Glucose-induced CRL4COP1-p53 axis amplifies glycometabolism to drive tumorigenesis.

The diabetes-cancer association remains underexplained. Here, we describe a glucose-signaling axis that reinforces glucose uptake and glycolysis to consolidate the Warburg effect and overcome tumor suppression. Specifically, glucose-dependent CK2 O-GlcNAcylation impedes its phosphorylation of CSN2, a modification required for the deneddylase CSN to sequester Cullin RING ligase 4 (CRL4). Glucose, therefore, elicits CSN-CRL4 dissociation to assemble the CRL4COP1 E3 ligase, which targets p53 to derepress glycolytic enzymes. A genetic or pharmacologic disruption of the O-GlcNAc-CK2-CSN2-CRL4COP1 axis abrogates glucose-induced p53 degradation and cancer cell proliferation. Diet-induced overnutrition upregulates the CRL4COP1-p53 axis to promote PyMT-induced mammary tumorigenesis in wild type but not in mammary-gland-specific p53 knockout mice. These effects of overnutrition are reversed by P28, an investigational peptide inhibitor of COP1-p53 interaction. Thus, glycometabolism self-amplifies via a glucose-induced post-translational modification cascade culminating in CRL4COP1-mediated p53 degradation. Such mutation-independent p53 checkpoint bypass may represent the carcinogenic origin and targetable vulnerability of hyperglycemia-driven cancer.

Secretin-dependent signals in the ventromedial hypothalamus regulate energy metabolism and bone homeostasis in mice.

Secretin, though originally discovered as a gut-derived hormone, is recently found to be abundantly expressed in the ventromedial hypothalamus, from which the central neural system controls satiety, energy metabolism, and bone homeostasis. However, the functional significance of secretin in the ventromedial hypothalamus remains unclear. Here we show that the loss of ventromedial hypothalamus-derived secretin leads to osteopenia in male and female mice, which is primarily induced by diminished cAMP response element-binding protein phosphorylation and upregulation in peripheral sympathetic activity. Moreover, the ventromedial hypothalamus-secretin inhibition also contributes to hyperphagia, dysregulated lipogenesis, and impaired thermogenesis, resulting in obesity in male and female mice. Conversely, overexpression of secretin in the ventromedial hypothalamus promotes bone mass accrual in mice of both sexes. Collectively, our findings identify an unappreciated secretin signaling in the central neural system for the regulation of energy and bone metabolism, which may serve as a new target for the clinical management of obesity and osteoporosis.

Effects of secretin gene knockout on the diversity, composition, and function of gut microbiota in adult male mice.

The gut microbiota plays a vital role in maintaining gastrointestinal homeostasis, however, whether it is influenced by gut hormones remains unknown. Secretin is a well-known gastrointestinal hormone produced by enteroendocrine S cells. This study utilized 16S rRNA amplicon sequencing to characterize the effect of SCT deficiency on the gut microbiota. Our results show that systemic SCT knockout alters the composition and abundance of the mouse gut microbiota but does not affect fecal short-chain fatty acids and lipids concentrations. At the genus level, the abundance of Turicibacter, Bacteroides, Ruminococcu, Romboutsia, Asaccharobacter, and Parasutterella increased in SCT-/- mice, whereas the abundance of Akkermansia and Escherichia decreased. Functional prediction results showed that lack of SCT reduced the abundance of carbohydrate metabolism-related pathways but increased the abundance of linoleic acid metabolism and branched-chain amino acid degradation. Overall, systemic SCT knockout had only minor effects on gut microbiota composition and function in adult male mice fed a standard chow diet.

A gut-brain axis mediates sodium appetite via gastrointestinal peptide regulation on a medulla-hypothalamic circuit.

Salt homeostasis is orchestrated by both neural circuits and peripheral endocrine factors. The colon is one of the primary sites for electrolyte absorption, while its potential role in modulating sodium intake remains unclear. Here, we revealed that a gastrointestinal hormone, secretin, is released from colon endocrine cells under body sodium deficiency and is indispensable for inducing salt appetite. As the neural substrate, circulating secretin activates specific receptors in the nucleus of the solitary tracts, which further activates the downstream paraventricular nucleus of the hypothalamus, resulting in enhanced sodium intake. These results demonstrated a previously unrecognized gut-brain pathway for the timely regulation of sodium homeostasis.

Secretin receptor deletion in the subfornical organ attenuates the activation of excitatory neurons under dehydration.

