Growth Hormone Gene Expression Research Articles

Creatine or guanidinoacetic acid? Which is more effective at enhancing growth, tissue creatine stores, quality of meat, and genes controlling growth/myogenesis in Mulard ducks

ABSTRACT This study aimed to determine the effectiveness of guanidinoacetic acid (GAA) with or without methionine (Met) compared to creatine (CREA) at enhancing duck’s performance, restoring tissue CREA and improving meat quality. Mulard ducklings (n = 250) were randomly assigned to control (without additives), or control plus CREA, GAA, GAA + 0.2%Met (GAAMet0.2) or GAA + 0.4%Met (GAAMet0.4) groups in a completely random experimental design. Dietary supplementation of CREA or GAA significantly increased (P < 0.05) overall weight gain and improved feed:gain ratio. Supplementation of GAA (especially GAAMet0.4 group) significantly increased (P < 0.05) carcass and breast yield. Meat pH values were higher (P < 0.05) with dietary GAA + Met or CREA. Providing of dietary GAA + Met led to higher levels of plasma CREA than dietary CREA itself. The molecular investigation indicated that dietary CREA or GAA with Met enhanced the gene expression of hepatic insulin-like growth factor-1, growth hormone and muscle myogenin. Finally, dietary GAA + Met was superior to CREA in improving duck’s performance based on molecular markers related to growth (IGF-1 and growth hormone) and myogenesis (upregulating myogenin and downregulating myostatin). Although, dietary GAA + Met enhanced muscle’s CREA loading than CREA, the long-term GAA supplementation in ducks may induce methyl-groups shortage for protein synthesis, this was resolved with Met addition in our study.

A Zebrafish Acromegaly Model Elevates DNA Damage and Impairs DNA Repair Pathways.

Acromegaly is a pathological condition due to excess growth hormone (GH) secretion. Acromegaly patients exhibit a deterioration of health and many associated complications, such as cardiovascular issues, arthritis, kidney diseases, muscular weakness, and colon cancer. Since these complications are generalized throughout the body, we investigated the effect of GH excess on cellular integrity. Here, we established stable acromegaly model zebrafish lines that overexpress tilapia GH and the red fluorescence protein (RFP) reporter gene for tracking GH gene expression throughout generations, and performed RNA-Seq data analysis from different organs. Intriguingly, heatmap and Expression2Kinases (X2K) analysis revealed the enrichment of DNA damage markers in various organs. Moreover, H2A.X immunostaining analysis in acromegaly zebrafish larvae and the adult acromegaly model brain and muscle showed a robust increase in the number of DNA-damaged cells. Using Gene Set Enrichment Analysis (GSEA), we found that the acromegaly zebrafish model had impaired DNA repair pathways in the liver, such as double-strand break (DSB), homologous recombination repair (HRR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), and translesion synthesis (TLS). Interestingly, the impairment of DNA repair was even more prominent in acromegaly model than in aged zebrafish (three years old). Thus, our study demonstrates that affection of cellular integrity is characteristic of acromegaly.

Cloning and differential expression of the growth hormone in Pampus argenteus

The growth hormone (GH) is a pluripotent hormone produced by the pituitary in vertebrates. It plays important roles in the growth, development, and metabolism of vertebrates.We cloned GH cDNA sequence of Pampus argenteus (GenBank: KT257176). Multi-sequence analysis revealed P. argenteus GH cDNA contained four conservative cysteine residues positions (Cys69, Cys177, Cys194, and Cys202) and shared more than 51.5% identity with homologues from other reported bony fish GHs, except that of Lepisosteus osseus. We used semi-quantitative RT-PCR and quantitative real-time PCR to detect GH expression in 10 tissues and GH expression levels in the pituitary at six different growth stages, and also detected GH content in serum at different growth stages. qPCR showed that GH mRNA was detected in the liver, muscle, kidney, intestine, pituitary, olfactory bulb, stomach, heart, gill, and ovary. The highest level of P. argenteus GH mRNA was observed in the pituitary (P < 0.01, n = 3). At different growth stages, P. argenteus GH expression first increased, decreased, and increased again. GH gene expression levels and the variations of serum GH levels of P. argenteus were consistent with the growth rate and associated with the sexual maturity. In addition, in situ hybridization was used to locate the GH expression in pituitary. In situ hybridization showed that the GH-positive cells were round, oval, or irregular and often gathered into groups or presented branches along the nerve fibers.

