Glyphosate-induced changes in the expression of galanin and GALR1, GALR2 and GALR3 receptors in the porcine small intestine wall.

Glyphosate is the active ingredient of glyphosate-based herbicides and the most commonly used pesticide in the world. The goal of the present study was to verify whether low doses of glyphosate (equivalent to the environmental exposure) evoke changes in galanin expression in intramural neurons in the small intestine in pigs and to quantitatively determine changes in the level of galanin receptor encoding mRNA (GALR1, GALR2, GALR3) in the small intestine wall. The experiment was conducted on 15 sexually immature gilts divided into three study groups: control (C)-animals receiving empty gelatin capsules; experimental 1 (G1)-animals receiving a low dose of glyphosate (0.05 mg/kg b.w./day); experimental 2 (G2)-animals receiving a higher dose of glyphosate (0.5 mg/kg b.w./day) orally in gelatine capsules for 28 days. Glyphosate ingestion led to an increase in the number of GAL-like immunoreactive intramural neurons in the porcine small intestine. The results of RT-PCR showed a significant increase in the expression of mRNA, which encodes the GAL-receptors in the ileum, a decreased expression in the duodenum and no significant changes in the jejunum. Additionally, intoxication with glyphosate increased the expression of SOD2-encoding mRNA in the duodenum and decreased it in the jejunum and ileum, but it did not affect SOD1 expression. The results suggest that it may be a consequence of the cytotoxic and/or neurotoxic properties of glyphosate and/or its ability to induce oxidative stress.

Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling.

Knowledge gaps that limit the development of therapies for polycystic ovary syndrome (PCOS) concern various environmental factors that impact clinical characteristics. Circadian dysrhythmia contributes to glycometabolic and reproductive hallmarks of PCOS. Here, we illustrated the amelioration of Limosilactobacillus reuteri (L. reuteri) on biorhythm disorder-ignited dyslipidemia of PCOS via a microbiota-metabolite-liver axis. A rat model of long-term (8 weeks) darkness treatment was used to mimic circadian dysrhythmia-induced PCOS. Hepatic transcriptomics certified by in vitro experiments demonstrated that increased hepatic galanin receptor 1 (GALR1) due to darkness exposure functioned as a critical upstream factor in the phosphoinositide 3-kinase (PI3K)/protein kinase B pathway to suppress nuclear receptors subfamily 1, group D, member 1 (NR1D1) and promoted sterol regulatory element binding protein 1 (SREBP1), inducing lipid accumulation in the liver. Further investigations figured out a restructured microbiome-metabolome network following L. reuteri administration to protect darkness rats against dyslipidemia. Notably, L. reuteri intervention resulted in the decrease of Clostridium sensu stricto 1 and Ruminococcaceae UCG-010 as well as gut microbiota-derived metabolite capric acid, which could further inhibit GALR1-NR1D1-SREBP1 pathway in the liver. In addition, GALR antagonist M40 reproduced similar ameliorative effects as L. reuteri to protect against dyslipidemia. While exogenous treatment of capric acid restrained the protective effects of L. reuteri in circadian disruption-induced PCOS through inhibiting GALR1-dependent hepatic lipid metabolism. These findings purport that L. reuteri could serve for circadian disruption-associated dyslipidemia. Manipulation of L. reuteri-capric acid-GALR1 axis paves way for clinical therapeutic strategies to prevent biorhythm disorder-ignited dyslipidemia in PCOS women.

Galanin Receptors (GALR1, GALR2, and GALR3) Immunoexpression in Enteric Plexuses of Colorectal Cancer Patients: Correlation with the Clinico-Pathological Parameters.

