Prevalence of growth hormone receptor gene polymorphisms and their association with milk production and fertility-related traits of cross-bred dairy cows in Sri Lanka.

This study investigated the association of selected growth hormone receptor (GHR) gene SNPs with selected fertility and milk production-related phenotypes of cross-bred dairy cows (n = 153) reared on three National Livestock Development Board farms in Sri Lanka. Selected cows were genetically screened for SNPs in the exon 08 (n = 153) and 5' upstream (n = 118) regions of the GHR gene using the target sequencing method. The relationships between different genotypes and fertility traits (average calving interval, average number of services per conception, and age at first calving) and milk production-related traits (average total lactation yield, average lactation length, and average milk yield) were analyzed using the General Linear Model in SPSS. Among the identified Four GHR SNPs, rs1099014416 was significantly associated with average calving interval and age at first calving. Cows with GG genotype exhibited younger age at first calving (918.51 ± 113.42 days) and longer calving intervals (543.41 ± 43.29 days) compared to cows with GT (1275.18 ± 38.31, 515.09 ± 24.49 days) and TT (1212.89 ± 88.22, 364.52 ± 54.01 days) genotypes. Other SNPs did not show associations with the studied traits. SNP rs109014416 has the potential to be used as a genetic marker for fertility-related traits in the selection of cross-bred dairy cows in Sri Lanka which should be validated with a larger population.

Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition.

Genome-wide association studies (GWAS) have identified a large number of candidate genes believed to affect longitudinal bone growth and bone mass. One of these candidate genes, TMEM263, encodes a poorly characterized plasma membrane protein. Single nucleotide polymorphisms in TMEM263 are associated with bone mineral density in humans and mutations are associated with dwarfism in chicken and severe skeletal dysplasia in at least one human fetus. Whether this genotype-phenotype relationship is causal, however, remains unclear. Here, we determine whether and how TMEM263 is required for postnatal growth. Deletion of the Tmem263 gene in mice causes severe postnatal growth failure, proportional dwarfism, and impaired skeletal acquisition. Mice lacking Tmem263 show no differences in body weight within the first 2 weeks of postnatal life. However, by P21 there is a dramatic growth deficit due to a disrupted growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, which is critical for longitudinal bone growth. Tmem263-null mice have low circulating IGF-1 levels and pronounced reductions in bone mass and growth plate length. The low serum IGF-1 in Tmem263-null mice is associated with reduced hepatic GH receptor (GHR) expression and GH-induced JAK2/STAT5 signaling. A deficit in GH signaling dramatically alters GH-regulated genes and feminizes the liver transcriptome of Tmem263-null male mice, with their expression profile resembling wild-type female, hypophysectomized male, and Stat5b-null male mice. Collectively, our data validates the causal role for Tmem263 in regulating postnatal growth and raises the possibility that rare mutations or variants of TMEM263 may potentially cause GH insensitivity and impair linear growth.

Food deprivation reduces sensitivity of liver Igf1 synthesis pathways to growth hormone in juvenile gopher rockfish (Sebastes carnatus).

