Growth Hormone Transgenic Mice Research Articles

Growth Hormone Receptor Antagonist Markedly Improves Gemcitabine Response in a Mouse Xenograft Model of Human Pancreatic Cancer.

Chemotherapy treatment against pancreatic ductal adenocarcinoma (PDAC) is thwarted by tumoral activation of multiple therapy resistance pathways. The growth hormone (GH)-GH receptor (GHR) pair is a covert driver of multimodal therapy resistance in cancer and is overexpressed in PDAC tumors, yet the therapeutic potential of targeting the same has not been explored. Here, we report that GHR expression is a negative prognostic factor in patients with PDAC. Combinations of gemcitabine with different GHR antagonists (GHRAs) markedly improve therapeutic outcomes in nude mice xenografts. Employing cultured cells, mouse xenografts, and analyses of the human PDAC transcriptome, we identified that attenuation of the multidrug transporter and epithelial-to-mesenchymal transition programs in the tumors underlie the observed augmentation of chemotherapy efficacy by GHRAs. Moreover, in human PDAC patients, GHR expression strongly correlates with a gene signature of tumor promotion and immune evasion, which corroborate with that in syngeneic tumors in wild-type vs. GH transgenic mice. Overall, we found that GH action in PDAC promoted a therapy-refractory gene signature in vivo, which can be effectively attenuated by GHR antagonism. Our results collectively present a proof of concept toward considering GHR antagonists to improve chemotherapeutic outcomes in the highly chemoresistant PDAC.

FRI023 Increased Fibrosis In White Adipose Tissue Of Male And Female bGH Transgenic Mice Appears Independent Of TGF-β Action

Abstract Disclosure: G. Lach: None. S. Bell: None. J.A. Young: None. E.O. List: None. R. Basu: None. D. Geitgey: None. K.Y. Lee: None. D. Swegan: None. L.J. Caggiano: None. S. Okada: None. J.J. Kopchick: None. D.E. Berryman: None. Fibrosis is a pathological state caused by excess deposition of extracellular matrix proteins, which can disrupt tissue structure and function. Previous work has shown that male bovine growth hormone (bGH) transgenic mice have high levels of circulating growth hormone (GH) and insulin-like growth factor 1 (IGF-1), marked decrease in lifespan, and increased fibrosis in several tissues including white adipose tissue (WAT), which is more pronounced in the subcutaneous (Sc) depot. The current study expanded on these findings, evaluating: 1) WAT fibrosis in female bGH mice, 2) levels of circulating factors implicated in fibrosis, and 3) the role of TGF-β, a central mediator of fibrogenesis, in the development of bGH WAT fibrosis. Our findings confirmed that female bGH mice exhibit increased WAT fibrosis, most prominent in the Sc depot, like male bGH mice, but also significantly increased in the perigonadal depot, unlike male bGH mice. bGH mice of both sexes have elevated circulating levels of several markers of collagen turnover (plasma P1NP, P3NP and ICTP) and FGF21 relative to controls but no genotype-dependent alteration in fibroblast activation protein (FAP). TGF-β and TGF-β signaling in serum or WAT was either unchanged or reduced in male and female bGH mice at both young and mature ages. The treatment of bGH mice with a TGF-β antagonist, pirfenidone, did not attenuate bGH WAT fibrosis. Contrary to the effects of the chronic exposure to excess GH, acute GH treatments in vivo or in vitro did elicit a modest increase in TGF-β signaling. Finally, single nucleus RNA sequencing confirmed no perturbation in TGF-β or its receptor gene expression in any WAT cell population within subcutaneous bGH WAT; however, a striking increase in B lymphocyte infiltration in Sc bGH WAT was observed. Overall, these data suggest that bGH WAT fibrosis is independent of the action of TGF-β and reveals an intriguing shift in immune cells, particularly B cells, in bGH WAT that should be further explored considering the increasing importance of B cell-mediated WAT fibrosis and aging. Acknowledgements:This work was sponsored, in part, by ASPIRE grant funding from Pfizer. This work was also supported by the State of Ohio’s Eminent Scholar Program to J. J. K. that includes a gift from Milton and Lawrence Goll and by the startup funds from the Heritage College of Osteopathic Medicine at Ohio University, Athens, OH. Presentation: Friday, June 16, 2023

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).

Increased Fibrosis in White Adipose Tissue of Male and Female bGH Transgenic Mice Appears Independent of TGF-β Action.