Effects of chronic thermal stress on growth performance, carcass traits, antioxidant indices and the expression of HSP70, growth hormone and superoxide dismutase genes in two broiler strains

The objective was to investigate the effects of genetic type and the duration of chronic thermal stress (36 °C) on the growing efficiency, carcass traits, antioxidant status, and the expression of liver heat shock protein 70 (HSP70), growth hormone (GH) and superoxide dismutase (SOD) genes. Two hundred and seventy one-day-old chicks (135 male chicks of each breed; Ross 308 and Cobb 500) were used in this work. On the 21st day of age, birds were allocated randomly into 3 equal groups till the 42 days of age (CON:raised in a thermoneutral condition; HS1 and HS2 groups were subjected to 4 and 6 h of daily thermal stress, respectively). Regardless of genetic type, thermal stress decreased the dressing percentage in broilers when compared with the thermoneutral conditions (p = 0.039). In both broiler strains, thermal stress for 6 h (HS2) increased the heterophil to lymphocyte ratio (p = 0.036) and the serum albumin, cholesterol and triglyceride levels (p = 0.023, 0.012 and 0.005, respectively) compared with the thermoneutral group. Under the thermonuteral and heat stress conditions, the Ross broiler chickens showed a significant lower serum triiodothyronine level compared with the Cobb boilers (p = 0.042). It is interesting to note that the expression of HSP70 in the liver of heat-stressed Ross broilers, either 4 or 6 h, was significantly (p = 0.002) higher than that reported in the heat-stressed Cobb broilers. In both broiler strains, the thermal stress for 6 h up-regulate the expression of SOD gene (p = 0.001), but down-regulate the expression of GH gene (p = 0.021) when compared with the CON group. In conclusion, chronic thermal stress down-regulate the mRNA expression of liver GH, concomitantly with an increase in the expression of HSP70 and SOD genes in both broiler strains. This could be useful in the identification of molecular genetic markers to assist in selecting broilers that are more tolerant to heat stress.

Expression of growth hormone gene in the baboon eye

The human growth hormone (GH) locus is comprised by two GH (GH1 and GH2) genes and three chorionic somatomammotropin (CSH1, CSH2 and CSH-L) genes. While GH1 is expressed in the pituitary gland, the rest are expressed in the placenta. However, GH1 is also expressed in several extrapituitary tissues, including the eye. So to understand the role of this hormone in the eye we used the baboon (Papio hamadryas), that like humans has a multigenic GH locus; we set up to investigate the expression and regulation of GH locus in adult and fetal baboon ocular tissues.We searched in baboon ocular tissues the expression of GH1, GH2, CSH1/2, Pit1 (pituitary transcription factor 1), GHR (growth hormone receptor), GHRH (growth hormone releasing hormone), GHRHR (growth hormone releasing hormone receptor), SST (somatostatin), SSTR1 (somatostatin receptor 1), SSTR2 (somatostatin receptor 2), SSTR3 (somatostatin receptor 3), SSTR4 (somatostatin receptor 4), and SSTR5 (somatostatin receptor 5) mRNA transcripts and derived proteins, by qPCR and immunofluorescence assays, respectively. The transcripts found were characterized by cDNA cloning and sequencing, having found only the one belonging to GH1 gene, mainly in the retina/choroid tissues. Through immunofluorescence assays the presence of GH1 and GHR proteins was confirmed in several retinal cell layers. Among the possible neuroendocrine regulators that may control local GH1 expression are GHRH and SST, since their mRNAs and proteins were found mainly in the retina/choroid tissues, as well as their corresponding receptors (GHRH and SSTR1-SSTR5). None of the ocular tissues express Pit1, so gene expression of GH1 in baboon eye could be independent of Pit1. We conclude that to understand the regulation of GH in the human eye, the baboon offers a very good experimental model.

Growth hormone transgenesis in coho salmon disrupts muscle immune function impacting cross-talk with growth systems.