Galanin (GAL) is an important neurotransmitter released by the enteric nervous system (ENS) neurons located in the muscularis externa and submucosa enteric plexuses that acts by binding to GAL receptors 1, 2 and 3 (GALR1, 2 and 3). In our previous studies, the GAL immunoexpression was compared in colorectal cancer (CRC) tissue and the adjacent parts of the large intestine wall including myenteric and submucosal plexuses. Recently we have also found that expression levels of GALR1 and GALR3 proteins are elevated in CRC tissue as compared with their expression in epithelial cells of unchanged mucosa. Moreover, higher GALR3 immunoreactivity in CRC cells correlated with better prognosis of CRC patients. To understand the distribution of GALRs in enteric plexuses distal and close to CRC invasion, in the present study we decided to evaluate GALRs expression within the myenteric and submucosal plexuses located proximally and distally to the cancer invasion and correlated the GALRs expression levels with the clinico-pathological data of CRC patients. The immunohistochemical and immunofluorescent methods showed only slightly decreased immunoexpression of GALR1 and GALR3 in myenteric plexuses close to cancer but did not reveal any correlation in the immunoexpression of all three GAL receptors in myenteric plexuses and tumour progression. No significant changes were found between the expression levels of GALRs in submucosal plexuses distal and close to the tumour. However, elevated GALR1 expression in submucosal plexuses in vicinity of CRC correlated with poor prognosis, higher tumour grading and shorter overall survival. When myenteric plexuses undergo morphological and functional alterations characteristic for atrophy, GALRs maintain or only slightly decrease their expression status. In contrast, the correlation between high expression of GALR1 in the submucosal plexuses and overall survival of CRC patients suggest that GAL and GALRs can act as a components of local neuro-paracrine pro-proliferative pathways accelerating the invasion and metastasis of cancer cell. The obtained results suggest an important role of GALR1 in submucosal plexuses function during the progression of CRC and imply that GALR1 expression in submucosal plexuses of ENS could be an important predictive factor for CRC progression.

Promoter hypermethylation of GALR1 acts as an early epigenetic susceptibility event in colorectal carcinogenesis.

Epigenetics play an essential role in colorectal neoplasia process. There is a need to determine the appropriateness of epigenetic biomarkers for early detection as well as expand our understanding of the carcinogenic process. Therefore, the aim of the study was to assess how DNA methylation pattern of GALR1 gene evolves in a sample set representing colorectal neoplastic progression. The study was designed into three phases. Firstly, Methylation status of GALR1 was assessed with genome-wide DNA methylation beadchip and pyrosequencing assays in colorectal lesions and paired normal tissues. Then, linear mixed-effects modeling analyses were applied to describe the trend of DNA methylation during the progression of colorectal neoplasia. In the third phase, quantitative RT-PCR was used to examine GALR1 expression in patients with precursor lesion and colorectal cancer. We found that significant hypermethylation of GALR1 promoter was a widely existent modification in CRCs (P < 0.001). When further examined methylation pattern of GALR1 during neoplastic progression of CRC, we found that DNA methylation level of GALR1 showed a significant stepwise increase from normal to hyperplastic polyps, to adenomas and to carcinoma samples (P < 0.001). Besides, loss of mRNA expression is a common accompaniment to adenomas and carcinomas. Public omics data analyses showed an inverse correlation between gene expression and DNA methylation (P < 0.001). Our findings indicate that epigenetic alteration of GALR1 promoter is gradually accumulated during the colorectal neoplastic progression. It can potentially be a promising biomarker used for screening and surveillance of colorectal cancer.

Galanin Receptors (GalR1, GalR2, and GalR3) Expression in Colorectal Cancer Tissue and Correlations to the Overall Survival and Poor Prognosis of CRC Patients.

