Adropin promotes testicular functions by modulating redox homeostasis in adult mouse.

PURPOSE: Adropin is an emerging metabolic hormone that has a role in regulating energy homeostasis. The present study aimed to explore the impact of adropin on redox homeostasis and its possible role in testicular functions in adult mouse testis. METHODS: Western blot, flow-cytometry, and TUNEL assay were performed to explore the impact of intra-testicular treatment of adropin (0.5 µg/testis) on testicular functions of adult mice. Hormonal assay was done by ELISA. Further, antioxidant enzyme activities were measured. RESULTS: Adropin treatment significantly increased the sperm count and testicular testosterone by increasing the expression of GPR19 and steroidogenic proteins. Also, adropin treatment reduced the oxidative/nitrosative stress by facilitating the translocation of NRF2 and inhibiting NF-κB into the nucleus of germ cells. Enhanced nuclear translocation of NRF2 leads to elevated biosynthesis of antioxidant enzymes, evident by increased HO-1, SOD, and catalase activity that ultimately resulted into declined LPO levels in adropin-treated mice testes. Furthermore, adropin decreased nuclear translocation of NF-κB in germ cells, that resulted into decreased NO production leading to decreased nitrosative stress. Adropin/GPR19 signaling significantly increased its differentiation, proliferation, and survival of germ cells by elevating the expression of PCNA and declining caspase 3, cleaved caspase 3 expression, Bax/Bcl2 ratio, and TUNEL-positive cells. FACS analysis revealed that adropin treatment enhances overall turnover of testicular cells leading to rise in production of advanced germ cells, notably spermatids. CONCLUSION: The present study indicated that adropin improves testicular steroidogenesis, spermatogenesis via modulating redox potential and could be a promising target for treating testicular dysfunctions.

Adropin, a novel hepatokine: localization and expression during postnatal development and its impact on testicular functions of pre-pubertal mice.

Adropin, a multifaceted peptide, was identified as a new metabolic hormone responsible for regulating gluco-lipid homeostasis. However, its role in the testicular function is not yet understood. We aimed to investigate the localization and expression of adropin and GPR19 during different phases of postnatal development. Immunohistochemical study revealed the intense reactivity of adropin in the Leydig cells during all phases of postnatal development, while GPR19 showed intense immunoreactivity in the pachytene spermatocytes and mild immunoreactivity in Leydig cells as well as primary and secondary spermatocytes. Western blot study revealed maximum expression of GPR19 in pre-pubertal mouse testis that clearly indicates maximum responsiveness of adropin during that period. So, we hypothesized that adropin may act as an autocrine/paracrine factor that regulates pubertal changes in mouse testis. To examine the effect of adropin on pubertal onset, we gave bilateral intra-testicular doses (0.5 and 1.5 µg/testis) to pre-pubertal mice. Adropin treatment promoted testicular testosterone synthesis by increasing the expression of StAR, 3ß-HSD, and 17ß-HSD. Adropin also promoted germ cell survival and proliferation by upregulating the expression of PCNA and downregulating the Bax/Bcl2 ratio and Caspase 3 expression resulting in fewer TUNEL-positive cells in adropin-treated groups. FACS analysis demonstrated that adropin treatment not only increases 1C to 4C ratio but also significantly increases the 1C (spermatid) and 1C to 2C ratio which demarcates accelerated germ cell differentiation and turnover of testicular cells. In conclusion, adropin promotes steroidogenesis, germ cell survival, as well as the proliferation in the pre-pubertal mouse testis that may hasten the pubertal transition in an autocrine/paracrine manner.

Adropin may promote insulin stimulated steroidogenesis and spermatogenesis in adult mice testes.