Growth hormone (Gh) regulates growth in part by stimulating the liver to synthesize and release insulin-like growth factor-1 (Igf1), which then promotes somatic growth. However, for fish experiencing food limitation, elevated blood Gh can occur even with low circulating Igf1 and slow growth, suggesting that nutritional stress can alter the sensitivity of liver Igf1 synthesis pathways to Gh. Here, we examined how recent feeding experience affected Gh regulation of liver Igf1 synthesis pathways in juvenile gopher rockfish (Sebastes carnatus) to illuminate mechanisms underlying the nutritional modulation of Igf1 production. Juvenile gopher rockfish were maintained under conditions of feeding or complete food deprivation (fasting) for 14 d and then treated with recombinant sea bream (Sparus aurata) Gh or saline control. Gh upregulated hepatic igf1 mRNA levels in fed fish but not in fasted fish. The liver of fasted rockfish also showed a lower relative abundance of gene transcripts encoding teleost Gh receptors 1 (ghr1) and 2 (ghr2), as well as reduced protein levels of phosphorylated janus tyrosine kinase 2 (pJak2) and signal transducer and activator of transcription 5 (pStat5), which function to induce igf1 gene transcription following Gh binding to Gh receptors. Relative hepatic mRNA levels for suppressors of cytokine signaling (Socs) genes socs2, socs3a, and socs3b were also lower in fasted rockfish. Socs2 can suppress Gh activation of Jak2/Stat5, and fasting-related variation in socs expression may reflect modulated inhibitory control of igf1 gene transcription. Fasted rockfish also had elevated liver mRNA abundances for lipolytic hormone-sensitive lipase 1 (hsl1) and Igf binding proteins igfbp1a, -1b and -3a, reduced liver mRNAs encoding igfbp2b and an Igfbp acid labile subunit-like (igfals) gene, and higher transcript abundances for Igf1 receptors igf1ra and igf1rb in skeletal muscle. Together, these findings suggest that food deprivation impacts liver Igf1 responsiveness to Gh via multiple mechanisms that include a downregulation of hepatic Gh receptors, modulation of the intracellular Jak2/Stat5 transduction pathway, and possible shifts in Socs-inhibitory control of igf1 gene transcription, while also demonstrating that these changes occur in concert with shifts in liver Igfbp expression and muscle Gh/Igf1 signaling pathway components.

Growth Axis Somatostatin, Growth Hormone Receptor, and Insulin-like Growth Factor-1 Genes Express and Are Affected by the Injection of Exogenous Growth Hormone in Chinemys reevesii.

In this study, to explore the effect of growth hormone changes on the related genes and regulatory roles of the turtle, PCR amplification, real-time fluorescence quantitative analysis, and enzyme cutting technology were used to clone and sequence the somatostatin (SS) gene, growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-I) sequence of Chinemys reevesii. The effects of human growth hormone on the mRNA expression of growth-axis-related genes SS, GHR, and IGF-1 in different sexes were observed. The study of the SS gene in turtles using real-time fluorescence quantitative PCR showed that the SS gene was mainly expressed in the nervous system and the digestive system, with the highest expression found in the brain, while the GHR gene and the IGF-I gene were expressed in all tissues of Chinemys reevesii. The SS gene was expressed in the brain, pituitary, liver, stomach, and intestine, with the highest expression in the brain and the lowest expression in the liver. Within 4 weeks of the injection of exogenous growth hormone, the expression level of the SS gene in the brain of both sexes first increased and then decreased, showing a parabolic trend, and the expression level of the experimental group was lower than that of the control group. After the injection of growth hormone (GH), the expression of the GHR gene in the liver of both sexes showed a significant increase in the first week, decreasing to the control group level in the second week, and then gradually increasing. Finally, a significant level of difference in the expression of the GHR gene was reached at 3 and 4 weeks. In terms of the IGF-I gene, the changing trend of the expression level in the liver was the same as that of the GHR gene. After the injection of exogenous growth hormone, although the expression of the SS gene increased the inhibition of the secretion of the GHR gene by the Reeves' turtle, exogenous growth hormone could replace the synthesis of GH and GHR, accelerating the growth of the turtle. The experiments showed that the injection of recombinant human growth hormone affects the expression of SS, GHR, and IGF-1 genes, and promotes the growth of the Reeves' turtle.

A Recurrent Mutation in Growth Hormone Receptor (GHR) Gene Underlying Laron-type Dwarfism in a Pakistani Family.

