Hepatic extracellular ATP/adenosine dynamics in zebrafish models of alcoholic and metabolic steatotic liver disease.

Steatotic liver disease (SLD) is a burgeoning health problem predominantly associated with excessive alcohol consumption, which causes alcohol-related liver disease (ALD), and high caloric intake, which results in metabolic dysfunction-associated SLD (MASLD). The pathogenesis of ALD and MASLD, which can progress from steatohepatitis to more severe conditions such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, is complicated by several factors. Recently, extracellular ATP and adenosine (Ado), as damage-associated molecular patterns, were reported to promote inflammation and liver fibrosis, contributing to SLD pathogenesis. Here, we explored the in vivo dynamics of hepatic extracellular ATP and Ado during the progression of steatohepatitis using a genetically encoded GPCR-activation-based sensor (GRAB) in zebrafish models. We established hepatocyte-specific GRABATP and GRABAdo in zebrafish and investigated the changes in in vivo hepatic extracellular ATP and Ado levels under ALD or MASLD conditions. Disease-specific changes in hepatocyte extracellular ATP and Ado levels were observed, clearly indicating a correlation between hepatocyte extracellular ATP/Ado dynamics and disease progression. Furthermore, clodronate, a vesicular nucleotide transporter inhibitor, alleviated the MASLD phenotype by reducing the hepatic extracellular ATP and Ado content. These findings provide deep insights into extracellular ATP/Ado dynamics in disease progression, suggesting therapeutic potential for ALD and MASLD.

Behavioral and neurotransmitter changes on antiepileptic drugs treatment in the zebrafish pentylenetetrazol-induced seizure model.

Epilepsy, a recurrent neurological disorder involving abnormal neurotransmitter kinetics in the brain, has emerged as a global health concern. The mechanism of epileptic seizures is thought to involve a relative imbalance between excitatory and inhibitory neurotransmitters. Despite the recent advances in clinical and basic research on the pathogenesis of epilepsy, the complex relationship between the neurotransmitter changes and behavior with and without antiepileptic drugs (AEDs) during seizures remains unclear. To investigate the effects of AEDs such as levetiracetam (LEV), carbamazepine (CBZ), and fenfluramine (FFR) on key neurotransmitters in the pentylenetetrazol (PTZ)-induced seizures in adult zebrafish, we examined the changes in glutamic acid, gamma-aminobutyric acid (GABA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), choline, acetylcholine, norepinephrine, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and adenosine. In this study, we observed that 5-HT and DA levels in the brain increased immediately after PTZ-induced seizures. Behavioral tests clearly showed that all of these AEDs suppressed the PTZ-induced seizures. Upon treatment of PTZ-induced seizures with these AEDs, CBZ decreased the glutamic acid and FFR increased the GABA levels; however, no neurotransmitter changes were observed in the brain after LEV administration. Thus, we demonstrated a series of neurotransmitter changes linked to behavioral changes during PTZ-induced epileptic seizures when LEV, CBZ, or FFR were administered. These findings will lead to a more detailed understanding of the pathogenesis of epilepsy associated with behavioral and neurotransmitter changes under AED treatment.

Chemogenetic Activation of Oxytocin Neurons Improves Pain in a Reserpine-induced Fibromyalgia Rat Model.

Fibromyalgia (FM) is a syndrome characterized by chronic pain with depression as a frequent comorbidity. However, efficient management of the pain and depressive symptoms of FM is lacking. Given that endogenous oxytocin (OXT) contributes to the regulation of pain and depressive disorders, herein, we investigated the role of OXT in an experimental reserpine-induced FM model. In FM model, OXT-monomeric red fluorescent protein 1 (OXT-mRFP1) transgenic rats exhibited increased depressive behavior and sensitivity in a mechanical nociceptive test, suggesting reduced pain tolerance. Additionally, the development of the FM-like phenotype in OXT-mRFP1 FM model rats was accompanied by a significant reduction in OXT mRNA expression in the magnocellular neurons of the paraventricular nucleus. OXT-mRFP1 FM model rats also had significantly fewer tryptophan hydroxylase (TPH)- and tyrosine hydroxylase (TH)-immunoreactive (ir) neurons as well as reduced serotonin and norepinephrine levels in the dorsal raphe and locus coeruleus. To investigate the effects of stimulating the endogenous OXT pathway, rats expressing OXT-human muscarinic acetylcholine receptor (hM3Dq)-mCherry designer receptors exclusively activated by designer drugs (DREADDs) were also assessed in the FM model. Treatment of these rats with clozapine-N-oxide (CNO), an hM3Dq-activating drug, significantly improved characteristic FM model-induced pathophysiological pain, but did not alter depressive-like behavior. The chemogenetically induced effects were reversed by pre-treatment with an OXT receptor antagonist, confirming the specificity of action via the OXT pathway. These results indicate that endogenous OXT may have analgesic effects in FM, and could be a potential target for effective pain management strategies for this disorder.