Fibrosis is a pathological state caused by excess deposition of extracellular matrix proteins in a tissue. Male bovine growth hormone (bGH) transgenic mice experience metabolic dysfunction with a marked decrease in lifespan and with increased fibrosis in several tissues including white adipose tissue (WAT), which is more pronounced in the subcutaneous (Sc) depot. The current study expanded on these initial findings to evaluate WAT fibrosis in female bGH mice and the role of transforming growth factor (TGF)-β in the development of WAT fibrosis. Our findings established that female bGH mice, like males, experience a depot-dependent increase in WAT fibrosis, and bGH mice of both sexes have elevated circulating levels of several markers of collagen turnover. Using various methods, TGF-β signaling was found unchanged or decreased-as opposed to an expected increase-despite the marked fibrosis in WAT of bGH mice. However, acute GH treatments in vivo, in vitro, or ex vivo did elicit a modest increase in TGF-β signaling in some experimental systems. Finally, single nucleus RNA sequencing confirmed no perturbation in TGF-β or its receptor gene expression in any WAT cell subpopulations of Sc bGH WAT; however, a striking increase in B lymphocyte infiltration in bGH WAT was observed. Overall, these data suggest that bGH WAT fibrosis is independent of the action of TGF-β and reveals an intriguing shift in immune cells in bGH WAT that should be further explored considering the increasing importance of B cell-mediated WAT fibrosis and pathology.

Excess Growth Hormone Triggers Inflammation-Associated Arthropathy, Subchondral Bone Loss, and Arthralgia

Growth hormone (GH) is a key mediator of skeletal growth. In humans, excess GH secretion due to pituitary adenoma, seen in patients with acromegaly, results in severe arthropathies. This study investigated the effects of long-term excess GH on the knee joint tissues. One year-old wild-type (WT) and bovine GH (bGH) transgenic mice were used as a model for excess GH. bGH mice showed increased sensitivity to mechanical and thermal stimuli, compared with WT mice. Micro-computed tomography analyses of the distal femur subchondral bone revealed significant reductions in trabecular thickness and significantly reduced bone mineral density of the tibial subchondral bone-plate associated with increased osteoclast activity in both male and female bGH compared with WT mice. bGH mice showed severe loss of matrix from the articular cartilage, osteophytosis, synovitis, and ectopic chondrogenesis. Articular cartilage loss in the bGH mice was associated with elevated markers of inflammation and chondrocyte hypertrophy. Finally, hyperplasia of synovial cells was associated with increased expression of Ki-67 and diminished p53 levels in the synovium of bGH mice. Unlike the low-grade inflammation seen in primary osteoarthritis, arthropathy caused by excess GH affects all joint tissues and triggers severe inflammatory response. Data from this study suggest that treatment of acromegalic arthropathy should involve inhibition of ectopic chondrogenesis and chondrocyte hypertrophy.

Growth Hormone Alters Circulating Levels of Glycine and Hydroxyproline in Mice

Growth hormone (GH) has established effects on protein metabolism, such as increasing protein synthesis and decreasing amino acid degradation, but its effects on circulating amino acid levels are less studied. To investigate this relationship, metabolomic analyses were used to measure amino acid concentrations in plasma and feces of mice with alterations to the GH axis, namely bovine GH transgenic (bGH; increased GH action) and GH receptor knockout (GHRKO; GH resistant) mice. To determine the effects of acute GH treatment, GH-injected GH knockout (GHKO) mice were used to measure serum glycine. Furthermore, liver gene expression of glycine metabolism genes was assessed in bGH, GHRKO, and GH-injected GHKO mice. bGH mice had significantly decreased plasma glycine and increased hydroxyproline in both sexes, while GHRKO mice had increased plasma glycine in both sexes and decreased hydroxyproline in males. Glycine synthesis gene expression was decreased in bGH mice (Shmt1 in females and Shmt2 in males) and increased in GHRKO mice (Shmt2 in males). Acute GH treatment of GHKO mice caused decreased liver Shmt1 and Shmt2 expression and decreased serum glycine. In conclusion, GH alters circulating glycine and hydroxyproline levels in opposing directions, with the glycine changes at least partially driven by decreased glycine synthesis.

Excess Growth Hormone Alters the Male Mouse Gut Microbiome in an Age-dependent Manner.