Laron syndrome (LS) is a rare autosomal recessively segregating disorder of severe short stature. The condition is characterized by short limbs, delayed puberty, hypoglycemia in infancy, and obesity. Mutations in growth hormone receptor (GHR) have been implicated in LS; hence, it is also known as growth hormone insensitivity syndrome (MIM-262500). Here we represent a consanguineous Pakistani family in which three siblings were afflicted with LS. Patients had rather similar phenotypic presentations marked with short stature, delayed bone age, limited extension of elbows, truncal obesity, delayed puberty, childish appearance, and frontal bossing. They also had additional features such as hypo-muscularity, early fatigue, large ears, widely-spaced breasts, and attention deficit behavior, which are rarely reported in LS. The unusual combination of the features hindered a straightforward diagnosis and prompted us to first detect the regions of shared homozygosity and subsequently the disease-causing variant by next generation technologies, like SNP genotyping and exome sequencing. A homozygous pathogenic variant c.508G>C (p.(Asp170His)) in GHR was detected. The variant is known to be implicated in LS, supporting the molecular diagnosis of LS. Also, we present detailed clinical, hematological, and hormonal profiling of the siblings.

Growth hormone insensitivity and adipose tissue: tissue morphology and transcriptome analyses in pigs and humans.

PURPOSE: Growth hormone receptor knockout (GHR-KO) pigs have recently been developed, which serve as a large animal model of Laron syndrome (LS). GHR-KO pigs, like individuals with LS, are obese but lack some comorbidities of obesity. The purpose of this study was to examine the histological and transcriptomic phenotype of adipose tissue (AT) in GHR-KO pigs and humans with LS. METHODS: Intraabdominal (IA) and subcutaneous (SubQ) AT was collected from GHR-KO pigs and examined histologically for adipocyte size and collagen content. RNA was isolated and cDNA sequenced, and the results were analyzed to determine differentially expressed genes that were used for enrichment and pathway analysis in pig samples. For comparison, we also performed limited analyses on human AT collected from a single individual with and without LS. RESULTS: GHR-KO pigs have increased adipocyte size, while the LS AT had a trend towards an increase. Transcriptome analysis revealed 55 differentially expressed genes present in both depots of pig GHR-KO AT. Many significant terms in the enrichment analysis of the SubQ depot were associated with metabolism, while in the IA depot, IGF and longevity pathways were negatively enriched. In pathway analysis, multiple expected and novel pathways were significantly affected by genotype, i.e. KO vs. controls. When GH related gene expression was analyzed, SOCS3 and CISH showed species-specific changes. CONCLUSION: AT of GHR-KO pigs has several similarities to that of humans with LS in terms of adipocyte size and gene expression profile that help describe the depot-specific adipose phenotype of both groups.

Growth hormone receptor gene disruption.

Much of our understanding of growth hormone's (GH)'s numerous activities stems from studies utilizing GH receptor (GHR) knockout mice. More recently, the role of GH action has been examined by creating mice with tissue-specific or temporal GHR disruption. To date, 37 distinct GHR knockout mouse lines have been created. Targeted tissues include fat, liver, muscle, heart, bone, brain, macrophage, intestine, hematopoietic stem cells, pancreatic ß cells, and inducible multi-tissue "global" disruption at various ages. In this chapter, a summary of each mouse line is provided with background information on the generation of the mouse line as well as important physiological outcomes resulting from GHR gene disruption. Collectively, these mouse lines provide unique insights into GH action and have resulted in the development of new hypotheses about the functions ascribed to GH action in particular tissues.

Growth Hormone Action in Somatostatin Neurons Regulates Anxiety and Fear Memory.

