article no.BJMMR.18659 2 Ninety two percent (n=166) of the children had thyroid stimulating hormone (TSH) values between 0.35-8.44 μIu/ml; 6.1% (n=11) had values >8.44 μIu/ml which are above normal while1.7% (n=3) had values <0.35 μIu/ml lower than normal. Triiodothyronine appears to increase from birth recording a peak within the first year of postnatal life, and then progressively declining to adult values while GH increases from birth attaining a peak about the age of nine then falling progressively to adult values. Children with apparent GH deficiency had significantly higher T3 and T4 levels and lower TSH, those with high GH values had correspondingly low T3 and T4 values and high TSH. The correlation coefficients of TSH and growth hormones in those with high and low GH (r=-0.05 and r=-0.130) respectively were both negative while that of TSH and GH in those with normal GH was positive (r=0.093), however, physical growth indexes are preserved across board. Conclusion: It was concluded from the study that growth failure may be due to failure of both hormones and may likely not occur when an unbroken synergy exist between thyroid and growth hormones during childhood.


INTRODUCTION
Atypical growth and development at childhood have been associated with a psycho-social burden and abnormal reproductive adulthood. Stunted growth due to growth hormone deficiency had been treated over the past two decades with recombinant human growth hormones. The biochemical changes and clinical symptoms of the effect of this practice on other hormonal axes had led to the realization of how the GH-insulin-like growth factor-I axis and the adrenal, gonadal and thyroid axes are connected and affect each other. A complex liaison exists between the GH-IGF-I and the Hypothalamic-Pituitary-Thyroid (HPT) axes. The interdependent rapport between growth and thyroid hormones seems to guarantee normal process of growth and development, since abundance of one hormone compensates for the deficiency of the other hormone. The synergy allows preservation of critical developmental processes such as the development of the brain and physical growth indexes even at reciprocal concentrations of these hormones especially in children. GH replacement unmasks central hypothyroidism in 36-47 per cent of subjects with normal thyroid, biochemically, with consequent reduction in the benefit of the replacement plan [1].
Thyroid and growth hormones, apart from their conventional pathways equally have the PI3K (phosphatidylinositol 3-kinase) and MAPK (MAPK: Mitogen-activated protein kinase) noncanonical pathways common to both. The relationship may be initiated from developments in the PI3K and MAPK, common downstream signal transduction pathways for these hormones. Since the pathway uses the same effectors, alteration in gene expression profile is the ultimate outcome; the abundant hormone compensate for the insufficient hormone by engaging the pathway segment common to both in implementing alteration in gene expression.
Thyroid and Growth hormones demonstrate a range of physiologic functions in metabolism, growth and development [2]. The anabolic chattels of growth hormones on tissues are exemplified by their influence on increased protein synthesis and decreased protein breakdown; the proteins may serve as enzymes or structural proteins.
Growth hormone stimulates IGF-1 mRNA synthesis [3] and thereby has both IGF-1independent and IGF-1 mediated measures through which it stimulates growth of skeletal system, and influences the accessibility of metabolic fuels. The growth promoting actions of GH are principally mediated through production of the insulin-like growth factor (IGF-I), which exert both an endocrine and paracrine effect [4]. IGF-1 stimulates the proliferation of chondrocytes in vitro [5], and remarkable decline in IGF-1 levels in young animals corresponds with decreased bone growth. The most typified anabolic action of GH growth in children is the stimulation of longitudinal bone, preceding closure of epiphyses. Bone growth is analogous to growth of other tissues and accrues from increased cell number, size, and extracellular ground substance. Longitudinal bone growth is mediated by a complex series of events that occur within the epiphyseal growth plate [6]. As long as the epiphyseal growth plates are able to produce chondrocytes, the bones continue to grow and linear growth continues. GH and IGF-1 have corresponding roles in stimulating bone growth, GH stimulates cartilage precursor cells to differentiate and become receptive to autocrine, paracrine and mitogenic effects of IGF-1 [7]. In this way GH and IGF-1 promote skeletal growth, maintenance, repair and regeneration. Deficiency of GH in adult results in decreased lean body mass and considerable improvement in muscle mass (weight) and BMI; have been observed upon GH replacement [8,9]. This may be owed to the increased protein synthetic and protein conservative ability of GH. The physiological effects of GH on growth and metabolism are achieved through alterations of gene expression profile and synthesis of IGF-1 mRNA [10].
Disruption of GH-IGF-1 axis in mouse models reliably yielded degeneration in parameters of bone health [11], with a striking decrease in cancellous bone volume, connectivity, trabecular number and spacing [12]. Induction of supraphysiological level of IGF-1 in osteoblast causes an increase in bone mineral density and trabecular bone volume [13]. Excess glucocorticoids affect growth by the lowering of local IGF-I production through IGF-I transcription inhibition, increased rate of apoptosis in growth plate chondrocytes and osteoblast cell lines, increased bone resorption, inhibit osteoblast activity, and reduce bone matrix production to cause growth retardation in children [14] and osteoporosis in adults [15]. The effect can be offset by GH in vitro and in vivo.
Thyroid hormones mediate cell proliferation and together with insulin-like growth factor-1 (IGF-1), insulin and glucocorticoids [16]; they regulate body protein metabolism, thereby, are intimately associated with the processes involved in growth and development. Growth occurs through synthesis of fresh protoplasm from nutrient materials. Caloric restriction and systemic illness obviously restrict growth, as can be deduced from malabsorption syndromes, metabolic acidosis, inflammatory diseases, renal failure and prolonged serious illness [17].
Thyroid hormones have general and specific effects on growth and development. Thyroid hormones affect growth by activation of nuclear transcription of large numbers of genes in practically all cells of the body consequently large number of protein enzymes, structural proteins, transport proteins and other substances are synthesized and available for the body's use. The early growth and development of most organs especially the brain, the metabolic models of most cells, direct manipulation on the linear growth through stimulation of DNA synthesis in osteoblasts are controlled by thyroid hormones [18].
Growth and development during childhood result from increased size of cell population; determined by the rates of cell proliferation, differentiation and cell death by apoptosis, this is put to check by a system of hormonal mechanism, the key hormone being triiodothyronine (T3) [19,20]. Thyroid hormone has the ability to stimulate the proliferation of IGF-1 mRNA in many tissues and has synergistic effect on GH, specifically in regulation of the growth hormone transcription gene.
GH and thyroid hormones are the major regulators of developmental and childhood growth. Regulations are achieved through their interactions at hypothalamus, pituitary and peripheral tissues such as the liver and most importantly at the epiphyseal growth plate the target organ for convergent hormone action [21]. The effects of hypothyroidism such as reduced growth plate width, articular cartilage, and trabecular bone volume were reversed by combination of GH and T4 in animal models [22].