Regulation of gonadotropin secretion and puberty onset by neuromedin U.

Neuromedin U (NMU), an anorexigenic peptide, was originally isolated from porcine spinal cord in 1985. As NMU is abundant in the anterior pituitary gland, we investigated the effects of NMU on gonadotropin secretion. Both NMU and its receptors, NMUR1 and NMUR2, were expressed in the pituitary gland. NMU suppressed LH and FSH releases from rat anterior pituitary cells. Moreover, NMU-deficient mice exhibit an early onset of vaginal opening. The LHbeta/FSHbeta ratio, which is an index of puberty onset, is high in young NMU-deficient mice. These results indicate that NMU suppresses gonadotropin secretion and regulates the onset of puberty.

Ghrelin directly regulates bone formation.

UNLABELLED: To clarify the role of ghrelin in bone metabolism, we examined the effect of ghrelin in vitro and in vivo. Ghrelin and its receptor, GHS-R1a, were identified in osteoblasts, and ghrelin promoted both proliferation and differentiation. Furthermore, ghrelin increased BMD in rats. Our results show that ghrelin directly affects bone formation. INTRODUCTION: Ghrelin is a gut peptide involved in growth hormone (GH) secretion and energy homeostasis. Recently, it has been reported that the adipocyte-derived hormone leptin, which also regulates energy homeostasis and opposes ghrelin's actions in energy homeostasis, plays a significant role in bone metabolism. This evidence implies that ghrelin may modulate bone metabolism; however, it has not been clarified. To study the role of ghrelin in skeletal integrity, we examined its effects on bone metabolism both in vitro and in vivo. MATERIALS AND METHODS: We measured the expression of ghrelin and growth hormone secretagogue receptor 1a (GHS-R1a) in rat osteoblasts using RT-PCR and immunohistochemistry (IHC). The effect of ghrelin on primary osteoblast-like cell proliferation was examined by recording changes in cell number and the level of DNA synthesis. Osteoblast differentiation markers (Runx2, collagen alpha1 type I [COLI], alkaline phosphatase [ALP], osteocalcin [OCN]) were analyzed using quantitative RT-PCR. We also examined calcium accumulation and ALP activity in osteoblast-like cells induced by ghrelin. Finally, to address the in vivo effects of ghrelin on bone metabolism, we examined the BMD of Sprague-Dawley (SD) rats and genetically GH-deficient, spontaneous dwarf rats (SDR). RESULTS: Ghrelin and GHS-R1a were identified in osteoblast-like cells. Ghrelin significantly increased osteoblast-like cell numbers and DNA synthesis in a dose-dependent manner. The proliferative effects of ghrelin were suppressed by [D-Lys(3)]-GHRP-6, an antagonist of GHS-R1a, in a dose-dependent manner. Furthermore, ghrelin increased the expression of osteoblast differentiation markers, ALP activity, and calcium accumulation in the matrix. Finally, ghrelin definitely increased BMD of both SD rats and SDRs. CONCLUSIONS: These observations show that ghrelin directly stimulates bone formation.

Molecular forms of hypothalamic ghrelin and its regulation by fasting and 2-deoxy-d-glucose administration.

Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a hormone expressed in stomach and other tissues, such as hypothalamus, testis, and placenta. This hormone acts at a central level to stimulate GH secretion and food intake. Little is known, however, about the molecular forms and physiological roles of ghrelin within the hypothalamus. In this report, we detail the molecular forms, mRNA expression patterns, and peptide contents of ghrelin within the rat hypothalamus. Using the combination of reverse-phase HPLC and ghrelin-specific RIA, we determined that the rat hypothalamus contains both n-octanoyl-modified and des-acyl ghrelins. Fasting for 24 and 48 h significantly decreased ghrelin mRNA expression in the hypothalamus to 24% and 28% of control values, respectively. Both n-octanoyl-modified and des-acyl ghrelin content in the hypothalamus decreased after 24 and 48 h of fasting. These results contrast the changes in gastric ghrelin after fasting, which decreased in content despite increased mRNA expression. Two hours after injection of 2-deoxy-d-glucose (2-DG), a selective blocker of carbohydrate metabolism, ghrelin peptide levels also decreased. Thus, induction of glucoprivic states, such as fasting and 2-DG treatment, decreased ghrelin gene expression and peptide content within the hypothalamus.