In mammals, thirst is strongly influenced by the subfornical organ (SFO), a forebrain structure that integrates circulating signals including osmotic pressure and sodium contents. Secretin (SCT), a classical gastrointestinal hormone, has been implicated as a humoral factor regulating body-fluid homeostasis. However, the neural mechanism of secretin in the central nervous system in managing thirst remains unclear. In this study, we report that the local ablation of SCT receptor (SCTR) in the SFO reduces water but not salt intake in dehydrated mice and this effect could not be rescued by exogenous SCT administration. Electrophysiology with single-cell RT-PCR indicates that SCT elicits inward currents in the SFO neuronal nitric oxide synthase (SFOnNOS) neurons via SCTR in the presence of glutamate receptor antagonists. We further show that the SCTR in the SFO permits the activation of SFOnNOS neurons under distinct thirst types. Projection-specific gene deletion of SCTR in SFO to the median preoptic nucleus (MnPO) pathway also reduces water intake in dehydrated animals. SCT signaling thus plays an indispensable role in driving thirst. These data not only expand the functional boundaries of SCTR but also provide insights into the central mechanisms of homeostatic regulation.

Unlocking the Non-IgE-Mediated Pseudo-Allergic Reaction Puzzle with Mas-Related G-Protein Coupled Receptor Member X2 (MRGPRX2).

Mas-related G-protein coupled receptor member X2 (MRGPRX2) is a class A GPCR expressed on mast cells. Mast cells are granulated tissue-resident cells known for host cell response, allergic response, and vascular homeostasis. Immunoglobulin E receptor (FcεRI)-mediated mast cell activation is a well-studied and recognized mechanism of allergy and hypersensitivity reactions. However, non-IgE-mediated mast cell activation is less explored and is not well recognized. After decades of uncertainty, MRGPRX2 was discovered as the receptor responsible for non-IgE-mediated mast cells activation. The puzzle of non-IgE-mediated pseudo-allergic reaction is unlocked by MRGPRX2, evidenced by a plethora of reported endogenous and exogenous MRGPRX2 agonists. MRGPRX2 is exclusively expressed on mast cells and exhibits varying affinity for many molecules such as antimicrobial host defense peptides, neuropeptides, and even US Food and Drug Administration-approved drugs. The discovery of MRGPRX2 has changed our understanding of mast cell biology and filled the missing link of the underlying mechanism of drug-induced MC degranulation and pseudo-allergic reactions. These non-canonical characteristics render MRGPRX2 an intriguing player in allergic diseases. In the present article, we reviewed the emerging role of MRGPRX2 as a non-IgE-mediated mechanism of mast cell activation in pseudo-allergic reactions. We have presented an overview of mast cells, their receptors, structural insight into MRGPRX2, MRGPRX2 agonists and antagonists, the crucial role of MRGPRX2 in pseudo-allergic reactions, current challenges, and the future research direction.

Formation of Kiss1R/GPER Heterocomplexes Negatively Regulates Kiss1R-mediated Signalling through Limiting Receptor Cell Surface Expression.

Kisspeptin receptor (Kiss1R) is an important receptor that plays central regulatory roles in reproduction by regulating hormone release in the hypothalamus. We hypothesize that the formation of heterocomplexes between Kiss1R and other hypothalamus G protein-coupled receptors (GPCRs) affects their cellular signaling. Through screening of potential interactions between Kiss1R and hypothalamus GPCRs, we identified G protein-coupled estrogen receptor (GPER) as one interaction partner of Kiss1R. Based on the recognised function of kisspeptin and estrogen in regulating the reproductive system, we investigated the Kiss1R/GPER heterocomplex in more detail and revealed that complex formation significantly reduced Kiss1R-mediated signaling. GPER did not directly antagonize Kiss1R conformational changes upon ligand binding, but it rather reduced the cell surface expression of Kiss1R. These results therefore demonstrate a regulatory mechanism of hypothalamic hormone receptors via receptor cooperation in the reproductive system and modulation of receptor sensitivity.

IP6-assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion.