The gut microbiome has an important role in host development, metabolism, growth, and aging. Recent research points toward potential crosstalk between the gut microbiota and the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis. Our laboratory previously showed that GH excess and deficiency are associated with an altered gut microbial composition in adult mice. Yet, no study to date has examined the influence of GH on the gut microbiome over time. Our study thus tracked the effect of excess GH action on the longitudinal changes in the gut microbial profile (ie, abundance, diversity/maturity, predictive metabolic function, and short-chain fatty acid [SCFA] levels) of bovine GH (bGH) transgenic mice at age 3, 6, and 12 months compared to littermate controls in the context of metabolism, intestinal phenotype, and premature aging. The bGH mice displayed age-dependent changes in microbial abundance, richness, and evenness. Microbial maturity was significantly explained by genotype and age. Moreover, several bacteria (ie, Lactobacillus, Lachnospiraceae, Bifidobacterium, and Faecalibaculum), predictive metabolic pathways (such as SCFA, vitamin B12, folate, menaquinol, peptidoglycan, and heme B biosynthesis), and SCFA levels (acetate, butyrate, lactate, and propionate) were consistently altered across all 3 time points, differentiating the longitudinal bGH microbiome from controls. Of note, the bGH mice also had significantly impaired intestinal fat absorption with increased fecal output. Collectively, these findings suggest that excess GH alters the gut microbiome in an age-dependent manner with distinct longitudinal microbial and predicted metabolic pathway signatures.

Growth hormone alters gross anatomy and morphology of the small and large intestines in age- and sex-dependent manners.

Growth hormone (GH) has an important role in intestinal barrier function, and abnormalities in GH action have been associated with intestinal complications. Yet, the impact of altered GH on intestinal gross anatomy and morphology remains unclear. This study investigated the influence of GH signaling on gross anatomy, morphology, and fibrosis by characterizing the small and large intestines in male and female bovine growth hormone transgenic (bGH) mice and GH receptor gene-disrupted (GHR-/-) mice at multiple timepoints. The length, weight, and circumference of the small and large intestines were increased in bGH mice and decreased in GHR-/- mice across all ages. Colon circumference was significantly increased in bGH mice in a sex-dependent manner while significantly decreased in male GHR-/- mice. Villus height, crypt depth, and muscle thickness of the small intestine were generally increased in bGH mice and decreased in GHR-/- mice compared to controls with age- and sex-dependent exceptions. Colonic crypt depth and muscle thickness in bGH and GHR-/- mice were significantly altered in an age- and sex-dependent manner. Fibrosis was increased in the small intestine of bGH males at 4months of age, but no significant differences were seen between genotypes at other timepoints. This study observed notable opposing findings in the intestinal phenotype between mouse lines with GH action positively associated with intestinal gross anatomy (i.e. length, weight, and circumference). Moreover, GH action appears to alter morphology of the small and large intestines in an age- and sex-dependent manner.

Growth Hormone Upregulates Mediators of Melanoma Drug Efflux and Epithelial-to-Mesenchymal Transition In Vitro and In Vivo.

Simple SummaryGrowth hormone (GH) action is strongly implicated in the progression and therapy resistance in several types of solid tumors which overexpress the GH receptor (GHR). The aim of our study was to characterize the effects of GH and its downstream effector insulin-like growth factor 1 (IGF-1) on melanoma using in vitro and in vivo models. We confirmed an IGF-1-independent role of elevated circulating GH in upregulating key mechanisms of therapy resistance and malignancy with analyses conducted at the molecular and cellular level. We identified that GH upregulates key mechanisms of therapy resistance and metastases in melanoma tumors in an IGF-1 dependent and independent manner by upregulating multidrug efflux pumps and EMT transcription factors. Our study reveals that GH action renders an intrinsic drug resistance phenotype to the melanoma tumors—a clinically crucial property of GH verifiable in other human cancers with GHR expression.Growth hormone (GH) and the GH receptor (GHR) are expressed in a wide range of malignant tumors including melanoma. However, the effect of GH/insulin-like growth factor (IGF) on melanoma in vivo has not yet been elucidated. Here we assessed the physical and molecular effects of GH on mouse melanoma B16-F10 and human melanoma SK-MEL-30 cells in vitro. We then corroborated these observations with syngeneic B16-F10 tumors in two mouse lines with different levels of GH/IGF: bovine GH transgenic mice (bGH; high GH, high IGF-1) and GHR gene-disrupted or knockout mice (GHRKO; high GH, low IGF-1). In vitro, GH treatment enhanced mouse and human melanoma cell growth, drug retention and cell invasion. While the in vivo tumor size was unaffected in both bGH and GHRKO mouse lines, multiple drug-efflux pumps were up regulated. This intrinsic capacity of therapy resistance appears to be GH dependent. Additionally, epithelial-to-mesenchymal transition (EMT) gene transcription markers were significantly upregulated in vivo supporting our current and recent in vitro observations. These syngeneic mouse melanoma models of differential GH/IGF action can be valuable tools in screening for therapeutic options where lowering GH/IGF-1 action is important.