Suppression of growth during infection may aid resource allocation towards effective immune function. Past work supporting this hypothesis in salmonid fish revealed an immune-responsive regulation of the insulin-like growth factor (IGF) system - an endocrine pathway downstream of growth hormone (GH). Skeletal muscle is the main target for growth and energetic storage in fish, yet little is known about how its growth is regulated during an immune response. We addressed this knowledge gap by characterising muscle immune responses in size-matched coho salmon (Oncorhynchus kisutch) achieving different growth rates. We compared a wild-type strain with two GH transgenic groups from the same genetic background achieving either maximal or suppressed growth - a design separating GH's direct effects from its influence on growth rate and nutritional state. Fish were sampled 30 h post-injection with phosphate-buffered saline (control) or mimics of bacterial or viral infection. We quantified mRNA expression levels for genes from the GH, GH receptor, IGF hormone, IGF1 receptor and IGF-binding protein families, along with immune genes involved in inflammatory or antiviral responses and muscle growth status marker genes. We demonstrate dampened immune function in GH transgenics compared with wild-type. The muscle of GH transgenics achieving rapid growth showed no detectable antiviral response, coupled with evidence of a constitutive inflammatory state. GH and IGF system gene expression was strongly altered by GH transgenesis and fast growth, both for baseline expression and responses to immune stimulation. Thus, GH transgenesis strongly disrupts muscle immune status and normal GH and IGF system expression responses to immune stimulation.

Effect of Dietary Calorie and Protein Content on Performance, Behaviour, Expression of some Growth-Related Genes and Economic of Broiler Chickens

The goal of this study was to evaluate the effects of variable dietary metabolizable energy and crude protein concentrations on the performance, behaviour, expression of growth related genes and economic impact of broiler chickens. A total of 250 Cobb broiler chicks were divided randomly into five groups of ten replicates (10 chicks/replicate). Five diets with five different combinations of calories and protein were formulated as the following; normal energy and normal protein (NENP), low energy and high protein (LEHP), high energy and low protein (HELP), normal energy and low protein (NELP), low energy and normal protein (LENP) during starter and finisher period. The results revealed that the highest body weight and body weight gain were observed in the HELP and NELP groups which were similar in improvement of feed utilization and protein efficiency ratio. The total feed intake was increased with decreased dietary energy as in NENP group. Nutrient digestibility was improved with increasing diet energy density as in HELP and NELP groups. The expression of IGF-1 and growth hormone genes were markedly higher in HELP and NELP groups, while the myogenin expression significantly increased in HELP, NELP and LEHP groups. Moreover, the final weight greatly correlated with the gene expression related to growth. Eating and drinking frequencies were highest in LENP and LEHP groups. Birds reared in HELP diet were more active, as expressed by greater walking, wing shaking, leg stretch, preening and flying. Briefly, our results suggested that the energy of diets greatly affected the broiler performance, behaviour and digestibility. Additionally, inclusion of low protein diet with addition of critical amino acids had a positive effect on economic efficiency of broilers. Thus, NELP and HELP diets are recommending for growth of Cobb broiler and could have a significant economic impact.

Effects of l-arginine on growth hormone and insulin-like growth factor 1.

l-Arginine has been reported to promote cellular and organismal growth. In this study, the effects of l-arginine on the expression of growth hormone (GH) and insulin-like growth factor 1 (IGF-1), the two key growth factors, are investigated in cultured GH3 pituitary epithelium and HepG2 cells, respectively. l-Arginine significantly induced the gene expression of GH and IGF-1 in GH3 pituitary epithelium and HepG2 hepatocytes respectively, and reduced IGF binding protein-1 gene expression in HepG2 cells assessed via quantitative polymerase chain reaction analysis. l-Arginine also significantly induced GH and IGF-1 hormone secretion from GH3 and HepG2 cells, respectively. In addition, the multi-target ELISA analysis conducted revealed that phosphorylation of p-38 MAPK, MEK, and JNK were significantly increased in HepG2 cells, suggesting l-arginine-induced activation of the MAPK signaling pathway. These results suggest that l-arginine promotes the synthesis and secretion of GH and IGF-1 in vitro and induces the MAPK signaling cascade in cultured hepatocytes.