COP1 and COP9 signalosome (CSN) are the substrate receptor and deneddylase of CRL4 E3 ligase, respectively. How they functionally interact remains unclear. Here, we uncover COP1-CSN antagonism during glucose-induced insulin secretion. Heterozygous Csn2WT/K70E mice with partially disrupted binding of IP6, a CSN cofactor, display congenital hyperinsulinism and insulin resistance. This is due to increased Cul4 neddylation, CRL4COP1 E3 assembly, and ubiquitylation of ETV5, an obesity-associated transcriptional suppressor of insulin secretion. Hyperglycemia reciprocally regulates CRL4-CSN versus CRL4COP1 assembly to promote ETV5 degradation. Excessive ETV5 degradation is a hallmark of Csn2WT/K70E, high-fat diet-treated, and ob/ob mice. The CRL neddylation inhibitor Pevonedistat/MLN4924 stabilizes ETV5 and remediates the hyperinsulinemia and obesity/diabetes phenotypes of these mice. These observations were extended to human islets and EndoC-ßH1 cells. Thus, a CRL4COP1-ETV5 proteolytic checkpoint licensing GSIS is safeguarded by IP6-assisted CSN-COP1 competition. Deregulation of the IP6-CSN-CRL4COP1-ETV5 axis underlies hyperinsulinemia and can be intervened to reduce obesity and diabetic risk.

Molecular evolution of CRH and CRHR subfamily before the evolutionary origin of vertebrate.

Corticotropin-releasing hormone (CRH) is well-cited for its important role in governing the stress responses via neuroendocrine system in vertebrates. After the identification of homologs of CRH receptor (CRHR) in both deuterostome and arthropod lineages, it was suggested that the ancestral homolog of CRH-CRHR molecular system is present in the bilaterian. However, homolog sequences from arthropods differ considerably from vertebrate CRH-like peptide sequences. Due to the significant difference between the biological system, as well as the gene regulatory network, of protostome and that of vertebrate, physiological studies on the protostomes may not provide important insight into the evolutionary history of vertebrate CRH system, while tunicate and amphioxus, two close relatives to vertebrate, which have diverged before two rounds of whole genome duplication (2WGDs) do. Given the identification of amphioxus CRH-like peptide by our group, this review aims to reexamine the current hypotheses on the evolution of CRH subfamily. It is generally accepted that paralogs of CRH and CRHR have been produced through 2WGDs, which occurred during the early vertebrate evolution. The identification of a single crh-like gene in amphioxi and tunicates by in silico search and the presence of two paralogons with a total of 5 crh-like genes in gnathostomes has shown that an additional duplication event might have happened to the ancestral crh-like gene before 2WGDs. On the other hand, the evolution of crhr gene subfamily appears to be mainly influenced by 2WGDs and only two receptor genes have been retained in the genomes of jawed vertebrates.

Corrigendum: Central Control of Feeding Behavior by the Secretin, PACAP, and Glucagon Family of Peptides.

[This corrects the article DOI: 10.3389/fendo.2017.00018.].

Central Control of Feeding Behavior by the Secretin, PACAP, and Glucagon Family of Peptides.

Constituting a group of structurally related brain-gut peptides, secretin (SCT), pituitary adenylate cyclase-activating peptide (PACAP), and glucagon (GCG) family of peptide hormones exert their functions via interactions with the class B1 G protein-coupled receptors. In recent years, the roles of these peptides in neuroendocrine control of feeding behavior have been a specific area of research focus for development of potential therapeutic drug targets to combat obesity and metabolic disorders. As a result, some members in the family and their analogs have already been utilized as therapeutic agents in clinical application. This review aims to provide an overview of the current understanding on the important role of SCT, PACAP, and GCG family of peptides in central control of feeding behavior.

Minimally Invasive Transverse Aortic Constriction in Mice.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

The Role of Endocrine G Protein-Coupled Receptors in Ovarian Cancer Progression.

Ovarian cancer is the seventh most common cancer in women and the most lethal gynecological cancer, causing over 151,000 deaths worldwide each year. Dysregulated production of endocrine hormones, known to have pluripotent effects on cell function through the activation of receptor signaling pathways, is believed to be a high-risk factor for ovarian cancer. An increasing body of evidence suggests that endocrine G protein-coupled receptors (GPCRs) are involved in the progression and metastasis of ovarian neoplasms. GPCRs are attractive drug targets because their activities are regulated by more than 25% of all drugs approved by the Food and Drug Administration. Therefore, understanding the role of endocrine GPCRs during ovarian cancer progression and metastasis will allow for the development of novel strategies to design effective chemotherapeutic drugs against malignant ovarian tumors. In this review, we address the signaling pathways and functional roles of several key endocrine GPCRs that are related to the cause, progression, and metastasis of ovarian cancer.

Excitatory effect of histamine on neuronal activity of rat globus pallidus by activation of H2 receptors in vitro.