Lifelong Excess in GH Elicits Sexually Dimorphic Effects on Skeletal Morphology and Bone Mechanical Properties.

Excess in growth hormone (GH) levels, seen in patients with acromegaly, is associated with increases in fractures. This happens despite wider bones and independent of bone mineral density. We used the bovine GH (bGH) transgenic mice, which show constitutive excess in GH and insulin-like growth factor 1 (IGF-1) in serum and tissues, to study how lifelong increases in GH and IGF-1 affect skeletal integrity. Additionally, we crossed the acid labile subunit (ALS) null (ALSKO) to the bGH mice to reduce serum IGF-1 levels. Our findings indicate sexually dimorphic effects of GH on cortical and trabecular bone. Male bGH mice showed enlarged cortical diameters, but with marrow cavity expansion and thin cortices as well as increased vascular porosity that were associated with reductions in diaphyseal strength and stiffness. In contrast, female bGH mice presented with significantly smaller-diameter diaphysis, with greater cortical bone thickness and with a slightly reduced tissue elastic modulus (by microindentation), ultimately resulting in overall stronger, stiffer bones. We found increases in C-terminal telopeptide of type 1 collagen and procollagen type 1N propeptide in serum, independent of circulating IGF-1 levels, indicating increased bone remodeling with excess GH. Sexual dimorphism in response to excess GH was also observed in the trabecular bone compartment, particularly at the femur distal metaphysis. Female bGH mice preserved their trabecular architecture during aging, whereas trabecular bone volume in male bGH mice significantly reduced and was associated with thinning of the trabeculae. We conclude that pathological excess in GH results in sexually dimorphic changes in bone architecture and gains in bone mass that affect whole-bone mechanical properties, as well as sex-specific differences in bone material properties. © 2022 American Society for Bone and Mineral Research (ASBMR).

Growth hormone deficiency and excess alter the gut microbiome in adult male mice

The gut microbiome has been implicated in host metabolism, endocrinology, and pathophysiology. Furthermore, several studies have shown that gut bacteria impact host growth, partially mediated through the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis. Yet, no study to date has examined the specific role of GH on the gut microbiome. Our study thus characterized the adult gut microbial profile and intestinal phenotype in GH gene-disrupted (GH-/-) mice (a model of GH deficiency) and bovine GH transgenic (bGH) mice (a model of chronic, excess GH action) at 6 months of age. Both the GH-/- and bGH mice had altered microbial signatures, in opposing directions at the phylum and genus levels. For example, GH-/- mice had significantly reduced abundance in the Proteobacteria, Campylobacterota, and Actinobacteria phyla, whereas bGH mice exhibited a trending increase in those phyla compared with respective controls. Analysis of maturity of the microbial community demonstrated that lack of GH results in a significantly more immature microbiome while excess GH increases microbial maturity. Several common bacterial genera were shared, although in opposing directions, between the 2 mouse lines (e.g., decreased in GH-/- mice and increased in bGH mice), suggesting an association with GH. Similarly, metabolic pathways like acetate, butyrate, heme B, and folate biosynthesis were predicted to be impacted by GH. This study is the first to characterize the gut microbiome in mouse lines with altered GH action and indicates that GH may play a role in the growth of certain microbiota thus impacting microbial maturation and metabolic function.

Differential gene signature in adipose tissue depots of growth hormone transgenic mice.