Obestatin stimulates the somatotrophic axis activity in sheep

The effects of obestatin (an anorexigenic peripheral peptide) on somatotrophic axis activity in ruminants have not yet been determined. The aim of this study was to investigate the consequence of intracerebroventricular infusions of obestatin on the activity of the somatotrophic axis in peripubertal female sheep. Animals were randomly divided into two groups: control group received intracerebroventricular infusions of the vehicle, and the obestatin group was infused with obestatin (25 µg/120 µL h−1). The series of four hourly infusions on three consecutive days were performed. The blood samples were collected on day 0 and on day 3. Immediately after the end of experiment sheep were slaughtered. Parts of the brains were fixed in situ for further immunohistochemical analysis, while the remaining brains were frozen for Real Time RT-qPCR analysis.Substantial changes in the activity of the somatotrophic axis were observed in obestatin-infused sheep. In those animals obestatin evoked an increase in growth hormone-releasing hormone (GHRH) mRNA expression and a decrease in somatostatin mRNA expression in the anterior hypothalamic area. Moreover, a decrease in somatostatin immunoreactivity in the periventricular nucleus and an increase in somatostatin immunopositive fibers in the median eminence were noted. Changes in the GHRH and somatostatin activity are associated with an increase in growth hormone (GH) gene expression and in the amount of GH immunoreactive material stored in the somatotrophic pituitary cells. Consequently, an increase in GH concentration in the peripheral blood, due to an increase in the number of pulses was observed.It was revealed that obestatin affects the somatostatin/GHRH/GH system at the level of protein synthesis, accumulation and release. It is suggested that obestatin participates in the mechanism modulating somatotrophic axis activity at the central level by stimulating GH release through suppression of somatostatin output. Thereby, it can be concluded that obestatin may be involved in the modulation of growth processes in sheep.

Exercise training reverses the negative effects of chronic L-arginine supplementation on insulin sensitivity

L-Arginine has emerged as an important supplement for athletes and non-athletes in order to improve performance. Arginine has been extensively used as substrate for nitric oxide synthesis, leading to increased vasodilatation and hormonal secretion. However, the chronic consumption of arginine has been shown to impair insulin sensitivity. In the present study, we aimed to evaluate whether chronic arginine supplementation associated with exercise training would have a beneficial impact on insulin sensitivity. We, therefore, treated Wistar rats for 4weeks with arginine, associated or not with exercise training (treadmill). We assessed the somatotropic activation, by evaluating growth hormone (GH) gene expression and protein content in the pituitary, as well is GH concentration in the serum. Additionally, we evaluate whole-body insulin sensitivity, by performing an insulin tolerance test. Skeletal muscle morpho-physiological parameters were also assessed. Insulin sensitivity was impaired in the arginine-treated rats. However, exercise training reversed the negative effects of arginine. Arginine and exercise training increased somatotropic axis function, muscle mass and body weight gain. The combination arginine and exercise training further decreased total fat mass. Our results confirm that chronic arginine supplementation leads to insulin resistance, which can be reversed in the association with exercise training. We provide further evidence that exercise training is an important tool to improve whole-body metabolism.

Tilapia adropin: the localization and regulation of growth hormone gene expression in pituitary cells

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.

Activin/follistatin system in grass carp pituitary cells: - Regulation by local release of growth hormone and luteinizing hormone and its functional role in growth hormone synthesis and secretion.

Gonadotrophin regulation by activin/follistatin system is well-documented, but the corresponding effect on growth hormone (GH) has not been fully characterized and with little information available in lower vertebrates, especially in fish models. In grass carp, local interactions of GH and luteinizing hormone (LH) can induce GH release and gene expression at pituitary level via autocrine/paracrine mechanisms. To shed light on the role of activin/follistatin system in GH regulation by local actions of GH and LH, grass carp activin βA and βB were cloned, shown to be single-copy genes expressed in the pituitary, and confirmed to encode activin proteins capable of transactivating promoter with activin-responsive elements. In grass carp pituitary cells, activin A and B were effective in reducing GH secretion and GH cell content with concurrent drop in GH mRNA level whereas the opposite was true for follistatin, the activin-binding protein known to neutralize the effects of endogenous activin. Treatment with activin A and B not only could suppress basal but also inhibit GH mRNA expression induced by GH and human chorionic gonadotropin (hCG), a functional analogue of LH in fish model. Apparently, down-regulation of GH mRNA by activin was mediated by reducing GH transcript stability with concurrent inhibition on GH promoter activity via the SMAD pathway. In reciprocal experiments, GH treatment was found to up-regulate activin βA, activin βB and follistatin mRNA levels in carp pituitary cells but the opposite was noted by removing endogenous GH with GH antiserum. Interestingly, parallel treatment with hCG could also inhibit basal as well as GH-induced activin βA, activin βB and follistatin gene expression. These results, as a whole, indicate that the pituitary activin/follistatin system can serve as a regulatory target for local interactions of GH and LH and contribute to GH regulation by autocrine/paracrine mechanisms in the carp pituitary.

