Dynamic Regulation of GH–IGF1 Signaling in Injury and Recovery in Hyperoxia-Induced Neonatal Lung Injury

Prematurely born infants often require supplemental oxygen that impairs lung growth and results in arrest of alveolarization and bronchopulmonary dysplasia (BPD). The growth hormone (GH)- and insulin-like growth factor (IGF)1 systems regulate cell homeostasis and organ development. Since IGF1 is decreased in preterm infants, we investigated the GH- and IGF1 signaling (1) in newborn mice with acute and prolonged exposure to hyperoxia as well as after recovery in room air; and (2) in cultured murine lung epithelial cells (MLE-12) and primary neonatal lung fibroblasts (pLFs) after treatment with GH, IGF1, and IGF1-receptor (IGF1-R) inhibitor or silencing of GH-receptor (Ghr) and Igf1r using the siRNA technique. We found that (1) early postnatal hyperoxia caused an arrest of alveolarization that persisted until adulthood. Both short-term and prolonged hyperoxia reduced GH-receptor expression and STAT5 signaling, whereas Igf1 mRNA and pAKT signaling were increased. These findings were related to a loss of epithelial cell markers (SFTPC, AQP5) and proliferation of myofibroblasts (αSMA+ cells). After recovery, GH-R-expression and STAT5 signaling were activated, Igf1r mRNA reduced, and SFTPC protein significantly increased. Cell culture studies showed that IGF1 induced expression of mesenchymal (e.g., Col1a1, Col4a4) and alveolar epithelial cell type I (Hopx, Igfbp2) markers, whereas inhibition of IGF1 increased SFTPC and reduced AQP5 in MLE-12. GH increased Il6 mRNA and reduced proliferation of pLFs, whereas IGF1 exhibited the opposite effect. In summary, our data demonstrate an opposite regulation of GH- and IGF1- signaling during short-term/prolonged hyperoxia-induced lung injury and recovery, affecting alveolar epithelial cell differentiation, inflammatory activation of fibroblasts, and a possible uncoupling of the GH-IGF1 axis in lungs after hyperoxia.

Towards Understanding the Direct and Indirect Actions of Growth Hormone in Controlling Hepatocyte Carbohydrate and Lipid Metabolism

Growth hormone (GH) is critical for achieving normal structural growth. In addition, GH plays an important role in regulating metabolic function. GH acts through its GH receptor (GHR) to modulate the production and function of insulin-like growth factor 1 (IGF1) and insulin. GH, IGF1, and insulin act on multiple tissues to coordinate metabolic control in a context-specific manner. This review will specifically focus on our current understanding of the direct and indirect actions of GH to control liver (hepatocyte) carbohydrate and lipid metabolism in the context of normal fasting (sleep) and feeding (wake) cycles and in response to prolonged nutrient deprivation and excess. Caveats and challenges related to the model systems used and areas that require further investigation towards a clearer understanding of the role GH plays in metabolic health and disease are discussed.

Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3–L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3–L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Ghrelin-Induced Food Intake, But not GH Secretion, Requires the Expression of the GH Receptor in the Brain of Male Mice

Ghrelin stimulates both growth hormone (GH) secretion and food intake. The orexigenic action of ghrelin is mainly mediated by neurons that co-express agouti-related protein (AgRP) and neuropeptide Y (NPY) in the arcuate nucleus (ARH). GH also stimulates food intake and, importantly, ARH AgRP/NPY neurons express GH receptor (GHR). Thus, ghrelin-induced GH secretion may contribute to the orexigenic effect of ghrelin. Here, we investigated the response to ghrelin in male mice carrying GHR ablation specifically in neurons (Brain GHR KO mice) or exclusively in ARH AgRP/NPY neurons (AgRP GHR KO mice). Although Brain GHR KO mice showed normal ghrelin-induced increase in plasma GH levels, these mutants lacked the expected orexigenic response to ghrelin. Additionally, Brain GHR KO mice displayed reduced hypothalamic levels of Npy and Ghsr mRNA and did not elicit ghrelin-induced c-Fos expression in the ARH. Furthermore, Brain GHR KO mice exhibited a prominent reduction in AgRP fiber density in the ARH and paraventricular nucleus of the hypothalamus (PVH). In contrast, AgRP GHR KO mice showed no changes in the hypothalamic Npy and Ghsr mRNAs and conserved ghrelin-induced food intake and c-Fos expression in the ARH. AgRP GHR KO mice displayed a reduced AgRP fiber density (~16%) in the PVH, but this reduction was less than that observed in Brain GHR KO mice (~61%). Our findings indicate that GHR signaling in the brain is required for the orexigenic effect of ghrelin, independently of GH action on ARH AgRP/NPY neurons.