Colorectal cancer (CRC) is the second most common cause of cancer in women and the third in men. The postoperative pathomorphological evaluation of patients with CRC is extremely important for future therapeutic decisions. Although our previous studies demonstrated high galanin (GAL) presence within tumor tissue and an elevated concentration of GAL in the serum of CRC patients, to date, there is a lack of data regarding GAL receptor (GalR) protein expression in CRC cells. Therefore, the aim of this study was to evaluate the presence of all three types of GalRs (GalR1, GalR2 and GalR3) within epithelial cells of the human colon and CRC tissue with the use of the immunohistochemical method and to correlate the results with the clinical-pathological data. We found stronger immunoreactivity of GalR1 and GalR3 in CRC cells compared to epithelial cells of the unchanged mucosa of the large intestine. No differences in the GalR2 protein immunoreactivity between the studied tissues were noted. We also found that the increased immunoexpression of the GalR3 in CRC tissue correlated with the better prognosis and longer survival (p < 0.0079) of CRC patients (n = 55). The obtained results suggest that GalR3 may play the role of a prognostic factor for CRC patients. Based on data from the TCGA-COAD project deposited in the GDC Data Portal, we also found that GalR mRNA in cancer samples and the adjacent normal tissue did not correlate with immunoexpression of the GalR proteins in CRC cells and epithelial cells of the unchanged mucosa.

Time-restricted feeding prevents metabolic diseases through the regulation of galanin/GALR1 expression in the hypothalamus of mice.

PURPOSE: Time-restricted feeding (TRF) reverses obesity and insulin resistance, yet the central mechanisms underlying its beneficial effects are not fully understood. Recent studies suggest a critical role of hypothalamic galanin and its receptors in the regulation of energy balance. It is yet unclear whether TRF could regulate the expression of galanin and its receptors in the hypothalamus of mice fed a high-fat diet. METHODS: To test this effect, we subjected mice to either ad lib or TRF of a high-fat diet for 8 h per day. After 4 weeks, galanin and many neuropeptides associated with the function of metabolism were examined. RESULTS: The present findings showed that mice under TRF consume equivalent calories from a high-fat diet as those with ad lib access, yet are protected against obesity and have improved glucose metabolism. Plasma galanin, orexin A, irisin and adropin levels were significantly reversed by TRF regimen. Besides, TRF regimen reversed the progression of metabolic disorders in mice by increasing GLUT4 and PGC-1α expression in skeletal muscles. Moreover, the levels of galanin and GALR1 expression were severely diminished in the hypothalamus of the TRF mice, whereas GALR2 was highly expressed. CONCLUSIONS: TRF diminished galanin and GALR1 expression, and increased GALR2 expression in the hypothalamus of mice fed a high-fat diet. The current studies provide additional evidence that TRF is effective in improving HFD-induced hyperglycemia and insulin resistance in mice, and this effect could be associated with TRF-induced changes of the galanin systems in the hypothalamus. LEVEL OF EVIDENCE: No level of evidence, animal studies.

Serotonin Heteroreceptor Complexes and Their Integration of Signals in Neurons and Astroglia-Relevance for Mental Diseases.

The heteroreceptor complexes present a novel biological principle for signal integration. These complexes and their allosteric receptor-receptor interactions are bidirectional and novel targets for treatment of CNS diseases including mental diseases. The existence of D2R-5-HT2AR heterocomplexes can help explain the anti-schizophrenic effects of atypical antipsychotic drugs not only based on blockade of 5-HT2AR and of D2R in higher doses but also based on blocking the allosteric enhancement of D2R protomer signaling by 5-HT2AR protomer activation. This research opens a new understanding of the integration of DA and 5-HT signals released from DA and 5-HT nerve terminal networks. The biological principle of forming 5-HT and other heteroreceptor complexes in the brain also help understand the mechanism of action for especially the 5-HT hallucinogens, including putative positive effects of e.g., psilocybin and the indicated prosocial and anti-stress actions of MDMA (ecstasy). The GalR1-GalR2 heterodimer and the putative GalR1-GalR2-5-HT1 heteroreceptor complexes are targets for Galanin N-terminal fragment Gal (1-15), a major modulator of emotional networks in models of mental disease. GPCR-receptor tyrosine kinase (RTK) heteroreceptor complexes can operate through transactivation of FGFR1 via allosteric mechanisms and indirect interactions over GPCR intracellular pathways involving protein kinase Src which produces tyrosine phosphorylation of the RTK. The exciting discovery was made that several antidepressant drugs such as TCAs and SSRIs as well as the fast-acting antidepressant drug ketamine can directly bind to the TrkB receptor and provide a novel mechanism for their antidepressant actions. Understanding the role of astrocytes and their allosteric receptor-receptor interactions in modulating forebrain glutamate synapses with impact on dorsal raphe-forebrain serotonin neurons is also of high relevance for research on major depressive disorder.