Expression of candidate genes of lipid metabolism in the Kazakhstani breeding simmental cattle

Abstract This research paper describes a gene polymorphism of growth hormone (GH), leptin (LEP) and diacylglycerol O-acyltransferase (DGAT) in Simmental cows of LLP “Galitskoe” of Pavlodar oblast. The frequency distribution of alleles and genotypes of the studied genes was held: at GH gene locus it was revealed that 19% of the 123 cows had VV genotype; 77% of the cows had VL genotype; 4% - LL genotype, at that the allele frequency was V - 58% and allele L - 42%.At Lep gene locus the genotype distribution was as follows: CC - 42%; CT - 48% and TT-10%. The frequency of allele C and T in the study was 66 and 34%. In DGAT gene locus genotype KK had 25%; genotype AA - 75%. The allelic frequency of K genotype was 63%, and A genotype - 37%.The study of GH gene expression showed that the minimum and maximum milk yield indicator of the test animals varied from 3089 to 8017 kg, the fat content ranged from 2.20 to 6.20%. Expression of Lep gene showed that the maximum milk yield was received from cows with CT genotype (9056 kg) and a minimum in the CC genotype (7407 kg), maximum fat content was observed in cows with CT genotype (6,1%), and the minimum in CC (2,2%) genotype. The variation of the studied milk yield indicators was from 2544 to 9056 kg of milk, fat content in the milk varied from 2.2 to 6.1%. According to DGAT gene milk production levels ranged from 3377 kg to 7985 kg of milk. The maximum yield of high fat content was obtained from cows with AK genotype (7985 kg - 5.9%), the minimum milk yield result was received from cows withKK genotype (3377 kg) and minimum fat cow result was received from cows with AK genotype (2.2%).

Dietary arginine requirement of juvenile hybrid sturgeon (Acipenser schrenckii♀ × Acipenser baerii♂)

An 8-week feeding trial was conducted to evaluate the dietary arginine requirement of juvenile hybrid sturgeon. Seven isonitrogenous and isolipidic diets were formulated to contain graded levels of dietary arginine ranging from 1.74% to 3.54% (dry weight). The results indicated that the fish fed with 1.76% arginine diet had lower specific growth rate (SGR) and feed efficiency ratio (FER) than the fish fed the 2.64% to 3.24% arginine diets (p < .05), and the fish fed the 2.64% arginine diet presented the highest SGR. The fish fed with 1.76% arginine diet had lower whole-body crude protein content than the fish fed the 2.64% or 2.93% arginine diets (p < .05). Compared with the 1.76% arginine diet, 2.36% to 2.93% arginine diets significantly increased the total amino acid (TAA) concentration and total nonessential amino acid (TNEAA) of carcasses. The fish fed with 1.76% arginine diet had lower activity of nitric oxide synthase (T-NOS) and content of nitric oxide (NO) than the fish fed the 2.05% to 2.93% arginine diets in the liver. The fish fed with 1.76%, 2.05% or 2.36% arginine diets had lower gene expression of growth hormone (GH) and insulin-like growth factor I (IGF-I) in the liver than the fish fed the 2.64% to 3.53% arginine diets in the liver (p < .05). A broken-line analysis between SGR against the dietary arginine levels provided estimates for the optimal dietary arginine requirement of 2.47% corresponding to 6.18% of the dietary protein on a dry weight basis.

Mode of GH administration and gene expression in the female rat brain.

The endogenous secretion of growth hormone (GH) is sexually dimorphic in rats with females having a more even and males a more pulsatile secretion and low trough levels. The mode of GH administration, mimicking the sexually dimorphic secretion, has different systemic effects. In the brains of male rats, we have previously found that the mode of GH administration differently affects neuron-haemoglobin beta (Hbb) expression whereas effects on other transcripts were moderate. The different modes of GH administration could have different effects on brain transcripts in female rats. Hypophysectomised female rats were given GH either as injections twice daily or as continuous infusion and GH-responsive transcripts were assessed by quantitative reverse transcription polymerase chain reaction in the hippocampus and parietal cortex (cortex). The different modes of GH-administration markedly increased Hbb and 5'-aminolevulinate synthase 2 (Alas2) in both brain regions. As other effects were relatively moderate, a mixed model analysis (MMA) was used to investigate general effects of the treatments. In the hippocampus, MMA showed that GH-infusion suppressed glia- and neuron-related transcript expression levels, whereas GH-injections increased expression levels. In the cortex, GH-infusion instead increased neuron-related transcripts, whereas GH-injections had no significant effect. Interestingly, this contrasts to previous results obtained from male rat cortex where GH-infusion generally decreased expression levels. In conclusion, the results indicate that there is a small but significant difference in response to mode of GH administration in the hippocampus as compared to the cortex. For both modes of GH administration, there was a robust effect on Hbb and Alas2.