Dysfunctions in growth hormone (GH) secretion increase the prevalence of anxiety and other neuropsychiatric diseases. GH receptor (GHR) signaling in the amygdala has been associated with fear memory, a key feature of posttraumatic stress disorder. However, it is currently unknown which neuronal population is targeted by GH action to influence the development of neuropsychiatric diseases. Here, we showed that approximately 60% of somatostatin (SST)-expressing neurons in the extended amygdala are directly responsive to GH. GHR ablation in SST-expressing cells (SSTΔGHR mice) caused no alterations in energy or glucose metabolism. Notably, SSTΔGHR male mice exhibited increased anxiety-like behavior in the light-dark box and elevated plus maze tests, whereas SSTΔGHR females showed no changes in anxiety. Using auditory Pavlovian fear conditioning, both male and female SSTΔGHR mice exhibited a significant reduction in fear memory. Conversely, GHR ablation in SST neurons did not affect memory in the novel object recognition test. Gene expression was analyzed in a micro punch comprising the central nucleus of the amygdala (CEA) and basolateral (BLA) complex. GHR ablation in SST neurons caused sex-dependent changes in the expression of factors involved in synaptic plasticity and function. In conclusion, GHR expression in SST neurons is necessary to regulate anxiety in males, but not female mice. GHR ablation in SST neurons also decreases fear memory and affects gene expression in the amygdala, although marked sex differences were observed. Our findings identified for the first time a neurochemically-defined neuronal population responsible for mediating the effects of GH on behavioral aspects associated with neuropsychiatric diseases.SIGNIFICANCE STATEMENT Hormone action in the brain regulates different neurological aspects, affecting the predisposition to neuropsychiatric disorders, like depression, anxiety, and posttraumatic stress disorder. Growth hormone (GH) receptor is widely expressed in the brain, but the exact function of neuronal GH action is not fully understood. Here, we showed that mice lacking the GH receptor in a group of neurons that express the neuropeptide somatostatin exhibit increased anxiety. However, this effect is only observed in male mice. In contrast, the absence of the GH receptor in somatostatin-expressing neurons decreases fear memory, a key feature of posttraumatic stress disorder, in males and females. Thus, our study identified a specific group of neurons in which GH acts to affect the predisposition to neuropsychiatric diseases.

Reporting a novel growth hormone receptor gene variant in an Iranian consanguineous pedigree with Laron syndrome: a case report.

BACKGROUND: Human growth hormone (hGH) plays a crucial role in growth by binding to growth hormone receptor (GHR) in target cells. Binding of GH molecules to their cognate receptors triggers downstream signaling pathways leading to the transcription of several genes, including insulin-like growth factor (IGF)-1. Pathogenic variants in the GHR gene can result in structural and functional defects in the GHR protein, leading to Laron Syndrome (LS) with the primary clinical manifestation of short stature. So far, around 100 GHR variants have been reported, mostly biallelic, as causing LS. CASE PRESENTATION: We report on three siblings from an Iranian consanguineous family who presented with dwarfism. Whole-exome sequencing (WES) was performed on the proband, revealing a novel homozygous missense variant in the GHR gene (NM_000163.5; c.610 T > A, p.(Trp204Arg)) classified as a likely pathogenic variant according to the recommendation of the American College of Medical Genetics (ACMG). Co-segregation analysis was investigated using Sanger sequencing. CONCLUSIONS: To date, approximately 400-500 LS cases with GHR biallelic variants, out of them 10 patients originating from Iran, have been described in the literature. Given the high rate of consanguineous marriages in the Iranian population, the frequency of LS is expected to be higher, which might be explained by undiagnosed cases. Early diagnosis of LS is very important, as treatment is available for this condition.

The GH/IGF1 axis in the kidney of young goats fed a protein-reduced diet.

Feeding approaches for ruminants are changing to reduce N excretion as a major source of pollution. Based on the ruminohepatic cycle of N, it was assumed that the metabolism of ruminants could tolerate a reduced-protein diet well. However, metabolic changes such as a reduction in hepatic IGF1 mRNA expression, resulting in lower blood IGF1 levels due to decreased hepatic growth hormone receptor (GHR) expression, were found. Therefore, the aim of the present study was to determine the effect of a low-protein diet on the expression of GHR and subsequent IGF1 signalling in the renal cortex of young male goats to verify whether organ-specific synthesis of IGF1 mRNA expression occurs. Kidney cortex samples were obtained from eight goats fed a control diet (20% CP) and nine animals fed a reduced-protein diet (9% CP). The expression of GHR in the kidneys was reduced, whereas the expression of Janus kinase 2 (JAK2), suppressor of cytokine signalling 3 and signal transducers and activators of transcription 3 (STAT3) increased significantly. The stimulated JAK2 expression could modulate the expression of STAT3, which led to increased renal IGF1 mRNA expression. These results suggest that this increase in IGF1 mRNA expression in the kidneys is tissue-specific. This could be due to the autocrine/paracrine IGF1 effect on renal cell metabolism during a protein-reduced diet. These signalling pathways need further investigation to understand how and why low levels of protein stimulate IGF1 synthesis differently in the kidney than in the liver.