Regulatory Influence of Galanin and GALR1/GALR2 Receptors on Inflamed Uterus Contractility in Pigs.

Uterine inflammation is a very common and serious pathology in domestic animals, the development and progression of which often result from disturbed myometrial contractility. We investigated the effect of inflammation on the protein expression of galanin (GAL) receptor subtypes (GALR)1 and GALR2 in myometrium and their role in the contractile amplitude and frequency of an inflamed gilt uterus. The gilts of the E. coli and SAL groups received E. coli suspension or saline in their uteri, respectively, and only laparotomy was performed (CON group). Eight days later, the E. coli group developed severe acute endometritis and lowered GALR1 protein expression in the myometrium. Compared to the pretreatment period, GAL (10-7 M) reduced the amplitude and frequency in myometrium and endometrium/myometrium of the CON and SAL groups, the amplitude in both stripes and frequency in endometrium/myometrium of the E. coli group. In this group, myometrial frequency after using GAL increased, and it was higher than in other groups. GALR2 antagonist diminished the decrease in amplitude in myometrium and the frequency in endometrium/myometrium (SAL, E. coli groups) induced by GAL (10-7 M). GALR1/GALR2 antagonist and GAL (10-7 M) reversed the decrease in amplitude and diminished the decrease in frequency in both examined stripes (CON, SAL groups), and diminished the drop in amplitude and abolished the rise in the frequency in the myometrium (E. coli group). In summary, the inflammation reduced GALR1 protein expression in pig myometrium, and GALR1 and GALR2 participated in the contractile regulation of an inflamed uterus.

Activation of Galanin Receptor 1 with M617 Attenuates Neuronal Apoptosis via ERK/GSK-3ß/TIP60 Pathway After Subarachnoid Hemorrhage in Rats.

Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease. Neuronal apoptosis plays an important pathological role in early brain injury after SAH. Galanin receptor 1 (GalR1) activation was recently shown to be anti-apoptotic in the setting of ischemic stroke. This study aimed to explore the anti-neuronal apoptosis effect of GalR1 activation after SAH, as well as the underlying mechanisms. GalR1 CRISPR and GalR1 selective agonist, M617, was administered, respectively. Extracellular-signal-regulated kinase (ERK) inhibitor (U0126) and glycogen synthase kinase 3-beta (GSK3-ß) CRISPR were administered to investigate the involvement of the ERK/GSK3-ß pathway in GalR1-mediated neuroprotection after SAH. Outcome assessments included neurobehavioral tests, western blot, and immunohistochemistry. The results showed that endogenous ligand galanin (Gal) and GalR1 were markedly increased in the ipsilateral brain hemisphere at 12 h and 24 h after SAH. GalR1 were expressed mainly in neurons, but expression was also observed in some astrocytes and microglia. GalR1 CRISPR knockdown exacerbated neurological deficits and neuronal apoptosis 24 h after SAH. Moreover, activation of GalR1 with M617 significantly improved short- and long-term neurological deficits but decreased neuronal apoptosis after SAH. Furthermore, GalR1 activation dysregulated the protein levels of phosphorylated ERK and GSK-3ß, but downregulated the phosphorylated Tat-interactive protein 60 (TIP60) and cleaved caspase-3 at 24 h after SAH. GalR1 CRISPR, U0126, and GSK-3ß CRISPR abolished the beneficial effects of GalR1 activation at 24 h after SAH in rats. Collectively, the present study demonstrated that activation of GalR1 using M617 attenuated neuronal apoptosis through the ERK/GSK-3ß/TIP60 pathway after SAH in rats. GalR1 may serve as a promising therapeutic target for SAH patients.

