Signaling in rat brainstem via Gpr160 is required for the anorexigenic and antidipsogenic actions of cocaine- and amphetamine-regulated transcript peptide.

Recent work identified Gpr160 as a candidate receptor for cocaine- and amphetamine-regulated transcript peptide (CARTp) and described its role in pain modulation. The aims of the present study were to determine if Gpr160 is required for the CARTp's ability to reduce food intake and water intake and to initially identify the distribution of Gpr160-like immunoreactivity (Gpr160ir) in the rat brain. A passive immunoneutralization approach targeting Gpr160 was used to block the behavioral effects of a pharmacological dose of CARTp in the fourth cerebroventricle (4V) of rats and to determine the importance of endogenously produced CARTp in the control of ingestive behaviors. Passive immunoneutralization of Gpr160 in the 4V blocked the actions of CARTp to inhibit food intake and water intake. Blockade of Gpr160 in the 4V, independent of pharmacological CART treatment, caused an increase in both overnight food intake and water intake. The decrease in food intake, but not water intake, caused by central injection of CARTp was demonstrated to be interrupted by prior administration of a glucagon-like peptide 1 (GLP-1) receptor antagonist. Gpr160ir was observed in several, distinct sites throughout the rat brain, where CARTp staining has been described. Importantly, Gpr160ir was observed to be present in both neuronal and nonneuronal cell types. These data support the hypothesis that Gpr160 is required for the anorexigenic actions of central CARTp injection and extend these findings to water drinking. Gpr160ir was observed in both neuronal and nonneuronal cell types in regions known to be important in the multiple pharmacological effects of CARTp, identifying those areas as targets for future compromise of function studies.

The Deductive Reasoning Strategy Enables Biomedical Breakthroughs.

G protein-coupled receptors (GPCRs) transmit the signals of a variety of hormones and neurotransmitters and are targets of more than 30% of all FDA-approved drugs. We developed an approach for identifying the endogenous ligands for a family of orphan GPCRs that enables the development of novel therapeutics for the potential treatment of a wide variety of disorders including pain, diabetes, appetitive behaviors, infertility and obesity. With this approach, we have deorphanized five previously orphaned GPCRs.

A novel regulator of thirst behavior: phoenixin.

There are examples of physiological conditions under which thirst is inappropriately exaggerated, and the mechanisms for these paradoxical ingestive behaviors remain unknown. We are interested in thirst mechanisms across the female life cycle and have identified a novel mechanism through which ingestive behavior may be activated. We discovered a previously unrecognized endogenous hypothalamic peptide, phoenixin (PNX), identified physiologically relevant actions of the peptide in brain and pituitary gland to control reproductive hormone secretion in female rodents, and in the process identified the previously orphaned G protein-coupled receptor Gpr173 to be a potential receptor for the peptide. Labeled PNX binding distribution in brain parallels areas known to be important in ingestive behaviors as well in areas where gonadal steroids feedback to control estrous cyclicity (Stein LM, Tullock CW, Mathews SK, Garcia-Galiano D, Elias CF, Samson WK, Yosten GLC, Am J Physiol Regul Integr Comp Physiol 311: R489-R496, 2016). We have demonstrated upregulation of Gpr173 during puberty, fluctuations across the estrous cycle, and, importantly, upregulation during the last third of gestation. It is during this hypervolemic, hyponatremic state that both vasopressin secretion and thirst are inappropriately elevated in humans. Here, we show that central administration of PNX stimulated water drinking in both males and females under ad libitum conditions, increased water drinking after overnight fluid deprivation, and increased both water and 1.5% NaCl ingestion under fed and hydrated conditions. Importantly, losartan pretreatment blocked the effect of PNX on water drinking, and knockdown of Gpr173 by use of short interfering RNA constructs significantly attenuated water drinking in response to overnight fluid deprivation. These actions, together with the stimulatory action of PNX on vasopressin secretion, suggest that this recently discovered neuropeptide may impact the recruitment of critically important neural circuits through which ingestive behaviors and endocrine mechanisms that maintain fluid and electrolyte homeostasis are regulated.

Expression and function of nesfatin-1 are altered by stage of the estrous cycle.