Coordinated transcriptional upregulation of oxidative metabolism proteins in long-lived endocrine mutant mice.

Caloric restriction (CR), which extends lifespan in rodents, leads to increased hepatic fatty acid ß-oxidation and oxidative phosphorylation (OXPHOS), with parallel changes in proteins and their mRNAs. Genetic mutants that extend lifespan, including growth hormone receptor knockout (GHRKO) and Snell dwarf (SD) mice, have lower respiratory quotient, suggesting increased reliance on fatty acid oxidation, but the molecular mechanism(s) of this metabolic shift have not yet been worked out. Here we show that both GHRKO and SD mice have significantly higher mRNA and protein levels of enzymes involved in mitochondrial and peroxisomal fatty acid ß-oxidation. In addition, multiple subunits of OXPHOS complexes I-IV are upregulated in GHRKO and SD livers, and Complex V subunit ATP5a is upregulated in liver of GHRKO mice. Expression of these genes is regulated by a group of nuclear receptors and transcription factors including peroxisome proliferator-activated receptors (PPARs) and estrogen-related receptors (ERRs). We found that levels of these nuclear receptors and their co-activator PGC-1α were unchanged or downregulated in liver of GHRKO and SD mice. In contrast, NCOR1, a co-repressor for the same receptors, was significantly downregulated in the two long-lived mouse models, suggesting a plausible mechanism for the changes in FAO and OXPHOS proteins. Hepatic levels of HDAC3, a co-factor for NCOR1 transcriptional repression, were also downregulated. The role of NCOR1 is well established in the contexts of cancer and metabolic disease, but may provide new mechanistic insights into metabolic control in long-lived mouse models.

The Mouse CircGHR Regulates Proliferation, Differentiation and Apoptosis of Hepatocytes and Myoblasts.

The anterior pituitary gland of animals secretes growth hormone (GH) to bind to the growth hormone receptor (GHR) on the liver cell membrane through the blood circulation, thereby promoting the downstream gene insulin-like growth factor-1 (IGF1) expression, which is the canonical GH-GHR-IGF1 signaling pathway. Therefore, the amount of GHR and the integrity of its structure will affect animal growth and development. In the previous study, we found that the mouse GHR gene can transcribe a circular transcript named circGHR. Our group cloned the full-length of the mouse circGHR and analyzed its spatiotemporal expression profile. In this study, we further predicted the open reading frame of circGHR with bioinformatics, subsequently constructed a Flag-tagged protein vector and preliminarily verified its coding potential with western blot. Additionally, we found that circGHR could inhibit the proliferation of NCTC469 cells and has a tendency to inhibit cell apoptosis, while for C2C12 cells, it showed a tendency to inhibit cell proliferation and promote its differentiation. Overall, these results suggested that the mouse circGHR had the potential to encode proteins and affect cell proliferation, differentiation and apoptosis.

GHR disruption in mature adult mice alters xenobiotic metabolism gene expression in the liver.