GENE TRANSFER ON Betta imbellis THROUGH TRANSFECTION METHOD WITH DIFFERENT DNA CONCENTRATION

Big size betta (Giant) have a high economic value compared to normal size betta, and over expression of growth hormone gene can produce giant fish. As an initial step of giant transgenic betta productions, this study was conducted in order to obtain DNA plasmid concentration which provide higher hatching and survival rate of betta larvae. Construction of PhGH pCcBA gene contains growth hormone gene of Siamese catfish (PhGH) and it is controlled by the CCBA promoter. Betta imbellis broodstocks were spawned naturally, and embryos were collected 1-2 minutes after spawning time. One hundred embryos were dipped in 2 mL of transfectan X-treme gene which containp CcBA-PhGH construction genes (50 µg/mL), on room temperature for about 30 minutes. Treatments on this study were different transfectant : DNA plasmid ratiosnamely:A (0,75 µL: 0,25 µL); B (0,75 µL : 0,50 µL); C (0,75 µL: 0,75 µL), D as Control 1(without transfectant, 0,25 µL DNA); E.as Control 2(0,75 µL transfectant, without DNA), and Fas control 3 (without transfectant and without DNA). Every treatments was repeated three times. Transfection embryos were hatched on a container (1L Volume). Study results showed that hatching rate and larvae survival rate (4 days after hatching) on treatment A were the same with the control, but slightly higher than B and C treatments. PCR analysis with DNA template showing that PhGH gene were found on embryos and larvae (pooled sample) of treatment A, B and C. Furthermore, RT-PCR analysis showing the existence of mRNA PhGH expression on embryos and larvae (pooled sample). Therefore, embryo transfection with transfectant ratio 0,75 µL and DNA 0,25 µLshowing the best results.

Irisin inhibition of growth hormone secretion in cultured tilapia pituitary cells

Irisin, the product of fibronectin type III domain-containing protein 5 (FNDC5) gene, is well-documented to be a regulator of energy metabolism. At present, not much is known about its biological function in non-mammalian species. In this study, a full-length tilapia FDNC5 was cloned and its tissue expression pattern has been confirmed. Based on the sequence obtained, we produced and purified recombinant irisin which could induce uncoupling protein 1 (UCP1) gene expression in tilapia hepatocytes. Further, the rabbit polyclonal irisin antiserum was produced and its specificity was confirmed by antiserum preabsorption. In tilapia pituitary cells, irisin inhibited growth hormone (GH) gene expression and secretion and triggered rapid phosphorylation of Akt, Erk1/2, and p38 MAPK. Furthermore, irisin-inhibited GH mRNA expression could be prevented by inhibiting PI3K/Akt, MEK1/2, and p38 MAPK, respectively. Apparently, fish irisin can act directly at the pituitary level to inhibit GH transcript expression via multiple signaling pathways.

Evidence for a Circadian Effect on the Reduction of Human Growth Hormone Gene Expression in Response to Excess Caloric Intake

Rhythmicity of biological functions is fundamental for optimal adaptations to environmental cues. Growth hormone (GH) is a major metabolic homeostatic factor that is secreted with a circadian pattern, but whether it is synthesized rhythmically is unknown. We used transgenic mice containing the human (h) GH gene (hGH1) locus to investigate the rhythmicity of hGH synthesis and secretion and to show that RNA and secreted protein levels oscillate over a 24-h cycle. Analysis of hGH1 promoter sequences revealed an enhancer motif (E-box) element that binds the circadian transcriptional machinery (Bmal1 and Clock). Furthermore, Bmal1/Clock were able to transactivate the hGH1 promoter, and mutation of this E-box element adversely affected basal activity after gene transfer. The ability of Bmal1 to bind the hGH1 promoter region containing the E-box element was confirmed in the hGH1 transgenic mouse pituitary in situ Occupancy was reduced in mice fed a high fat diet during the light (inactive) stage of the daily cycle in mice and corresponded to a decrease in hGH1 RNA levels. The decreases in occupancy and RNA levels were not seen, however, during the dark (active) stage. A chromatin loop required for efficient postnatal hGH1 expression was negatively affected by the high fat diet in the light but not dark stage similar to the pattern observed with Bmal1 association with the promoter region. This is the first evidence that hGH synthesis follows a diurnal rhythm and of dynamic associations of the circadian machinery with a component of a chromosomal structure of the hGH1 locus that is essential for efficient expression.