Involvement of Scratch2 in GalR1-mediated depression-like behaviors in the rat ventral periaqueductal gray.

Galanin receptor1 (GalR1) transcript levels are elevated in the rat ventral periaqueductal gray (vPAG) after chronic mild stress (CMS) and are related to depression-like behavior. To explore the mechanisms underlying the elevated GalR1 expression, we carried out molecular biological experiments in vitro and in animal behavioral experiments in vivo. It was found that a restricted upstream region of the GalR1 gene, from -250 to -220, harbors an E-box and plays a negative role in the GalR1 promoter activity. The transcription factor Scratch2 bound to the E-box to down-regulate GalR1 promoter activity and lower expression levels of the GalR1 gene. The expression of Scratch2 was significantly decreased in the vPAG of CMS rats. Importantly, local knockdown of Scratch2 in the vPAG caused elevated expression of GalR1 in the same region, as well as depression-like behaviors. RNAscope analysis revealed that GalR1 mRNA is expressed together with Scratch2 in both GABA and glutamate neurons. Taking these data together, our study further supports the involvement of GalR1 in mood control and suggests a role for Scratch2 as a regulator of depression-like behavior by repressing the GalR1 gene in the vPAG.

Spexin Promotes the Proliferation and Differentiation of C2C12 Cells In Vitro-The Effect of Exercise on SPX and SPX Receptor Expression in Skeletal Muscle In Vivo.

SPX (spexin) and its receptors GalR2 and GalR3 (galanin receptor subtype 2 and galanin receptor subtype 3) play an important role in the regulation of lipid and carbohydrate metabolism in human and animal fat tissue. However, little is still known about the role of this peptide in the metabolism of muscle. The aim of this study was to determine the impact of SPX on the metabolism, proliferation and differentiation of the skeletal muscle cell line C2C12. Moreover, we determined the effect of exercise on the SPX transduction pathway in mice skeletal muscle. We found that increased SPX, acting via GalR2 and GalR3 receptors, and ERK1/2 phosphorylation stimulated the proliferation of C2C12 cells (p < 0.01). We also noted that SPX stimulated the differentiation of C2C12 by increasing mRNA and protein levels of differentiation markers Myh, myogenin and MyoD (p < 0.01). SPX consequently promoted myoblast fusion into the myotubule (p < 0.01). Moreover, we found that, in the first stage (after 2 days) of myocyte differentiation, GalR2 and GalR3 were involved, whereas in the last stage (day six), the effect of SPX was mediated by the GalR3 isoform. We also noted that exercise stimulated SPX and GalR2 expression in mice skeletal muscle as well as an increase in SPX concentration in blood serum. These new insights may contribute to a better understanding of the role of SPX in the metabolism of skeletal muscle.

A Three Protein-Coding Gene Prognostic Model Predicts Overall Survival in Bladder Cancer Patients.

Bladder cancer (BLCA) is the most common urinary tract tumor and is the 11th most malignant cancer worldwide. With the development of in-depth multisystem sequencing, an increasing number of prognostic molecular markers have been identified. In this study, we focused on the role of protein-coding gene methylation in the prognosis of BLCA. We downloaded BLCA clinical and methylation data from The Cancer Genome Atlas (TCGA) database and used this information to identify differentially methylated genes and construct a survival model using lasso regression. We assessed 365 cases, with complete information regarding survival status, survival time longer than 30 days, age, gender, and tumor characteristics (grade, stage, T, M, N), in our study. We identified 353 differentially methylated genes, including 50 hypomethylated genes and 303 hypermethylated genes. After annotation, a total of 227 genes were differentially expressed. Of these, 165 were protein-coding genes. Three genes (zinc finger protein 382 (ZNF382), galanin receptor 1 (GALR1), and structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1)) were selected for the final risk model. Patients with higher-risk scores represent poorer survival than patients with lower-risk scores in the training set (HR = 2.37, 95% CI 1.43-3.94, p = 0.001), in the testing group (HR = 1.85, 95% CI 1.16-2.94, p = 0.01), and in the total cohort (HR = 2.06, 95% CI 1.46-2.90, p < 0.001). Further univariate and multivariate analyses using the Cox regression method were conducted in these three groups, respectively. All the results indicated that risk score was an independent risk factor for BLCA. Our study screened the different methylation protein-coding genes in the BLCA tissues and constructed a robust risk model for predicting the outcome of BLCA patients. Moreover, these three genes may function in the mechanism of development and progression of BLCA, which should be fully clarified in the future.