BACKGROUND: Lifelong reduction of growth hormone (GH) action extends lifespan and improves healthspan in mice. Moreover, congenital inactivating mutations of GH receptor (GHR) in mice and humans impart resistance to age-associated cancer, diabetes, and cognitive decline. To investigate the consequences of GHR disruption at an adult age, we recently ablated the GHR at 6-months of age in mature adult (6mGHRKO) mice. We found that both, male and female 6mGHRKO mice have reduced oxidative damage, with males 6mGHRKO showing improved insulin sensitivity and cancer resistance. Importantly, 6mGHRKO females have an extended lifespan compared to controls. OBJECTIVE AND METHODS: To investigate the possible mechanisms leading to health improvements, we performed RNA sequencing using livers from male and female 6mGHRKO mice and controls. RESULTS: We found that disrupting GH action at an adult age reduced the gap in liver gene expression between males and females, making gene expression between sexes more similar. However, there was still a 6-fold increase in the number of differentially expressed genes when comparing male 6mGHRKO mice vs controls than in 6mGHRKO female vs controls, suggesting that GHR ablation affects liver gene expression more in males than in females. Finally, we found that lipid metabolism and xenobiotic metabolism pathways are activated in the liver of 6mGHRKO mice. CONCLUSION: The present study shows for the first time the specific hepatic gene expression profile, cellular pathways, biological processes and molecular mechanisms that are driven by ablating GH action at a mature adult age in males and females. Importantly, these results and future studies on xenobiotic metabolism may help explain the lifespan extension seen in 6mGHRKO mice.

High growth hormone serum partially protects mice against Trypanosoma cruzi infection.

Chagas disease (CD) is one of the most devasting parasitic diseases in the Americas, affecting 7-8 million people worldwide. In vitro and in vivo experiments have demonstrated that growth hormone (GH) serum levels decrease as CD progresses. Interestingly, inactivating mutations in the GH receptor in humans result in Laron syndrome (LS), a clinical entity characterized by increased serum levels of GH and decreased insulin growth factor-1 (IGF-1). The largest cohort of LS subjects lives in the southern provinces of Ecuador. Remarkably, no clinical CD cases have been reported in these individuals despite living in highly endemic areas. In the current ex vivo study, we employed serum from GHR-/- mice, also known as LS mice (a model of GH resistance with high GH and low IGF-1 levels), and serum from bovine GH (bGH) transgenic mice (high GH and IGF-1), to test the effect on Trypanosoma cruzi infection. We infected mouse fibroblast L-cells with T. cruzi (etiological CD infectious agent) and treated them with serum from each mouse type. Treatment with GHR-/- serum (LS mice) significantly decreased L-cell infection by 28% compared with 48% from control wild-type mouse serum (WT). Treatment with bGH mouse serum significantly decreased infection of cells by 41% compared with 54% from WT controls. Our results suggest that high GH and low IGF-1 in blood circulation, as typically seen in LS individuals, confer partial protection against T. cruzi infection. This study is the first to report decreased T. cruzi infection using serum collected from two modified mouse lines with altered GH action (GHR-/- and bGH).

Direct and systemic actions of growth hormone receptor (GHR)-signaling on hepatic glycolysis, de novo lipogenesis and insulin sensitivity, associated with steatosis.