Nesfatin-1 is a peptide derived from the nucleobindin 2 (Nucb2) precursor protein that has been shown to exert potent effects on appetite and cardiovascular function in male animals. Sex hormones modulate the expression of Nucb2 in several species, including goldfish, mouse, and rat, and human studies have revealed differential expression based on male or female sex. We therefore hypothesized that the ability of nesfatin-1 to increase mean arterial pressure (MAP) would be influenced by stage of the estrous cycle. Indeed, we found that in cycling female Sprague-Dawley rats, nesfatin-1 induced an increase in MAP on diestrus, when both estrogen and progesterone levels are low but not on proestrus or estrus. The effect of nesfatin-1 on MAP was dependent on functional central melanocortin receptors, because the nesfatin-1-induced increase in MAP was abolished by pretreatment with the melanocortin 3/4 receptor antagonist, SHU9119. We previously reported that nesfatin-1 inhibited angiotensin II-induced water drinking in male rats but found no effect of nesfatin-1 in females in diestrus. However, nesfatin-1 enhanced angiotensin II-induced elevations in MAP in females in diestrus but had no effect on males. Finally, in agreement with previous reports, the expression of Nucb2 mRNA in hypothalamus was significantly reduced in female rats in proestrus compared with rats in diestrus. From these data we conclude that the function and expression of nesfatin-1 are modulated by sex hormone status. Further studies are required to determine the contributions of chromosomal sex and individual sex hormones to the cardiovascular effects of nesfatin-1.

Novel regulator of vasopressin secretion: phoenixin.

The newly described hypothalamic peptide, phoenixin, is produced in the hypothalamus and adenohypophysis, where it acts to control reproductive hormone secretion. Both phoenixin and its receptor GPR173 are expressed in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, suggesting additional, nonreproductive effects of the peptide to control vasopressin (AVP) or oxytocin (OT) secretion. Hypothalamo-neurohypophysial explants released AVP but not OT in response to phoenixin. Intracerebroventricular administration of phoenixin into conscious, unrestrained male and female rats significantly increased circulating AVP, but not OT, levels in plasma, and it increased immediate early gene expression in the supraoptic nuclei of male rats. Bath application of phoenixin in hypothalamic slice preparations resulted in depolarization of PVN neurons, indicating a direct, neural action of phoenixin in the hypothalamus. Our results suggest that the newly described, hypothalamic peptide phoenixin, in addition to its effects on hypothalamic and pituitary mechanisms controlling reproduction, may contribute to the physiological mechanisms regulating fluid and electrolyte homeostasis.

A 5'-upstream short open reading frame encoded peptide regulates angiotensin type 1a receptor production and signalling via the ß-arrestin pathway.

AUG sequences and short open reading frames are commonly present in the 5'-leader sequence of G protein-coupled receptor mRNAs. The presence of these upstream AUG sequences has been demonstrated to inhibit downstream receptor translation efficiency and, most recently, receptor signal transduction. A seven amino acid peptide encoded by a short open reading frame in exon 2 of the angiotensin type 1a receptor has been shown to inhibit non-G protein-coupled signalling of angiotensin II, without altering the classical G protein-coupled pathway activated by the ligand. This finding may lead to the development of a new class of angiotensin receptor antagonists with activities biased for one, but not all, of the signalling cascades activated by angiotensin II, which could have therapeutic implications for the myriad hormones and neurotransmitters that signal through G protein-coupled receptors.

The Physiology of Proinsulin C-Peptide: Unanswered Questions and a Proposed Model.

C-peptide is produced, processed, and secreted with insulin, and appears to exert separate but intimately related effects. In this review, we address the existence of the C-peptide receptor, the interaction between C-peptide and insulin, and the potential physiological significance of proinsulin C-peptide.

Adropin acts in brain to inhibit water drinking: potential interaction with the orphan G protein-coupled receptor, GPR19.

Adropin, a recently described peptide hormone produced in the brain and liver, has been reported to have physiologically relevant actions on glucose homeostasis and lipogenesis, and to exert significant effect on endothelial function. We describe a central nervous system action of adropin to inhibit water drinking and identify a potential adropin receptor, the orphan G protein-coupled receptor, GPR19. Reduction in GPR19 mRNA levels in medial basal hypothalamus of male rats resulted in the loss of the inhibitory effect of adropin on water deprivation-induced thirst. The identification of a novel brain action of adropin and a candidate receptor for the peptide should extend and accelerate the study of the potential therapeutic value of adropin or its mimetics for the treatment of metabolic disorders.