Previous studies have revealed distribution of histaminergic fibers and presence of histamine receptors in globus pallidus (GP). In this study, the brain slice preparation of adult rats was used to examine the effect of histamine on the spontaneous unitary discharge of GP neurons and the underlying receptor mechanism. Ninety-five GP neurons were extracellularly recorded from 42 slices containing the GP, of which 87 (91.6%) were excited by the stimulation of histamine. The histamine-induced excitation was concentration-dependent and persisted in low Ca2+/high Mg2+ medium (n = 9), demonstrating that the action of histamine on the GP neurons was postsynaptic. The excitatory effect of histamine on the GP neurons was not blocked by selective histamine H1 receptor antagonist triprolidine (n = 16) or chlorpheniramine (n = 6), but was effectively suppressed by ranitidine, a highly selective histamine H2 receptor antagonist (n = 21). On the other hand, highly selective histamine H2 receptor agonist dimaprit mimicked the excitatory effect of histamine on the GP neurons (n = 23), while histamine H1 receptor agonists, including 2-pyridylethylamine (n = 22), 2-thiazolyethylamine (n = 9) and betahistine (n = 9), did not cause GP neurons any response. The dimaprit-induced GP neuronal excitation was effectively antagonized by selective histamine H2 receptor antagonist ranitidine (n = 14) but not influenced by selective histamine H1 receptor antagonist triprolidine (n = 12). Moreover, adenylate cyclase (AC) activator forskolin (n = 7) was observed to evoke GP neurons an excitatory response, whereas the histamine-induced excitation was effectively reduced by H-89 (n = 9), a selective and potent inhibitor of protein kinase A (PK(A)). Finally, it was noted that neurons of both subdivisions of the GP, the internal (GPi, n = 35) and external (GPe, n = 60) segment, showed no differences in their responses to stimulations of the tested histaminergic reagents. These results demonstrated that histamine excited GP (including GPi and GPe) neurons via histamine H2 receptors and H2 receptors linked intracellular G-protein-AC-PK(A) signaling pathway, suggesting that the hypothalamic histaminergic afferent fibers innervating GP may play an important modulatory role in motor control through its excitatory effect on GP neurons.

CpG methylation and transcription factors Sp1 and Sp3 regulate the expression of the human secretin receptor gene.

The human secretin receptor (hSR) is an important glycoprotein receptor for regulating the secretion of pancreatic bicarbonate, water, and electrolytes. In this study we investigated the transcriptional regulation of the hSR gene. A minimal 106-bp promoter was identified, and it contains two GC boxes (GC box-A, -240 to -226; and GC box-B, -203 to -194, from the translation start site). EMSA and supershift analyses showed that both GC boxes interact with Sp1 and Sp3 transcription factors. Transient transfection in pancreas-derived human pancreatic ductule carcinoma (PANC)-1 and bovine pancreatic duct-1 cells showed that mutation of either GC box-A or -B reduced the promoter strength by 56-67%, whereas mutation of both GC boxes caused more than 90% reduction of promoter activity. Cotransfections of the hSR promoter with Sp1 and Sp3 expression vectors in Sp-deficient Drosophila SL-2 Schneider cells further demonstrated that the ratio of Sp1 to Sp3 is the key mechanism to modulate hSR gene expression. The methylation statuses of 27 CpG sites within the promoter region (-400 to -151 bp) were assessed in various human pancreas and liver cell lines. The hSR promoter is unmethylated (CAPAN-1, human pancreatic adenocarcinoma) or partially methylated (PANC-1 and HPAC, human pancreatic adenocarcinoma) in hSR-expressing cell lines but is completely methylated in hSR nonexpressing HepG2 cells. Methyltransferase inhibitor 5-aza-2'deoxycytidine increased hSR gene expression level in PANC-1 cells and induced hSR gene expression in HepG2 cells. Together, our study shows that, in addition to Sp1 and Sp3, promoter methylation also plays a role in the regulation of hSR gene expression.

Regulation of the human secretin gene is controlled by the combined effects of CpG methylation, Sp1/Sp3 ratio, and the E-box element.

To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (-11 to -341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (-336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5'-Aza-2' deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

Identification of a potential receptor for both peptide histidine isoleucine and peptide histidine valine.