GH Receptor Exon 3 Genotype and Echocardiographic Abnormalities in Patients With Active Acromegaly

Background: The GH receptor (GHR) exon 3 polymorphism occurs at a genomic level. Approximately 50-60% of the population is homozygous for the exon-3 containing genotype (+3/+3), 30-40% are heterozygous (+3/-3) and 10-20% are homozygous for the exon-3 lacking genotype (-3/-3). Some studies suggest that children homo- and heterozygous for the GHR exon 3 lacking genotype (-more efficient 3/-3 and +3/-3, respectively) respond better to treatment with exogenous rhGH and there is also in vitro evidence showing a more efficient signal transduction through this exon 3 deleted isoform. Some studies have found that patients with acromegaly harboring the exon 3-deleted genotype may have a higher prevalence of diabetes and hypertension. Hypothesis and Objective: Patients with active acromegaly harboring the exon 3-lacking GHR genotype may have more echocardiographic abnormalities than those who are homozygous for the exon 3 containing genotype. Patients and Methods: This is a cross-sectional study of patients with active acromegaly, defined by an IGF-1 level > 1.3 times the upper limit of normal (x ULN), who underwent transthoracic echocardiography. Exon-3 GHR genotype was determined by PCR using previously described sense and antisense primers. Results: The cohort consisted of 28 patients, 54% female, with a mean age of 51 ± 12 years. Mean disease duration at the time of echocardiographic examination was 4.48 ± 4.7 years; median basal GH and IGF-1 were 12 ± 26 ng/mL and 2.4 ± 1.04 x ULN. The prevalence of hypertension and diabetes were 43% and 36%, respectively. Fifty three percent of the patients were homozygous for the exon 3-containaing genotype (+3/+3), 18% were homozygous for the exon 3-lacking genotype (-3/-3) and 29% were heterozygous (+3/-3). Clinical and biochemical features did not differ between patients with the different GHR genotypes, except for hypertension that was more prevalent in the +3/+3 genotype group (60% vs 23%, p= 0.04). The frequency of the different echocardiographic parameters was similar among groups (left ventricular hypertrophy 33% vs 15%, p= 0.27; diastolic dysfunction 47% vs 31%, p= 0.39; subclinical systolic dysfunction 42% vs 54%, p= 0.54; left ventricular ejection fraction 59±10% vs 60±16%, p= 0.83); aortic valve abnormalities 19% vs 15%, p=0.63; mitral valve abnormalities 46% vs 15%, p=0.07). Conclusions: Echocardiographic abnormalities in patients with active acromegaly do not differ among patients with the different GHR exon 3 genotypes. The clinical spectrum of acromegaly varies considerably. Although such variability is usually related to the severity of the hypersomatotropinemia, in many patients this is not the case.

Hypothalamic GHR—SIRT1 Axis in Fasting

Many aspects of physiological functions are controlled by the hypothalamus, a brain region that connects the neuroendocrine system to whole-body metabolism. Growth hormone (GH) and the GH receptor (GHR) are expressed in hypothalamic regions known to participate in the regulation of feeding and whole-body energy homeostasis. Sirtuin 1 (SIRT1) is the most conserved mamma-lian nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase that plays a key role in controlling life span and sensing nutrient availability in the hypothalamus in response to caloric restriction. However, the interaction between GHR signaling and SIRT1 in the hypothal-amus is not established. In the arcuate nucleus (ARC) of the hypothalamus, the anorexigenic proopiomelanocortin (POMC)-expressing neurons and the orexigenic agouti-related protein (AgRP)-expressing neurons are the major regulators of feeding and energy expenditure. We show that in the ARC, the majority of GHR-expressing neurons also express SIRT1 and respond to fasting by upregulating SIRT1 expression. Accordingly, hypothalamic upregulation of SIRT1 in response to fasting is blunted in animals with GHR deletion in the AgRP neurons (AgRPEYFPΔGHR). Our data thus reveal a novel interaction between GH and SIRT1 in responses to fasting.

A Balanced Act: The Effects of GH–GHR–IGF1 Axis on Mitochondrial Function

Mitochondrial function is multifaceted in response to cellular energy homeostasis and metabolism, with the generation of adenosine triphosphate (ATP) through the oxidative phosphorylation (OXPHOS) being one of their main functions. Selective elimination of mitochondria by mitophagy, in conjunction with mitochondrial biogenesis, regulates mitochondrial function that is required to meet metabolic demand or stress response. Growth hormone (GH) binds to the GH receptor (GHR) and induces the JAK2/STAT5 pathway to activate the synthesis of insulin-like growth factor 1 (IGF1). The GH–GHR–IGF1 axis has been recognized to play significant roles in somatic growth, including cell proliferation, differentiation, division, and survival. In this review, we describe recent discoveries providing evidence for the contribution of the GH–GHR–IGF1 axis on mitochondrial biogenesis, mitophagy (or autophagy), and mitochondrial function under multiple physiological conditions. This may further improve our understanding of the effects of the GH–GHR–IGF1 axis on mitochondrial function, which may be controlled by the delicate balance between mitochondrial biogenesis and mitophagy. Specifically, we also highlight the challenges that remain in this field.

Growth Hormone Signaling Pathway Leading to the Induction of DNA Synthesis and Proliferation in Primary Cultured Hepatocytes of Adult Rats

Background: We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Methods: Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams’ medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Results: Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10−6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10−6 M), RTK (AG538, 10−6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10−6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10−6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. Conclusions: The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

The Role of Growth Hormone Receptor Isoforms and Their Effects in Bone Metabolism and Skeletal Fragility

Acromegaly and Growth Hormone Deficiency (GHD) are associated with skeletal fragility and with an increased prevalence of Vertebral Fractures (VFs). In the most recent years, several authors tried to investigate surrogate markers that may predict the risk of bone fragility in these endocrine disorders. The aim of this review is to evaluate the role of GH receptor polymorphisms in skeletal fragility in patients affected by GHD and acromegaly. In fact, until now, two different isoforms of the GH Receptor (GHR) were described, that differ for the presence or the absence of transcription of the exon 3 of the GHR gene. Both the isoforms produce a functioning receptor, but the exon 3-deleted isoforms (d3-GHR) has a higher sensitivity to endogenous and recombinant GH as compared to the full-length isoform (fl-GHR).