Adropin is a versatile peptide which was discovered as a novel metabolic hormone that is involved in the regulation of lipid and glucose homeostasis. However, its possible role in the testicular function is not yet understood. The aim of our study was to explore the distribution pattern of adropin and GPR19 in various cell types and its possible role in testicular functions of adult mice. Immunohistochemical study revealed the intense immunoreactivity of adropin in the Leydig cells, while GPR19 showed intense immunoreactivity in the pachytene spermatocytes and mild immunoreactivity in Leydig cells and primary as well as secondary spermatocytes in mouse testis. Enho mRNA was also found to be expressed in the mouse testis. These findings suggested that adropin-GPR19 signaling may act in autocrine/paracrine manner to modulate testicular functions. Furthermore, to find out the direct role of adropin in the testicular function, in vitro study was performed in which testicular slices were cultured with adropin alone (10 and 100 ng/mL) and in combination with insulin (5 µg/mL). Adropin alone inhibited testicular testosterone synthesis by inhibiting the expression of P450-SCC, 3ß-HSD, and 17ß-HSD while along with insulin stimulated the testicular testosterone synthesis by increasing the expression of GPR19, IR, StAR, P450-SCC, 3ß-HSD, and 17ß-HSD. Adropin alone or in combination with insulin promoted germ cell survival and proliferation by upregulating the expression of PCNA, Bcl2, and pERK1/2. Thus, it can be concluded that adropin-GPR19 signaling promotes insulin stimulated steroidogenesis and germ cell survival as well as proliferation in the mice testes in an autocrine/paracrine manner.

Ontogeny of adropin and its receptor expression during postnatal development and its pro-gonadal role in the ovary of pre-pubertal mouse.

Adropin, a highly conserved multifunctional peptide hormone, has a beneficial effect on the maintenance of gluco-lipid homeostasis, endothelial and cardiovascular functions. However, the expression and potential role of adropin in ovarian function are not fully elucidated. The present study aimed to investigate the expression of adropin and GPR19 in the mice ovary during various stages of postnatal development. This study also explored whether the treatment of adropin can modulate the timing of puberty, for which pre-pubertal mice were treated with adropin. The result showed the intense immunoreactivity of adropin in TICs, while GPR19 immunoreactivity was noted in GCs in infantile, pre-pubertal, and pubertal mice ovary. Also, adropin and GPR19 are highly expressed in the CL of the ovary of reproductively active mice. The fact that adropin expression in the ovary at different stages of postnatal development positively correlated with circulating progesterone and estradiol indicated that it has a role in the production of steroid hormones. Furthermore, the results of in vivo studies in pre-pubertal mice showed that adropin promotes early folliculogenesis by enhancing the proliferation (PCNA) of GCs of cortical ovarian follicles and promotes estradiol production by enhancing the expression of GPR19, StAR, CYP11A1 and aromatase proteins. Also, adropin treatment increases the Bax/Bcl2 ratio and expression of cleaved caspase-3 and ERα proteins, which may result in increased apoptosis of medullary follicles leading to the formation of a well-developed interstitium with interstitial glandular cells. Collectively, these findings indicate that adropin may be a factor that accelerates pubertal development in the ovary and could be utilized as a therapeutic approach for treating pubertal delay.

Adropin may regulate corpus luteum formation and its function in adult mouse ovary.

BACKGROUND: Adropin, a unique peptide hormone, has been associated with the regulation of several physiological processes, including glucose homeostasis, fatty acid metabolism, and neovascularization. However, its possible role in ovarian function is not understood. Our objective was to examine the expression of adropin and its putative receptor, GPR19, in the ovaries of mice at various phases of the estrous cycle. METHODS: Immunohistochemistry and western blot analysis were performed to explore the localization and changes in expression of adropin and GPR19 in the ovaries during different phases of the estrous cycle in mice. Hormonal assays were performed with ELISA. An in vitro study was performed to examine the direct effect of adropin (10, 100 ng/ml) on ovarian function. RESULTS: A western blot study showed that adropin and GPR19 proteins were maximum during the estrus phase of the estrous cycle. Interestingly, adropin and GPR19 displayed intense immunoreactivity in granulosa cells of large antral follicles and corpus luteum. This suggested the possible involvement of adropin in corpus luteum formation. Adropin treatment stimulated progesterone synthesis by increasing GPR19, StAR, CYP11A1, and 3ß-HSD expressions, while it decreased estrogen synthesis by inhibiting 17ß-HSD and aromatase protein expressions. Moreover, adropin treatment upregulated the cell cycle arrest-CDK inhibitor 1B (p27kip1), pERK1/2, and angiogenic protein (EG VEGF) that are involved in the process of luteinization. CONCLUSIONS: Adropin GPR19 signaling promotes the synthesis of progesterone and upregulates the expression of p27kip1, EG VEGF, and erk1/2, resulting in cell cycle arrest and neovascularization, which ultimately leads to corpus luteum formation.

GPR19 Coordinates Multiple Molecular Aspects of Stress Responses Associated with the Aging Process.