Peptide histidine isoleucine (PHI), peptide histidine valine (PHV), and vasoactive intestinal polypeptide (VIP) are cosynthesized from the same precursor and share high levels of structural similarities with overlapping biological functions. In this study, the first PHI/PHV receptor was isolated and characterized in goldfish. To study this receptor using homologous peptides, we have also characterized the goldfish prepro-PHI/VIP, and, surprisingly, a shorter transcript lacking the VIP coding region was isolated. A PHI/VIP precursor without the VIP coding sequence has never before been reported. Initial functional expression of the PHI/PHV receptor in Chinese hamster ovary cells revealed that it could be activated by human PHV [50% effective concentration (EC(50)): 43 nM] and to a lesser extent human PHI (EC(50): 133 nM) and helodermin (EC(50): 166 nM) but not fish and mammalian pituitary adenylate cyclase-activating polypeptides and VIPs. Subsequent studies indicated that, similar to the pituitary adenylate cyclase-activating polypeptide receptors (PAC1-R, VPAC1-R, and VPAC2-R), the receptor isolated in this study is able to interact with goldfish PHI and its C-terminally extended form, PHV with EC(50) values 93 and 43 nM, respectively. Northern blot and RT-PCR/Southern blot analyses revealed that the PHI/VIP gene is expressed in the intestine, brain, and gall bladder and the PHI/PHV receptor gene is primarily expressed in the pituitary and to a lesser extend in the intestine and gall bladder, suggesting that PHI/PHV may play a role, notably in the regulation of pituitary function. In conclusion, our results demonstrate for the first time the existence of a PHI/PHV receptor, indicating that the functions of PHI and PHV could be mediated by their own receptor in addition to VIP receptors.

The knockout of secretin in cerebellar Purkinje cells impairs mouse motor coordination and motor learning.

Secretin (SCT) was first considered to be a gut hormone regulating gastrointestinal functions when discovered. Recently, however, central actions of SCT have drawn intense research interest and are supported by the broad distribution of SCT in specific neuronal populations and by in vivo physiological studies regarding its role in water homeostasis and food intake. The direct action of SCT on a central neuron was first discovered in cerebellar Purkinje cells in which SCT from cerebellar Purkinje cells was found to potentiate GABAergic inhibitory transmission from presynaptic basket cells. Because Purkinje neurons have a major role in motor coordination and learning functions, we hypothesize a behavioral modulatory function for SCT. In this study, we successfully generated a mouse model in which the SCT gene was deleted specifically in Purkinje cells. This mouse line was tested together with SCT knockout and SCT receptor knockout mice in a full battery of behavioral tasks. We found that the knockout of SCT in Purkinje neurons did not affect general motor ability or the anxiety level in open field tests. However, knockout mice did exhibit impairments in neuromuscular strength, motor coordination, and motor learning abilities, as shown by wire hanging, vertical climbing, and rotarod tests. In addition, SCT knockout in Purkinje cells possibly led to the delayed development of motor neurons, as supported by the later occurrence of key neural reflexes. In summary, our data suggest a role in motor coordination and motor learning for SCT expressed in cerebellar Purkinje cells.

Central and peripheral administration of secretin inhibits food intake in mice through the activation of the melanocortin system.

Secretin (Sct) is released into the circulation postprandially from the duodenal S-cells. The major functions of Sct originated from the gastrointestinal system are to delay gastric emptying, stimulate fluid secretion from pancreas and liver, and hence optimize the digestion process. In recent years, Sct and its receptor (Sctr) have been identified in discrete nuclei of the hypothalamus, including the paraventricular nucleus (PVN) and the arcuate nucleus (Arc). These nuclei are the primary brain sites that are engaged in regulating body energy homeostasis, thus providing anatomical evidence to support a functional role of Sct in appetite control. In this study, the effect of Sct on feeding behavior was investigated using wild-type (wt), Sct(-/-), and secretin receptor-deficient (Sctr(-/-)) mice. We found that both central and peripheral administration of Sct could induce Fos expression in the PVN and Arc, suggesting the activation of hypothalamic feeding centers by this peptide. Consistent with this notion, Sct was found to increase thyrotropin-releasing hormone and melanocortin-4 receptor (Mc4r) transcripts in the PVN, and augment proopiomelanocortin, but reduces agouti-related protein mRNA expression in the Arc. Injection of Sct was able to suppress food intake in wt mice, but not in Sctr(-/-) mice, and that this effect was abolished upon pretreatment with SHU9119, an antagonist for Mc4r. In summary, our data suggest for the first time that Sct is an anorectic peptide, and that this function is mediated by the melanocortin system.