Beneficial effects of galanin system on diabetic peripheral neuropathic pain and its complications.

Diabetic peripheral neuropathic pain (DPNP) is a distal spontaneous pain, caused by lesion of sensory neurons and accompanied by depression and anxiety frequently, which reduce life quality of patients and increase society expenditure. To date, antidepressants, serotonin-noradrenaline reuptake inhibitors and anticonvulsants are addressed as first-line therapy to DPNP, alone or jointly. It is urgently necessary to develop novel agents to treat DPNP and its complications. Evidences indicate that neuropeptide galanin can regulate multiple physiologic and pathophysiological processes. Pain, depression and anxiety may upregulate galanin expression. In return, galanin can modulate depression, anxiety, pain threshold and pain behaviors. This article provides a new insight into regulative effects of galanin and its subtype receptors on antidepressant, antianxiety and against DPNP. Through activating GALR1, galanin reinforces depression-like and anxiogenic-like behaviors, but exerts antinociceptive roles. While via activating GALR2, galanin is referred to as anti-depressive and anti-anxiotropic compounds, and at low and high concentration facilitates and inhibits nociceptor activity, respectively. The mechanism of the galanin roles is relative to increase in K+ currents and decrease in Ca2+ currents, as well as neurotrophic and neuroprotective roles. These data are helpful to develop novel drugs to treat DPNP and its complications.

Further evidence for the association of GAL, GALR1 and NPY1R variants with opioid dependence.

Aim: Heroin addiction is a chronic, relapsing disease that has genetic and environmental, including drug-induced, contributions. Stress influences the development of addictions. This study was conducted to determine if variants in stress-related genes are associated with opioid dependence (OD). Patients & methods: One hundred and twenty variants in 26 genes were analyzed in 597 Dutch subjects. Patients included 281 OD in methadone maintenance with or without heroin-assisted treatment and 316 controls. Results: Twelve SNPs in seven genes showed a nominally significant association with OD. Experiment-wise significant associations (p < 0.05) were found for three SNP pairs, through an interaction effect: NPY1R/GAL rs4691910/rs1893679, NPY1R/GAL rs4691910/rs3136541 and GALR1/GAL rs9807208/rs3136541. Conclusion: This study lends more evidence to previous reports of association of stress-related variants with heroin dependence.

Galanin System in Human Glioma and Pituitary Adenoma.

Expression of neuropeptides and their corresponding receptors has been demonstrated in different cancer types, where they can play a role in tumor cell growth, invasion, and migration. Human galanin (GAL) is a 30-amino-acid regulatory neuropeptide which acts through three G protein-coupled receptors, GAL1-R, GAL2-R, and GAL3-R that differ in their signal transduction pathways. GAL and galanin receptors (GALRs) are expressed by different tumors, and direct involvement of GAL in tumorigenesis has been shown. Despite its strong expression in the central nervous system (CNS), the role of GAL in CNS tumors has not been extensively studied. To date, GAL peptide expression, GAL receptor binding and mRNA expression have been reported in glioma, meningioma, and pituitary adenoma. However, data on the cellular distribution of GALRs are sparse. The aim of the present study was to examine the expression of GAL and GALRs in different brain tumors by immunohistochemistry. Anterior pituitary gland (n = 7), pituitary adenoma (n = 9) and glioma of different WHO grades I-IV (n = 55) were analyzed for the expression of GAL and the three GALRs with antibodies recently extensively validated for specificity. While high focal GAL immunoreactivity was detected in up to 40% of cells in the anterior pituitary gland samples, only one pituitary adenoma showed focal GAL expression, at a low level. In the anterior pituitary, GAL1-R and GAL3-R protein expression was observed in up to 15% of cells, whereas receptor expression was not detected in pituitary adenoma. In glioma, diffuse and focal GAL staining was noticed in the majority of cases. GAL1-R was observed in eight out of nine glioma subtypes. GAL2-R immunoreactivity was not detected in glioma and pituitary adenoma, while GAL3-R expression was significantly associated to high-grade glioma (WHO grade IV). Most interestingly, expression of GAL and GALRs was observed in tumor-infiltrating immune cells, including neutrophils and glioma-associated macrophages/microglia. The presence of GALRs on tumor-associated immune cells, especially macrophages, indicates that GAL signaling contributes to homeostasis of the tumor microenvironment. Thus, our data indicate that GAL signaling in tumor-supportive myeloid cells could be a novel therapeutic target.