G protein-coupled receptors (GPCRs) play a significant role in controlling biological paradigms such as aging and aging-related disease. We have previously identified receptor signaling systems that are specifically associated with controlling molecular pathologies associated with the aging process. Here, we have identified a pseudo-orphan GPCR, G protein-coupled receptor 19 (GPR19), that is sensitive to many molecular aspects of the aging process. Through an in-depth molecular investigation process that involved proteomic, molecular biological, and advanced informatic experimentation, this study found that the functionality of GPR19 is specifically linked to sensory, protective, and remedial signaling systems associated with aging-related pathology. This study suggests that the activity of this receptor may play a role in mitigating the effects of aging-related pathology by promoting protective and remedial signaling systems. GPR19 expression variation demonstrates variability in the molecular activity in this larger process. At low expression levels in HEK293 cells, GPR19 expression regulates signaling paradigms linked with stress responses and metabolic responses to these. At higher expression levels, GPR19 expression co-regulates systems involved in sensing and repairing DNA damage, while at the highest levels of GPR19 expression, a functional link to processes of cellular senescence is seen. In this manner, GPR19 may function as a coordinator of aging-associated metabolic dysfunction, stress response, DNA integrity management, and eventual senescence.

Intersection of the Orphan G Protein-Coupled Receptor, GPR19, with the Aging Process.

G protein-coupled receptors (GPCRs) represent one of the most functionally diverse classes of transmembrane proteins. GPCRs and their associated signaling systems have been linked to nearly every physiological process. They also constitute nearly 40% of the current pharmacopeia as direct targets of remedial therapies. Hence, their place as a functional nexus in the interface between physiological and pathophysiological processes suggests that GPCRs may play a central role in the generation of nearly all types of human disease. Perhaps one mechanism through which GPCRs can mediate this pivotal function is through the control of the molecular aging process. It is now appreciated that, indeed, many human disorders/diseases are induced by GPCR signaling processes linked to pathological aging. Here we discuss one such novel member of the GPCR family, GPR19, that may represent an important new target for novel remedial strategies for the aging process. The molecular signaling pathways (metabolic control, circadian rhythm regulation and stress responsiveness) associated with this recently characterized receptor suggest an important role in aging-related disease etiology.

Ciliary signaling proteins are mislocalized in the brains of Bardet-Biedl syndrome 1-null mice.

In the brain, primary cilia are found on most, if not all, central neurons. The importance of neuronal cilia is underscored by the fact that human diseases caused by primary cilia dysfunction, which are known as ciliopathies, are associated with neuropathologies, including neuropsychiatric disorders and learning and memory deficits. Neuronal cilia are enriched for certain G protein-coupled receptors and their downstream effectors, suggesting they sense and respond to neuromodulators in the extracellular milieu. GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome, with GPCRs failing to localize to cilia, indicating the Bardet-Biedl syndrome proteins are required for trafficking of G protein-coupled receptors into neuronal cilia. Yet, dopamine receptor 1 accumulates in cilia in the absence of Bardet-Biedl syndrome proteins, suggesting Bardet-Biedl syndrome proteins are required for normal ciliary import and export. To further explore the roles of the Bardet-Biedl syndrome proteins in neuronal cilia, we examined localization of ciliary signaling proteins in a new constitutive Bbs1 knockout mouse model. Interestingly, we find that two additional ciliary G protein-coupled receptors (Gpr161 and Gpr19) abnormally accumulate in cilia on Bardet-Biedl syndrome neurons. In addition, we find that the GPCR signaling protein ß-arrestin accumulates in a subset of cilia in the brain, suggesting the presence of additional unidentified ciliary G protein-coupled receptors. These results confirm the importance of the Bardet-Biedl syndrome proteins in establishing ciliary GPCR pathways and indicate that loss of Bbs1 leads to complex changes in the localization of signaling proteins in the brain.

Adropin Stimulates Proliferation and Inhibits Adrenocortical Steroidogenesis in the Human Adrenal Carcinoma (HAC15) Cell Line.