Bovine growth hormone (bGH) transgenic mice mimic the clinical condition of acromegaly, having high circulating growth hormone (GH) levels. These mice are giant, have decreased adipose tissue (AT) mass, impaired glucose metabolism and a shortened lifespan. The detrimental effects of excess GH have been suggested, in part, to be a result of its depot-specific actions on AT. To investigate this relationship, we evaluated gene expression, biological mechanisms, cellular pathways and predicted microRNA (miRNA) in two AT depots (subcutaneous [Subq] and epididymal [Epi]) from bGH and littermate controls using RNA sequencing analysis. Two analyses on the differentially expressed genes (DEG) were performed: (i) comparison of the same AT depot between bGH and wild-type (WT) mice (genotype comparison) and (ii) comparison of Subq and Epi AT depots within the same genotype (depot comparison). For the genotype comparison, we found a higher number of significant DEG in the Subq AT depot of bGH mice compared to WT controls, corroborating previous reports that GH has a greater impact on the Subq depot. Furthermore, most of the DEG in bGH mice were not shared by WT mice, suggesting that excess GH induces the expression of genes not commonly present in AT. Through gene ontology and pathway analysis, the genotype comparison revealed that the DEG of the Subq depot of bGH mice relate to fatty acid oxidation, branched-chain amino acid degradation and the immune system. Additionally, the AT depot comparison showed that the immune cell activation and T-cell response appear up-regulated in the Subq compared to the Epi AT depot. The miRNA prediction also suggested a modulation of T-cell-related biological process in Subq. In summary, the present study provides a unique resource for understanding the specific differences in gene expression that are driven by both excess GH action and AT depot location.

SAT-281 Chronic, Excess Growth Hormone Action Alters the Development and Aging of the Microbial Community in the Mouse Gut

Emerging evidence proposes that the gut microbiome has an vital role in host growth, metabolism and endocrinology. That is, gut microbes impact growth by potentially altering the growth hormone (GH)/insulin-like growth factor-1 axis. Our previous research has also shown that GH - in states of absence and excess - is associated with altered gut microbial composition, maturity and predictive metabolic function in mice. Moreover, both GH and the gut microbiome are implicated in development and aging. Yet, it is unknown how GH impacts the longitudinal microbiome. This study thus aimed to characterize the longitudinal changes in the gut microbial profile of bovine GH transgenic mice (a model of chronic, excess GH action and accelerated aging). Microbial composition was quantified from fecal pellets of the same bGH and control mice at 3, 6 and 12 months of age through 16S rRNA gene sequencing and QIIME 2. Additional bioinformatic analyses assessed the unique signature and predictive metabolic function of the microbiome. The bGH mice had a distinct microbial profile compared to controls longitudinally. At 3 months, bGH mice had increased Firmicutes and Actinobacteria, decreased Bacteroidetes, Proteobacteria and Campylobacterota, and a significant reduction in microbial richness and evenness. By 6 months, all of the aforesaid phyla were increased with the exception of Firmicutes. By 12 months, bGH mice exhibited dysbiosis with increased Firmicutes and Proteobacteria and reduced Bacteroidetes, microbial richness and evenness. Moreover, abundance in Firmicutes, Bacteroidetes and Campylobacterota were significantly explained by the combined effect of genotype and age (p = 0.006, 0.005 and 0.02, respectively). Across all timepoints, bGH mice had a significantly different microbiome compared to controls (p = 0.002), and the development of microbial richness and evenness were also significantly different in bGH mice (p = 0.034 and 0.023). Bacterial genera Lactobacillus, Ruminococcaceae and Lachnospiraceae were identified as a unique candidates in bGH mice across all timepoints. Likewise, metabolic pathways involved in biosynthesis of heme b, menaquinol, acetate and butyrate differentiated the longitudinal bGH microbiome. Collectively, these results show that chronic, excess GH impacts the development and aging of the gut microbiome. Notably, several of the stated bacterial genera and metabolic pathways were associated with GH in our previous study, suggesting that GH may influence the longitudinal presence of certain gut microbes and metabolic functions. Additional studies will be performed to further explore the GH-associated gut microbiome and its impact on host health. Research was partially funded by the John J. Kopchick MCB/TBS Fellowship, a fellowship from the Osteopathic Heritage Foundation and the MMPC at UC, Davis (NIH grant U240DK092993).

GHR-/- Mice are protected from obesity-related white adipose tissue inflammation.