Isolation and characterization of four VIP-related peptides from red-bellied newt, Cynops pyrrhogaster.

Four novel bioactive peptides were isolated from the red-bellied newt, Cynops pyrrhogaster, using a bioassay system monitoring the rectum contraction of the Japanese quail, Coturnix japonica. As these peptides are structurally related to vasoactive intestinal polypeptide (VIP), we termed these peptides newt VIP-related peptides 1, 2, 3, and 4 (NVRP-1, -2, -3, and -4). The primary sequences of these peptides were determined to be HSDAVFTDNYSRLLGKTALKNYLDGALKKE (NVRP-1), HSDAVFTDNYSRLLAKTALKNYLDGALKKE (NVRP-2), HSDAVFT-DNYSRLLGKIALKNYLDEALKKE (NVRP-3), and HSDAVFTDNYSRLLGKT-ALKNYLDSALKKE (NVRP-4). The N-terminal regions of these NVRPs possessed homology at the amino-acid level to various VIP, while the NVRP C-termini differed from VIPs significantly. All of the VIP consist of 28 amino-acid residues with amidated forms at the C-termini, whereas NVRPs possess 30 amino-acid residues and have free forms at the C-termini. NVRPs exert relaxant activities on isolated quail rectums in a dose-dependent manner, with threshold concentrations between 1 x 10(-8) and 3 x 10(-8) M. NVRPs also exhibited potent relaxant activities acting on the newt duodenum at 3 x 10(-8) M. As yet, this is the first isolation of biologically active VIP-related peptides from urodele.

The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system.

Immunohistochemical analysis revealed the presence of a gut-brain peptide, neuromedin U (NMU), in the suprachiasmatic nucleus (SCN), which is the site of the master circadian oscillator. The expression of NMU mRNA exhibited a circadian rhythm, with the peak expression in the SCN occurring at CT4-8h. The two NMU-binding receptors (NMU-R1 and NMU-R2) were also expressed in the SCN, but their phase angles were different. Intracerebroventricular injection (ICV) of NMU induced the expression of Fos protein in the SCN cells and caused a phase-dependent phase shift of the circadian locomotor activity rhythm. The magnitude of the phase shift was dose dependent. This NMU-induced phase shift was of the nonphotic type. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed increases in the expression in the SCN of immediate early genes, such as c-fos, NGFI-A, NGFI-B, and JunB. Furthermore, ICV injection of NMU increased the expression of Per1, but not Per2, in the SCN. These results indicate that NMU may play some important role in the circadian oscillator by exerting an autocrine or paracrine action in the SCN.

A novel amphibian hypothalamic neuropeptide: isolation, localization, and biological activity.

Neuropeptides similar to the molluscan cardioexcitatory Phe-Met-Arg-Phe-NH2 have been identified in several vertebrates and characterized by the RFa motif at their C terminus (RFa peptides). In this study, we sought to identify an amphibian hypothalamic RFa peptide that may regulate secretion of hormones by the anterior pituitary gland. An acid extract of bullfrog hypothalami was passed through C-18 reversed-phase cartridges, and then the retained material was subjected to HPLC, initially using a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by a dot immunoblot assay employing an antiserum raised against RFa. Immunoreactive fractions were subjected to further cation exchange and reversed-phase HPLC purification. The isolated peptide was a novel RFa peptide and shown to have the sequence Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2. The cell bodies and terminals containing this peptide were localized immunohistochemically in the suprachiasmatic nucleus and median eminence, respectively. This RFa peptide stimulated, in a dose-related way, the release of GH from cultured pituitary cells, its threshold concentration ranging between 10(-9) and 10(-8) M. This peptide did not have any appreciable effect on the secretion of PRL and gonadotropins. It was ascertained that the peptide was also effective in elevating the circulating GH level when administered systemically. Thus, the amphibian hypothalamus was revealed to contain a novel functional RFa peptide that stimulates GH release. This peptide was designated frog GH-releasing peptide.