Adropin is a multifunctional peptide hormone encoded by the ENHO (energy homeostasis associated) gene. It plays a role in mechanisms related to increased adiposity, insulin resistance, as well as glucose, and lipid metabolism. The low adropin levels are strongly associated with obesity independent insulin resistance. On the other hand, overexpression or exogenous administration of adropin improves glucose homeostasis. The multidirectional, adropin-related effects associated with the regulation of metabolism in humans also appear to be attributable to the effects of this peptide on the activity of various elements of the endocrine system including adrenal cortex. Therefore, the main purpose of the present study was to investigate the effect of adropin on proliferation and secretory activity in the human HAC15 adrenal carcinoma cell line. In this study, we obtained several highly interesting findings. First, GPR19, the main candidate sensitizer of adrenocortical cells to adropin, was expressed in HAC15 cells. Moreover, GPR19 expression was relatively stable and not regulated by ACTH, forskolin, or adropin itself. Our findings also suggest that adropin has the capacity to decrease expression levels of steroidogenic genes such as steroidogenic acute regulatory protein (StAR) and CYP11A1, which then led to a statistically significant inhibition in cortisol and aldosterone biosynthesis and secretion. Based on whole transcriptome study and research involving transforming growth factor (TGF)-ß type I receptor kinase inhibitor we demonstrated that attenuation of steroidogenesis caused by adropin is mediated by the TGF-ß signaling pathway likely to act through transactivation mechanism. We found that HAC15 cells treated with adropin presented significantly higher proliferation levels than untreated cells. Using specific intracellular inhibitors, we showed that adropin stimulate proliferation via ERK1/2 and AKT dependent signaling pathways. We have also demonstrated that expression of GPR19 is elevated in adrenocortical carcinoma in relation to normal adrenal glands. High level of GPR19 expression in adrenocortical carcinoma may constitute a negative prognostic factor of disease progression.

Inhibition of the LncRNA Gpr19 attenuates ischemia-reperfusion injury after acute myocardial infarction by inhibiting apoptosis and oxidative stress via the miR-324-5p/Mtfr1 axis.

Reperfusion therapy after acute myocardial infarction (AMI) can effectively restore the blood supply and nutritional support of ischemic myocardium and save the dying myocardium. However, myocardial ischaemia-reperfusion (I/R) injury has become a new threat to reperfusion therapy for AMI. Many long-chain noncoding RNAs (lncRNAs) are dysregulated by I/R damage. Of these dysregulated lncRNAs, Gpr19 was selected as a potential gene of interest based on its high expression change. We aimed to explore the functional role and molecular mechanism of Gpr19 in I/R injury of AMI. C57BL/6 mice underwent I/R injury as in vivo models. Neonatal rat ventricular cardiomyocytes (NRCMs) exposed to an oxygen glucose deprivation/recovery (OGD/R) system were used as an in vitro model. A TUNEL assay, western blot, and oxidative stress analysis were conducted in this study to determine apoptosis and oxidative stress levels. Our results indicated that inhibition of Gpr19 improves cardiac function and reduces apoptosis and myocardial fibrosis scar formation in vivo. Suppression of Gpr19 attenuates oxidative stress and apoptosis in NRCMs exposed to OGD/R. We further demonstrated that inhibition of Gpr19 decreases oxidative stress and apoptosis in OGD/R-induced NRCMs by regulating miR-324-5p and mitochondrial fission regulator 1 (Mtfr1). We elucidated the functional role and potential molecular mechanism of Gpr19 in I/R injury of AMI, provided a theoretical basis for the importance of Gpr9 in I/R injury, and provided a new perspective for the clinical treatment of I/R injury of AMI.

G protein-coupled receptor GPR19 regulates E-cadherin expression and invasion of breast cancer cells.

Dysregulation of G protein-coupled receptors (GPCRs) is known to be involved in the pathogenesis of a variety of diseases, including cancer initiation and progression. Within this family, approximately 140 GPCRs have no known endogenous ligands and these "orphan" GPCRs remain poorly characterized. The orphan GPCR GPR19 was identified and cloned 2 decades ago, but relatively little is known about its physio-pathological relevance. We observed its expression to be elevated in breast cancers and therefore sought to investigate its potential role in breast cancer pathology. In this work, we show that overexpression of GPR19 drives mesenchymal-like breast cancer cells to adopt an epithelial-like phenotype, as demonstrated by the upregulation in E-cadherin expression and changes in functional behavior. We confirm a previous report that a peptide, adropin, is an endogenous ligand for GPR19. We further show that adropin-mediated activation of GPR19 activates the MAPK/ERK1/2 pathway, which is essential for the observed upregulation in E-cadherin and accompanying phenotypic changes. The recapitulation of epithelial characteristics at the secondary tumor sites is now understood to be an essential step in the colonization process. Taken together our work shows for the first time that GPR19 plays a potential role in metastasis by promoting the mesenchymal-epithelial transition (MET) through the ERK/MAPK pathway, thus facilitating colonization of metastatic breast tumor cells.