MATERIALS AND METHODS
This study was conducted in Calabar, Cross River State, Nigeria. Ethical approval was obtained from the Research Ethical Committee of the Cross River State Ministry of Health. The cooperations of the heads of the various Schools were obtained and informed consent sought after from parents of the children before being enlisted in the study, an assent was equally given by each child whose consent had been given by the parents. The study recruited 180 (The sample size of 180 was determined using using the formula- Where Z=degree of reliability (level of confidence) 95% standard at 1.96, P= estimated prevalence (13%= 0.13), D= margin of error 0.05%, n= required sample size) children in the ages between 6-10 years, attending both private and public primary schools in Calabar. A standard questionnaire was designed and administered to each of the participants. Anthropometric characteristics were described for all the participants and BMI calculated.
A standard venepuncture method was used to obtain three milliliters (3 ml) of venous blood from all the children under aseptic condition; dispensed into plain containers. The samples were allowed to clot and retract then spun at 3000 rpm for 5 minutes. The supernatants obtained were stored frozen till analysis. GH and thyroxin were analyzed by Enzyme Linked Immunosorbent Assay (ELISA) while TSH and Triiodothyronine T3 were determined by Enzyme immunoassay (EIA) and the analyses read using STAT FAX 303 microtitre plate reader; all reagents were obtained from DRG international Inc. USA. Statistical analysis: data generated in this study were analyzed for level of significance (P = .05) using SPSS (version 20) statistical software. Variations among groups were determined via ANOVA, student's t-test and Pearson's correlation.

RESULTS
This research determined growth hormone, thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormones (TSH) in 180 participants of the study. Table 1 shows physical and biochemical parameter of the various age groups in our study population. Table 2 shows physical and biochemical parameters between those with low, normal and high levels of growth hormones, the compensation for low hormone by the high circulating level of the other hormone resulting in the protection of physical growth indexes among the various groups may be seen. Fig. 1 shows a scatter plot of the means of GH against ages in years, the progressive increase in GH before the attainment of the peak may be seen. Fig. 2 shows a scatter plot of the means of T3 against ages in years; the progressive decrease in T3 towards adult values may be seen. Figs. 3, 4 and 5 show correlations of GH and TSH in children with normal, low and high GH correspondingly.