Growth hormone (GH) excess in bovine (b)GH transgenic mice has been shown to alter white adipose tissue (WAT) immune cell populations. The present study aimed to evaluate the effects of GH resistance on WAT immune cell populations using GH receptor knockout (GHR-/- ) mice. Eight- and 24-month-old, male GHR-/- and wild-type mice were used. Body composition and tissue weights were determined, and systemic inflammation was assessed by measuring serum cytokine levels. The stromal vascular fraction (SVF) was isolated from three distinct WAT depots, and immune cell populations were quantified using flow cytometry. GHR-/- mice at both ages had decreased body weight but were obese. Although no significant changes were observed in serum levels of the measured cytokines, SVF cell alterations were seen and differed from depot to depot. Total SVF cells were decreased in epidydimal (Epi) depots, whereas SVF cells per gram adipose tissue weight were increased in mesenteric (Mes) depots of GHR-/- mice relative to controls. T cells and T helper cells were increased in Mes at 8months old, whereas cytotoxic T cells were decreased in subcutaneous(SubQ) at 24months old. Other cells were unchanged at both ages measured. The present study demonstrates that removal of GH action results in modest and depot-specific changes to several immune cell populations in WAT of intra-abdominal depots (Epi and Mes), which are somewhat surprising resultsbecause the SubQ has the largest change in size, whereas the Mes has no size change. Taken together with previous results from bovine GH transgenic mice, these data suggest that GH induces changes in the immune cell population of WAT in a depot-specific manner. Notably, GHR-/- mice appear to be protected from age-related WAT inflammation and immune cell infiltration despite obesity.

Growth Hormone Deficiency and Excess Alter the Gut Microbiome in Adult Male Mice.

The gut microbiome has been implicated in host metabolism, endocrinology, and pathophysiology. Furthermore, several studies have shown that gut bacteria impact host growth, partially mediated through the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis. Yet, no study to date has examined the specific role of GH on the gut microbiome. Our study thus characterized the adult gut microbial profile and intestinal phenotype in GH gene-disrupted (GH-/-) mice (a model of GH deficiency) and bovine GH transgenic (bGH) mice (a model of chronic, excess GH action) at 6 months of age. Both the GH-/- and bGH mice had altered microbial signatures, in opposing directions at the phylum and genus levels. For example, GH-/- mice had significantly reduced abundance in the Proteobacteria, Campylobacterota, and Actinobacteria phyla, whereas bGH mice exhibited a trending increase in those phyla compared with respective controls. Analysis of maturity of the microbial community demonstrated that lack of GH results in a significantly more immature microbiome while excess GH increases microbial maturity. Several common bacterial genera were shared, although in opposing directions, between the 2 mouse lines (e.g., decreased in GH-/- mice and increased in bGH mice), suggesting an association with GH. Similarly, metabolic pathways like acetate, butyrate, heme B, and folate biosynthesis were predicted to be impacted by GH. This study is the first to characterize the gut microbiome in mouse lines with altered GH action and indicates that GH may play a role in the growth of certain microbiota thus impacting microbial maturation and metabolic function.

Growth Hormone Upregulates Melanocyte-Inducing Transcription Factor Expression and Activity via JAK2-STAT5 and SRC Signaling in GH Receptor-Positive Human Melanoma.

Growth hormone (GH) facilitates therapy resistance in the cancers of breast, colon, endometrium, and melanoma. The GH-stimulated pathways responsible for this resistance were identified as suppression of apoptosis, induction of epithelial-to-mesenchymal transition (EMT), and upregulated drug efflux by increased expression of ATP-binding cassette containing multidrug efflux pumps (ABC-transporters). In extremely drug-resistant melanoma, ABC-transporters have also been reported to mediate drug sequestration in intracellular melanosomes, thereby reducing drug efficacy. Melanocyte-inducing transcription factor (MITF) is the master regulator of melanocyte and melanoma cell fate as well as the melanosomal machinery. MITF targets such as the oncogene MET, as well as MITF-mediated processes such as resistance to radiation therapy, are both known to be upregulated by GH. Therefore, we chose to query the direct effects of GH on MITF expression and activity towards conferring chemoresistance in melanoma. Here, we demonstrate that GH significantly upregulates MITF as well as the MITF target genes following treatment with multiple anticancer drug treatments such as chemotherapy, BRAF-inhibitors, as well as tyrosine-kinase inhibitors. GH action also upregulated MITF-regulated processes such as melanogenesis and tyrosinase activity. Significant elevation in MITF and MITF target gene expression was also observed in mouse B16F10 melanoma cells and xenografts in bovine GH transgenic (bGH) mice compared to wild-type littermates. Through pathway inhibitor analysis we identified that both the JAK2-STAT5 and SRC activities were critical for the observed effects. Additionally, a retrospective analysis of gene expression data from GTEx, NCI60, CCLE, and TCGA databases corroborated our observed correlation of MITF function and GH action. Therefore, we present in vitro, in vivo, and in silico evidence which strongly implicates the GH–GHR axis in inducing chemoresistance in human melanoma by driving MITF-regulated and ABC-transporter-mediated drug clearance pathways.