Galanin Signaling in the Brain Regulates Color Pattern Formation in Zebrafish.

Color patterns are prominent features of many animals and are of high evolutionary relevance. In basal vertebrates, color patterns are composed of specialized pigment cells that arrange in multilayered mosaics in the skin. Zebrafish (Danio rerio), the preeminent model system for vertebrate color pattern formation, allows genetic screens as powerful approaches to identify novel functions in a complex biological system. Adult zebrafish display a series of blue and golden horizontal stripes, composed of black melanophores, silvery or blue iridophores, and yellow xanthophores. This stereotyped pattern is generated by self-organization involving direct cell contacts between all three types of pigment cells mediated by integral membrane proteins [1-5]. Here, we show that neuropeptide signaling impairs the striped pattern in a global manner. Mutations in the genes coding either for galanin receptor 1A (npm/galr1A) or for its ligand galanin (galn) result in fewer stripes, a pale appearance, and the mixing of cell types, thus resembling mutants with thyroid hypertrophy [6]. Zebrafish chimeras obtained by transplantations of npm/galr1A mutant blastula cells indicate that mutant pigment cells of all three types can contribute to a normal striped pattern in the appropriate host. However, loss of galr1A expression in a specific region of the brain is sufficient to cause the mutant phenotype in an otherwise wild-type fish. Increased thyroid hormone levels in mutant fish suggest that galanin signaling through Galr1A in the pituitary is an upstream regulator of the thyroid hormone pathway, which in turn promotes precise interactions of pigment cells during color pattern formation.

Coordinated Targeting of Galanin Receptors on Cholangiocytes and Hepatic Stellate Cells Ameliorates Liver Fibrosis in Multidrug Resistance Protein 2 Knockout Mice.

Galanin (Gal) is a peptide with a role in neuroendocrine regulation of the liver. In this study, we assessed the role of Gal and its receptors, Gal receptor 1 (GalR1) and Gal receptor 2 (GalR2), in cholangiocyte proliferation and liver fibrosis in multidrug resistance protein 2 knockout (Mdr2KO) mice as a model of chronic hepatic cholestasis. The distribution of Gal, GalR1, and GalR2 in specific liver cell types was assessed by laser-capture microdissection and confocal microscopy. Galanin immunoreactivity was detected in cholangiocytes, hepatic stellate cells (HSCs), and hepatocytes. Cholangiocytes expressed GalR1, whereas HSCs and hepatocytes expressed GalR2. Strategies were used to either stimulate or block GalR1 and GalR2 in FVB/N (wild-type) and Mdr2KO mice and measure biliary hyperplasia and hepatic fibrosis by quantitative PCR and immunostaining of specific markers. Galanin treatment increased cholangiocyte proliferation and fibrogenesis in both FVB/N and Mdr2KO mice. Suppression of GalR1, GalR2, or both receptors in Mdr2KO mice resulted in reduced bile duct mass and hepatic fibrosis. In vitro knockdown of GalR1 in cholangiocytes reduced α-smooth muscle actin expression in LX-2 cells treated with cholangiocyte-conditioned media. A GalR2 antagonist inhibited HSC activation when Gal was administered directly to LX-2 cells, but not via cholangiocyte-conditioned media. These data demonstrate that Gal contributes not only to cholangiocyte proliferation but also to liver fibrogenesis via the coordinate activation of GalR1 in cholangiocytes and GalR2 in HSCs.