Acute excess caloric intake negatively affects the rhythmic expression of the human growth hormone gene and its association with the circadian‐linked transcription factor Bmal1

Rhythmicity of biological functions is fundamental for optimal adaptations to environmental cues. Growth hormone (GH) is a major metabolic homeostatic factor that is secreted with a circadian pattern, but whether it is produced rhythmically is unknown. We used transgenic (TG) mice containing the intact human GH gene locus (human GH/CS‐TG) to investigate the rhythmicity of human GH gene (hGH‐N) expression. Human GH RNA expression oscillates over a 24‐hour cycle. Moreover, we have previously reported that excess caloric intake is associated with a decrease in human GH synthesis with reduced hGH‐N promoter function. Sequence analysis of the hGH‐N promoter revealed the presence of an enhancer motif (E‐box) and as such a putative binding site for circadian transcriptional machinery (Bmal1 and Clock). Results from electrophoretic mobility shift assay support participation of Bmal1/Clock and the hGH‐N E‐box DNA element in a common complex. Transfection studies combined with site‐directed mutagenesis revealed that this E‐box DNA element contributes to basal hGH‐N promoter activity. Furthermore, Bmal1/Clock were able to transactivate the hGH‐N promoter. The ability of Bmal1 to associate with the hGH‐N promoter region containing the E‐box element was confirmed in the human GH/CS‐TG mouse pituitary in situ by chromatin immunoprecipitation assay. Occupancy was reduced in mice fed a high fat diet during the light cycle. This decrease was not seen, however, during the dark cycle. Initial assessment of the mechanism suggests an impact of excess caloric intake on the chromatin and/or DNA structure. In summary, the presence of an E‐box DNA element as well as association of Bmal1 with the hGH‐N promoter region in situ supports the notion that hGH‐N is a cyclic gene. Acute excess caloric intake for three days was sufficient to reprogram the circadian rhythm mediated by chromatin remodeling of the hGH‐N promoter region and interfer with recruitment of Bmal1 to the E‐box element. Further investigation of the rhythmic expression of human GH may provide insight into the changes in GH availability under pathophysiological conditions such as metabolic changes associated with overeating and obesity.Support or Funding InformationFunded by the Canadian Institutes of Health Research

Effects of retinoic acid on growth hormone-releasing hormone receptor, growth hormone secretagogue receptor gene expression and growth hormone secretion in rat anterior pituitary cells.

Retinoic acid (RA) is an important signaling molecule in embryonic development and adult tissue. The actions of RA are mediated by the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR), which regulate gene expression. RAR and RXR are widely expressed in the anterior pituitary gland. RA was reported to stimulate growth hormone (GH) gene expression in the anterior pituitary cells. However, current evidence is unclear on the role of RA in gene expression of growth hormone-releasing hormone receptor (Ghrh-r), growth hormone secretagogue receptor (Ghs-r) and somatostatin receptors (Sst-rs). Using isolated anterior pituitary cells of rats, we examined the effects of RA on gene expression of these receptors and GH release. Quantitative real-time PCR revealed that treatment with all-trans retinoic acid (ATRA; 10(-6) M) for 24 h increased gene expression levels of Ghrh-r and Ghs-r; however, expressions of Sst-r2 and Sst-r5 were unchanged. Combination treatment with the RAR-agonist Am80 and RXR-agonist PA024 mimicked the effects of ATRA on Ghrh-r and Ghs-r gene expressions. Exposure of isolated pituitary cells to ATRA had no effect on basal GH release. In contrast, ATRA increased growth hormone-releasing hormone (GHRH)- and ghrelin-stimulated GH release from cultured anterior pituitary cells. Our results suggest that expressions of Ghrh-r and Ghs-r are regulated by RA through the RAR-RXR receptor complex and that RA enhances the effects of GHRH and ghrelin on GH release from the anterior pituitary gland.