MON-473 Growth Hormone (GH) Impacts the Gut Microbial Profile: the Presence of Unique Bacterial Genera in Mouse Lines with Altered GH Action

Several recent studies have shown that gut microbiota can regulate the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, or specifically modulate endocrine IGF-1. Yet, the impact of GH on the gut microbiome has not been evaluated. This study characterized the fecal microbial profile in GH gene disrupted (GH-/-) mice and bovine GH transgenic (bGH) mice at 6 months of age compared to their respective littermate controls. Microbial abundance and diversity were quantified from fecal pellets of these four groups through 16S rRNA sequencing. Additional bioinformatics analyses assessed the unique microbial signature and predictive biological function of the microbiome. Both GH-/- and bGH mice exhibited a unique microbial profile. In terms of microbial abundance, GH-/- and bGH mice resulted in a similar significant shift in the ratio of Firmicutes to Bacteroidetes. However, GH-/- and bGH mice differed in abundance of the Proteobacteria and Epsilonbacteraeota phyla with a significant reduction in abundance in GH-/- mice and significantly increased abundance in bGH mice. The GH-/- microbiome exhibited a distinct microbial signature with microbial immaturity and a predictive biological function associated with increased carbohydrate metabolism and decreased antimicrobial synthesis. The bGH microbiome also had a unique microbial signature with increased microbial richness and evenness. Moreover, both mouse lines shared common bacterial candidates. Common bacterial genera like Turicibacter, Ruminococcaceae and Faecalibaculum had similar abundance profiles in the bGH mice and GH-/- mice (i.e. either upregulated or downregulated with both increased and decreased GH action). Interestingly, however, other bacteria, such as Parasutterella, Lachnospiraceae and Rikenellaceae, followed opposite trends between bGH and GH-/- mice (i.e. upregulated with increased GH action and downregulated with decreased GH action). Collectively, these results demonstrate that GH - in both states of excess and deficiency - yields a distinct microbial profile, including microbial abundance, diversity and potential function. More importantly, the common bacterial candidates like Parasutterella suggest that GH action influences the presence of certain gut microbiota. Additional studies will be conducted to further explore these unique GH-associated bacterial candidates and their impact on metabolic and intestinal health of the host. This work was partially funded by the John J. Kopchick Molecular and Cellular Biology/Translational Biomedical Sciences Research Fellowship, the Osteopathic Heritage Foundation, Dual Degree Program at Ohio University Heritage College of Osteopathic Medicine and the Mouse Metabolic Phenotyping Center at University of California, Davis (NIH grant U240DK092993).

MON-479 Gross and Microscopic Intestinal Morphology of Bovine Growth Hormone Transgenic Mice Longitudinally

Growth hormone (GH) has been shown to have important effects on the intestines, demonstrated by its use in treatment of short bowel syndrome and the increase in colon polyps seen in acromegaly. Accordingly, GH has been shown to affect intestinal morphology at the gross and microscopic levels, including increased intestinal length and increased villus height in situations of excess GH aged 2 months. However, the effect of excess GH on intestinal morphology longitudinally or at advanced ages has not been extensively examined. This study measured the gross and microscopic intestinal morphology at multiple ages in bovine GH (bGH) transgenic mice, a mouse model of acromegaly. Adult bGH mice and littermate controls aged 6, 11, or 13 months were used for this study. The intestines were dissected, mesenteric fat was removed, and the small intestine (SI) was separated from the large intestine (LI). The length and circumference of the SI and the LI were measured. Intestinal portions were processed using the Swiss roll method, paraffin embedded, sectioned, and stained. The images of the sections were then used to measure the villus height, crypt depth and muscle thickness. In accordance with previous reports in humans and mice, chronic GH excess causes increased intestinal length. This difference was present in both the SI and LI and at 6, 11, and 13 months of age. bGH mice at all three ages had increased SI weight. LI weight was increased in bGH mice at 6 and 13 months of age, but there was no increase at 11 months of age. At 13 months of age, the LI circumference was increased in bGH mice, and the SI circumference at 6 months of age was also increased. At the microscopic level, the 6-month-old bGH mice had significantly increased crypt depth in the duodenum and jejunum, and a trend in the ileum. There is also a trend towards increased villus height in the 6-month bGH mice in all three sections of the SI that did not reach significance. The only change observed in the 11-month bGH mice was increased muscle thickness in the duodenum. The results of this study indicate that chronic GH excess indeed increases intestinal length, a change that persists longitudinally. The previously reported increase in intestinal villus height in response to GH excess was not observed in this experiment, but increased crypt depth was observed in the youngest cohort. The oldest group had an increase in SI circumference that coincided with increased muscle thickness in the 11-month bGH mice. In summary, our data show a potent trophic effect of GH on the intestinal tract, as well as an effect on the microscopic morphology of the intestines; however, the microscopic changes differ from those previously reported and do not seem to persist over the lifespan of a mouse. Acknowledgements: This work was supported by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by AMVETS, and by the Diabetes Institute at Ohio University