Neuronostatin acts via GPR107 to increase cAMP-independent PKA phosphorylation and proglucagon mRNA accumulation in pancreatic α-cells.

Neuronostatin (NST) is a recently described peptide that is produced from the somatostatin preprohormone in pancreatic δ-cells. NST has been shown to increase glucagon secretion from primary rat pancreatic islets in low-glucose conditions. Here, we demonstrate that NST increases proglucagon message in α-cells and identify a potential mechanism for NST's cellular activities, including the phosphorylation of PKA following activation of the G protein-coupled receptor, GPR107. GPR107 is abundantly expressed in the pancreas, particularly, in rodent and human α-cells. Compromise of GPR107 in pancreatic α-cells results in failure of NST to increase PKA phosphorylation and proglucagon mRNA levels. We also demonstrate colocalization of GPR107 and NST on both mouse and human pancreatic α-cells. Taken together with our group's observation that NST infusion in conscious rats impairs glucose clearance in response to a glucose challenge and that plasma levels of the peptide are elevated in the fasted compared with the fed or fasted-refed state, these studies support the hypothesis that endogenous NST regulates islet cell function by interacting with GPR107 and initiating signaling in glucagon-producing α-cells.

Hypothalamic action of phoenixin to control reproductive hormone secretion in females: importance of the orphan G protein-coupled receptor Gpr173.

Sexual maturation and maintenance of reproductive function are regulated by neurohormonal communication between the hypothalamus, pituitary, and gonads (referred to as the HPG axis). Phoenixin (PNX) is a newly identified, endogenous peptide abundantly produced in the hypothalamus and shown to be an important mediator of ovarian cyclicity. However, the underlying mechanisms by which phoenixin functions within the HPG axis are unknown. Previous in vitro studies demonstrated a direct action of PNX on gonadotrophs to potentiate gonadotrophin-releasing hormone (GnRH) induced luteinizing hormone (LH) secretion. Therefore, we hypothesized that centrally derived phoenixin regulates the preovulatory LH surge required for ovarian cyclicity. We observed a significant dose-related increase in the level of plasma LH in diestrous, female rats that were given an intracerebroventricular injection of PNX compared with vehicle-treated controls. While this suggests that even under low-estrogen conditions, PNX acts centrally to stimulate the HPG axis, further characterization is contingent on the elucidation of its cognate receptor. Using the "deductive ligand receptor matching strategy," we identified the orphan G protein-coupled receptor, Gpr173, as our top candidate. In cultured pituitary cells, siRNA-targeted compromise of Gpr173 abrogated PNX's action to potentiate GnRH-stimulated LH secretion. In addition, siRNA-mediated knockdown of endogenous Gpr173, which localized to several hypothalamic sites related to reproductive function, not only significantly extended the estrous cycle but also prevented the PNX-induced LH secretion in diestrous, female rats. These studies are the first to demonstrate a functional relationship between PNX and Gpr173 in reproductive physiology and identify a potential therapeutic target for ovulatory dysfunction.

Aging-related impairment of urine-concentrating mechanisms correlates with dysregulation of adrenocortical angiotensin type 1 receptors in male Fischer rats.

To investigate age-associated impairments in fluid homeostasis, 4-mo (young) and 32-mo (old) Fischer 344/BN male rats were studied before and after a dietary sodium load. Transferring young rats from a low-sodium (LS) to a high-sodium (HS) diet increased water intake and urine volume by 1.9- and 3.0-fold, respectively, while urine osmolality and plasma aldosterone decreased by 33 and 98%. Concomitantly, adrenocortical angiotensin type 1 receptor (AT1R) density decreased by 35%, and AT1bR mRNA decreased by 39%; no changes were observed in AT1aR mRNA. In contrast, the increase in water intake (1.4-fold) was lower in the old rats, and there was no effect of the HS diet on urine volume or urine osmolality. AT1R densities were 29% less in the old rats before transferring to the HS diet, and AT1R densities were not reduced as rapidly in response to a HS diet compared with the young animals. After 6 days on the HS diet, plasma potassium was lowered by 26% in the old rats, whereas no change was detected in the young rats. Furthermore, while plasma aldosterone was substantially decreased after 2 days on the HS diet in both young and old rats, plasma aldosterone was significantly lower in the old compared with the young animals after 2 wk on the LS diet. These findings suggest that aging attenuates the responsiveness of the adrenocortical AT1R to a sodium load through impaired regulation of AT1bR mRNA, and that this dysregulation contributes to the defects in water and electrolyte homeostasis observed in aging.