DISCUSSION
Growth and thyroid hormones are the principal hormones concerned with the regulation of growth and development during childhood. They are essential for normal development, growth and metabolism through coordinated actions on different tissues, including liver, adipose tissue, skeletal system and bone [23]. The mean values of T3 and T4 in our locality; 225 ng/dL and 11.34 µg/dL were observed to be higher than 2.20 nmol/L (143.22 ng/dL) and 115 nmol/L (8.98 µg/dL) correspondingly, established in the pediatric reference range, for children in this age bracket [24] (Table 1). This may be due to differences in diet affecting the availability of the precursors of these hormones and their binding proteins. Children generally gain height and weight as they grow from one year to the next, but the biochemical changes that accompany the physical growth are variable.
Thyroid hormones economy during childhood is quite inconsistent, a few hours after birth perhaps due to temperature changes plasma TSH increases strikingly in normal newborns, with a peak in the first 24 hours of life. Plasma T4 increases shallowly in rejoinder, peaking during  This level of T3 during infancy may likely be the highest in normal life, the level keep decreasing progressively tending toward adult values after the age of 10 years, as growth hormone (GH) assumes a more dominant physiological role. GH on the other hand increases progressively with peaks occurring at about 9 years, and then beginning to decline to adult levels. This developmental age 9 years, corresponds to the middle of the linear growth period, when GH and IGF-1 play key roles in growth and development ( Figs. 1 and 2).
Thyroid and GH hormones have a converging point in their actions, they stimulate IGF-1 mRNA synthesis in several tissues and this allows them to function in synergy. Their interdependent relationship may be referred to as "reciprocal

Fig. 4. Correlation of GH and TSH in those with low GH
potentiation" as the physical growth indices among children with low, normal and high GH are not significantly different, the variations in GH being compensated for by corresponding increases or decreases in thyroid hormones (Table 2). Also, the correlation coefficients of TSH and GH in those with low and high GH correspondingly are negative (Figs. 4 & 5), while that of TSH and GH in those with normal GH, is positive (Fig. 3), however, the physical growth indices are not significantly different across board (Table 2), implying a synergistic effect and a 'reciprocal potentiation' between the hormones. The hormones reciprocally potentiate one another by that interplay between T3, T4, and GH at all concentrations. Children with low T3 and T4 had high GH recompensing for the low T3 and a high TSH working to make more thyroid hormones, while those with low GH had high T3 and T4 and low THS due to the negative

Fig. 5. Correlation of GH and TSH in those with high
feedback inhibition from the high T3 and T4, this guarantees the preservation of the growth process. These observations are not in line with those of [25], who demonstrated an association of hyperthyroidism with increase means 24-hour serum GH concentration and secretion rates, and [26], who found association between hypothyroidism with an attenuation of GH response to secretagogues and decreased GH pulses. Many potential explanations had been proffered; all in an attempt to elucidate the observed interdependence between the hormones. One mechanism suggested the presence of atypical TRH receptors on somatotropes, perhaps in the pituitaries of acromegalic subjects [27], another proposed an impairment of the inhibitory hypothalamic control of GH secretion [28] or secretion from fetal, neonatal, and prepubertal animals in which the hypothalamic control of GH secretion is presumably functionally immature [29]. Downstream signaling pathways for these hormones may be important in the explanation of the synergy between these hormones.
Apart from the classical pathways for these hormones they equally have noncanonical signaling pathways that lead to gene transcription. Thyroid hormone action on gene expression now includes nonclassical actions of T3 and T4, T3 has been demonstrated to activate PI3K through the thyroid receptors, eventually increasing transcription of certain genes, also, both T3 and thyroxine (T4) can bind to a membrane integrin, αvβ3, which leads to activation of the PI3K and MAPK signal transduction pathways, finally increasing gene transcription [23]. In spite of the JAK2/STAT conventional pathways for GH action several intracellular signal transduction pathways are activated by GH [30]. JAK2 phosphorylation of non-receptor protein tyrosine kinase (src) initiates a cascade of the MAPK pathway with activation of ERK, whereas phosphorylation of insulin receptor substrate-1(IRS-1) triggers the PI3K pathway. Noncanonical signaling independent of JAK2 through src leading to activation of extracellular signal-regulated kinase (ERK) has also been demonstrated. These pathways can then activate signaling effectors such as transcription factors to directly influence gene regulation. The PI3K and MAPK signal transduction pathways are common to both hormones. The "reciprocal potentiation" referred to above may be due to sequential dedication and engagement of the pathway by the abundant hormone which then yield end products common to the separate actions of the two hormones, thereby reciprocally potentiating one another's effect. Since the pathways lead to alterations of gene expression, the engagement of the pathway by the abundant hormones may have the same net effect of altered gene expression profile that the separate hormones would, when present and acting separately at physiological levels. And by this way the hormones make up for one another's shortfall, the benefit of this rapport is continued normal growth and

CONCLUSION
The processes of growth and development in children are salient for normal social and reproductive adulthood, the synergy between growth and thyroid hormones serve as a double mechanism to ensure proper development.
Understanding of the molecular mechanisms underlying growth and thyroid hormone actions may have significant implication in human health and diseases. Therefore, in addressing the feasibilities of pituitary hormone replacement; the reciprocal effects that replacement of thyroid and growth hormones may have on the GH-IGF-I and the thyroid axes, respectively should be considered. It was concluded from the study that growth failure may be due to failure of both hormones and may likely not occur when an unbroken synergy exist between thyroid and growth hormones during childhood.