Metabolic adaptation of short-living growth hormone transgenic mice to methionine restriction and supplementation.

Extension of mammalian health and life span has been achieved using various dietary interventions. We previously reported that restricting dietary methionine (MET) content extends life span only when growth hormone signaling is intact (no life span increase in GH deficiency or GH resistance). To understand the metabolic responses of altered dietary MET in the context of accelerated aging (high GH), the current study evaluated MET and related pathways in short-living GH transgenic (GH Tg) and wild-type mice following 8weeks of restricted (0.16%), low (0.43%), or enriched (1.3%) MET consumption. Liver MET metabolic enzymes were suppressed in GH Tg compared to diet-matched wild-type mice. MET metabolite levels were differentially affected by GH status and diet. SAM:SAH ratios were markedly higher in GH Tg mice. Glutathione levels were lower in both genotypes consuming 0.16% MET but reduced in GH Tg mice when compared to wild type. Tissue thioredoxin and glutaredoxin were impacted by diet and GH status. The responsiveness to the different MET diets is reflected across many metabolic pathways indicating the importance of GH signaling in the ability to discriminate dietary amino acid levels and alter metabolism and life span.

Increased fibrosis: A novel means by which GH influences white adipose tissue function

ObjectiveWhite adipose tissue (WAT) fibrosis – the buildup of extracellular matrix (ECM) proteins, primarily collagen – is now a recognized hallmark of tissue dysfunction and is increased with obesity and lipodystrophy. While growth hormone (GH) is known to increase collagen in several tissues, no previous research has addressed its effect on ECM in WAT. Thus, the purpose of this study is to determine if GH influences WAT fibrosis. DesignThis study examined WAT from four distinct strains of GH-altered mice (bGH and GHA transgenic mice as well as two tissue specific GH receptor gene disrupted lines, fat growth hormone receptor knockout or FaGHRKO and liver growth hormone receptor knockout or LiGHRKO mice). Collagen content and adipocyte size were studied in all cohorts and compared to littermate controls. In addition, mRNA expression of fibrosis-associated genes was assessed in one cohort (6month old male bovine GH transgenic and WT mice) and cultured 3T3-L1 adipocytes treated with GH. ResultsCollagen stained area was increased in WAT from bGH mice, was depot-dependent, and increased with age. Furthermore, increased collagen content was associated with decreased adipocyte size in all depots but more dramatic changes in the subcutaneous fat pad. Notably, the increase in collagen was not associated with an increase in collagen gene expression or other genes known to promote fibrosis in WAT, but collagen gene expression was increased with acute GH administration in 3T3-LI cells. In contrast, evaluation of 6month old GH antagonist (GHA) male mice showed significantly decreased collagen in the subcutaneous depot. Lastly, to assess if GH induced collagen deposition directly or indirectly (via IGF-1), fat (Fa) and liver (Li) specific GHRKO mice were evaluated. Decreased fibrosis in FaGHRKO and increased fibrosis in LiGHRKO mice suggest GH is primarily responsible for the alterations in collagen. ConclusionsOur results show that GH action is positively associated with an increase in WAT collagen content as well as a decrease in adipocyte size, particularly in the subcutaneous depot. This effect appears to be due to GH and not IGF-1 and reveals a novel means by which GH regulates WAT accumulation.