Physiological effects and therapeutic potential of proinsulin C-peptide.

Connecting Peptide, or C-peptide, is a product of the insulin prohormone, and is released with and in amounts equimolar to those of insulin. While it was once thought that C-peptide was biologically inert and had little biological significance beyond its role in the proper folding of insulin, it is now known that C-peptide binds specifically to the cell membranes of a variety of tissues and initiates specific intracellular signaling cascades that are pertussis toxin sensitive. Although it is now clear that C-peptide is a biologically active molecule, controversy still remains as to the physiological significance of the peptide. Interestingly, C-peptide appears to reverse the deleterious effects of high glucose in some tissues, including the kidney, the peripheral nerves, and the vasculature. C-peptide is thus a potential therapeutic agent for the treatment of diabetes-associated long-term complications. This review addresses the possible physiologically relevant roles of C-peptide in both normal and disease states and discusses the effects of the peptide on sensory nerve, renal, and vascular function. Furthermore, we highlight the intracellular effects of the peptide and present novel strategies for the determination of the C-peptide receptor(s). Finally, a hypothesis is offered concerning the relationship between C-peptide and the development of microvascular complications of diabetes.

Neuronostatin inhibits glucose-stimulated insulin secretion via direct action on the pancreatic α-cell.

Neuronostatin is a recently described peptide hormone encoded by the somatostatin gene. We previously showed that intraperitoneal injection of neuronostatin into mice resulted in c-Jun accumulation in pancreatic islets in a pattern consistent with the activation of glucagon-producing α-cells. We therefore hypothesized that neuronostatin could influence glucose homeostasis via a direct effect on the α-cell. Neuronostatin enhanced low-glucose-induced glucagon release in isolated rat islets and in the immortalized α-cell line αTC1-9. Furthermore, incubation with neuronostatin led to an increase in transcription of glucagon mRNA, as determined by RT-PCR. Neuronostatin also inhibited glucose-stimulated insulin secretion from isolated islets. However, neuronostatin did not alter insulin release from the ß-cell line INS 832/13, indicating that the effect of neuronostatin on insulin secretion may be secondary to a direct action on the α-cell. In agreement with our in vitro data, intra-arterial infusion of neuronostatin in male rats delayed glucose disposal and inhibited insulin release during a glucose challenge. These studies suggest that neuronostatin participates in maintaining glucose homeostasis through cell-cell interactions between α-cells and ß-cells in the endocrine pancreas, leading to attenuation in insulin secretion.

Neural circuitry underlying the central hypertensive action of nesfatin-1: melanocortins, corticotropin-releasing hormone, and oxytocin.

Nesfatin-1 is produced in the periphery and in the brain where it has been demonstrated to regulate appetite, stress hormone secretion, and cardiovascular function. The anorexigenic action of central nesfatin-1 requires recruitment of neurons producing the melanocortins and centrally projecting oxytocin (OT) and corticotropin-releasing hormone (CRH) neurons. We previously have shown that two components of this pathway, the central melanocortin and oxytocin systems, contribute to the hypertensive action of nesfatin-1 as well. We hypothesized that the cardiovascular effect of nesfatin-1 also was dependent on activation of neurons expressing CRH receptors, and that the order of activation of the melanocortin-CRH-oxytocin circuit was preserved for both the anorexigenic and hypertensive actions of the peptide. Pretreatment of male rats with the CRH-2 receptor antagonist astressin2B abrogated nesfatin-1-induced increases in mean arterial pressure (MAP). Furthermore, the hypertensive action of CRH was blocked by pretreatment with an oxytocin receptor antagonist ornithine vasotocin (OVT), indicating that the hypertensive effect of nesfatin-1 may require activation of oxytocinergic (OTergic) neurons in addition to recruitment of CRH neurons. Interestingly, we found that the hypertensive effect of α-melanocyte stimulating hormone (α-MSH) itself was not blocked by either astressin2B or OVT. These data suggest that while α-MSH-producing neurons are part of a core melanocortin-CRH-oxytocin circuit regulating food intake, and a subpopulation of melanocortin neurons activated by nesfatin-1 do mediate the hypertensive action of the peptide, α-MSH can signal independently from this circuit to increase MAP.