BACKGROUND: Evidence is accumulating that growth hormone (GH) protects against the development of steatosis and progression of non-alcoholic fatty liver disease (NAFLD). GH may control steatosis indirectly by altering systemic insulin sensitivity and substrate delivery to the liver and/or by the direct actions of GH on hepatocyte function. APPROACH: To better define the hepatocyte-specific role of GH receptor (GHR) signaling on regulating steatosis, we used a mouse model with adult-onset, hepatocyte-specific GHR knockdown (aHepGHRkd). To prevent the reduction in circulating insulin-like growth factor 1 (IGF1) and the subsequent increase in GH observed after aHepGHRkd, subsets of aHepGHRkd mice were treated with adeno-associated viral vectors (AAV) driving hepatocyte-specific expression of IGF1 or a constitutively active form of STAT5b (STAT5bCA). The impact of hepatocyte-specific modulation of GHR, IGF1 and STAT5b on carbohydrate and lipid metabolism was studied across multiple nutritional states and in the context of hyperinsulinemic:euglycemic clamps. RESULTS: Chow-fed male aHepGHRkd mice developed steatosis associated with an increase in hepatic glucokinase (GCK) and ketohexokinase (KHK) expression and de novo lipogenesis (DNL) rate, in the post-absorptive state and in response to refeeding after an overnight fast. The aHepGHRkd-associated increase in hepatic KHK, but not GCK and steatosis, was dependent on hepatocyte expression of carbohydrate response element binding protein (ChREBP), in re-fed mice. Interestingly, under clamp conditions, aHepGHRkd also increased the rate of DNL and expression of GCK and KHK, but impaired insulin-mediated suppression of hepatic glucose production, without altering plasma NEFA levels. These effects were normalized with AAV-mediated hepatocyte expression of IGF1 or STAT5bCA. Comparison of the impact of AAV-mediated hepatocyte IGF1 versus STAT5bCA in aHepGHRkd mice across multiple nutritional states, indicated the restorative actions of IGF1 are indirect, by improving systemic insulin sensitivity, independent of changes in the liver transcriptome. In contrast, the actions of STAT5b are due to the combined effects of raising IGF1 and direct alterations in the hepatocyte gene program that may involve suppression of BCL6 and FOXO1 activity. However, the direct and IGF1-dependent actions of STAT5b cannot fully account for enhanced GCK activity and lipogenic gene expression observed after aHepGHRkd, suggesting other GHR-mediated signals are involved. CONCLUSION: These studies demonstrate hepatocyte GHR-signaling controls hepatic glycolysis, DNL, steatosis and hepatic insulin sensitivity indirectly (via IGF1) and directly (via STAT5b). The relative contribution of these indirect and direct actions of GH on hepatocytes is modified by insulin and nutrient availability. These results improve our understanding of the physiologic actions of GH on regulating adult metabolism to protect against NAFLD progression.

The crosstalk between FGF21 and GH leads to weakened GH receptor signaling and IGF1 expression and is associated with growth failure in very preterm infants.

Background: Fibroblast growth factor 21 (FGF21) is an essential metabolic regulator that adapts to changes in nutritional status. Severe childhood undernutrition induces elevated FGF21 levels, contributing to growth hormone (GH) resistance and subsequent linear growth attenuation potentially through a direct action on chondrocytes. Methods: In this study, we assessed expression of the components of both GH and FGF21 pathways in rare and unique human growth plates obtained from children. Moreover, we investigated the mechanistic interplay of FGF21 on GH receptor (GHR) signaling in a heterologous system. Results: Chronic FGF21 exposure increased GH-induced GHR turnover and SOCS2 expression, leading to the inhibition of STAT5 phosphorylation and IGF-1 expression. The clinical significance of FGF21 signaling through GH receptors was tested in nutritionally driven growth failure seen in very preterm (VPT) infants right after birth. VPT infants display an immediate linear growth failure after birth followed by growth catch-up. Consistent with the in vitro model data, we show that circulating FGF21 levels were elevated during deflection in linear growth compared to catch-up growth and were inversely correlated with the length velocity and circulating IGF1 levels. Conclusions: This study further supports a central role of FGF21 in GH resistance and linear growth failure and suggests a direct action on the growth plate.

Effects of GHR Deficiency and Juvenile Hypoglycemia on Immune Cells of a Porcine Model for Laron Syndrome.

Laron syndrome (LS) is a rare genetic disorder characterized by low levels of insulin-like growth factor 1 (IGF1) and high levels of growth hormone (GH) due to mutations in the growth hormone receptor gene (GHR). A GHR-knockout (GHR-KO) pig was developed as a model for LS, which displays many of the same features as humans with LS-like transient juvenile hypoglycemia. This study aimed to investigate the effects of impaired GHR signaling on immune functions and immunometabolism in GHR-KO pigs. GHR are located on various cell types of the immune system. Therefore, we investigated lymphocyte subsets, proliferative and respiratory capacity of peripheral blood mononuclear cells (PBMCs), proteome profiles of CD4- and CD4+ lymphocytes and IFN-α serum levels between wild-type (WT) controls and GHR-KO pigs, which revealed significant differences in the relative proportion of the CD4+CD8α- subpopulation and in IFN-α levels. We detected no significant difference in the respiratory capacity and the capacity for polyclonal stimulation in PBMCs between the two groups. But proteome analysis of CD4+ and CD4- lymphocyte populations revealed multiple significant protein abundance differences between GHR-KO and WT pigs, involving pathways related to amino acid metabolism, beta-oxidation of fatty acids, insulin secretion signaling, and oxidative phosphorylation. This study highlights the potential use of GHR-KO pigs as a model for studying the effects of impaired GHR signaling on immune functions.

GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system.

BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.

Characterization of dominant-negative growth hormone receptor variants reveals a potential therapeutic target for short stature.

OBJECTIVE: Growth hormone insensitivity (GHI) encompasses growth restriction, normal/elevated growth hormone (GH), and low insulin-like growth factor I (IGF1). "Nonclassical" GHI is poorly characterized and is rarely caused by heterozygous dominant-negative (DN) variants located in the intracellular or transmembrane domains of the GH receptor (GHR). We sought to determine the molecular mechanisms underpinning the growth restriction in 2 GHI cases. METHODS AND DESIGN: A custom-made genetic investigative pipeline was exploited to identify the genetic cause of growth restriction in patients with GHI. Nanoluc binary technology (NanoBiT), in vitro splicing assays, western blotting, and flow cytometry, characterized the novel GHR variants. RESULTS: Novel heterozygous GHR variants were identified in 2 unrelated patients with GHI. In vitro splicing assays indicated both variants activated the same alternative splice acceptor site resulting in aberrant splicing and exclusion of 26 base pairs of GHR exon 9. The GHR variants produced truncated receptors and impaired GH-induced GHR signaling. NanoBiT complementation and flow cytometry showed increased cell surface expression of variant GHR homo/heterodimers compared to wild-type (WT) homodimers and increased recombinant human GH binding to variant GHR homo/heterodimers and GH binding protein (GHBP) cleaved from the variant GHRs. The findings demonstrated increased variant GHR dimers and GHBP with resultant GH sequestration. CONCLUSION: We identified and characterized 2 novel, naturally occurring truncated GHR gene variants. Intriguingly, these DN GHR variants act via the same cryptic splice acceptor site, highlighting impairing GH binding to excess GHBP as a potential therapeutic approach.

Resistance to mild cold stress is greater in both wild-type and long-lived GHR-KO female mice.

Adapting to stress, including cold environmental temperature (eT), is crucial for the survival of mammals, especially small rodents. Long-lived mutant mice have enhanced stress resistance against oxidative and non-oxidative challenges. However, much less is known about the response of those long-lived mice to cold stress. Growth hormone receptor knockout (GHR-KO) mice are long-lived with reduced growth hormone signaling. We wanted to test whether GHR-KO mice have enhanced resistance to cold stress. To examine the response of GHR-KO mice to cold eT, GHR-KO mice were housed at mild cold eT (16 °C) immediately following weaning. Longevity results showed that female GHR-KO and wild-type (WT) mice retained similar lifespan, while both male GHR-KO and WT mice had shortened lifespan compared to the mice housed at 23 °C eT. Female GHR-KO and WT mice housed at 16 °C had upregulated fibroblast growth factor 21 (FGF21), enhanced energy metabolism, reduced plasma triglycerides, and increased mRNA expression of some xenobiotic enzymes compared to females housed at 23 °C and male GHR-KO and WT mice housed under the same condition. In contrast, male GHR-KO and WT mice housed at 16 °C showed deleterious effects in parameters which might be associated with their shortened longevity compared to male GHR-KO and WT mice housed at 23 °C. Together, this study suggests that in response to mild cold stress, sex plays a pivotal role in the regulation of longevity, and female GHR-KO and WT mice are more resistant to this challenge than the males.