Fronto-temporal galanin modulates impulse control.

RATIONALE: The neuropeptide galanin has been implicated in a wide range of pathological conditions in which frontal and temporal structures are compromised. It works through three subtypes of G-protein-coupled receptors. One of these, the galanin receptor 1 (Gal-R1) subtype, is densely expressed in the ventral hippocampus (vHC) and ventral prefrontal cortex (vPFC); two brain structures that have similar actions on behavioral control. We hypothesize that Gal-R1 contributes to cognitive-control mechanisms that require hippocampal-prefrontal cortical circuitry. OBJECTIVE: To examine the effect of local vHC and vPFC infusions of M617, a Gal-R1 agonist, on inhibitory mechanisms of response control. METHODS: Different cohorts of rats were implanted with bilateral guide cannulae targeting the vPFC or the vHC. Following infusion of the Gal-R1 agonist, we examined the animals' behavior using a touchscreen version of the 5-choice reaction time task (5-choice task). RESULTS: The Gal-R1 agonist produced opposing behaviors in the vPFC and vHC, leading to disruption of impulse control when infused in the vPFC but high impulse control when infused into the vHC. This contrast between areas was accentuated when we added variability to the timing of the stimulus, which led to long decision times and reduced accuracy in the vPFC group but a general improvement in performance accuracy in the vHC group. CONCLUSIONS: These results provide the first evidence of a selective mechanism of Gal-R1-mediated modulation of impulse control in prefrontal-hippocampal circuitry.

San-Huang-Tang protects obesity/diabetes induced NAFLD by upregulating PGC-1α/PEPCK signaling in obese and galr1 knockout mice models.

ETHNOPHARMACOLOGICAL RELEVANCE: San-Huang-Tang (ST), a classic prescription, has been clinically used to cure diabetes and diabetes-associated metabolic disorders. Established studies have reported that ST can alleviate inflammation, obesity, hyperglycemia and insulin resistance. AIM OF THE STUDY: To the best of our knowledge, here, we reported for the first time the underlying mechanistic therapeutic efficacy of the ST against nonalcoholic fatty liver disease (NAFLD) in high-fat induced obese and galr1-deficient diabetic mice. MATERIALS AND METHODS: The obese and galr1-deficient mice were treated with ST at a dose of 10 g/kg every day for three weeks. Then food intake, body weight and insulin resistance indexes were measured. Western blotting, qRT-PCR, and plasma biochemical analyses were applied. RESULTS: ST reduced food intake, body weight, blood glucose level and insulin resistance, improved glucose tolerance in obese and galr1-deficient mice. Mechanistically, we confirmed that ST protected against NAFLD through activation of PGC-1α and its downstream signaling pathways as shown by the attenuated hepatic adipogenesis and lipid accumulation, increased hepatic fatty acid oxidation, regulated plasma lipid parameters, and increased energy expenditure and metabolic function in fat and muscle. CONCLUSIONS: Reduction in food intake produced by ST may contribute to the observed metabolic effects. Our findings strongly suggest that ST might be a potential novel therapeutic drug against obesity/diabetes-induced NAFLD and other metabolic disorders.

Opioid-galanin receptor heteromers mediate the dopaminergic effects of opioids.

Identifying non-addictive opioid medications is a high priority in medical sciences, but µ-opioid receptors mediate both the analgesic and addictive effects of opioids. We found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of µ-opioid receptors with galanin Gal1 receptors, rendering a profound decrease in the potency of methadone. This was explained by methadone's weaker proficiency to activate the dopaminergic system as compared to morphine and predicted a dissociation of therapeutic versus euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-report of "high" in methadone-maintained patients. These results suggest that µ-opioid-Gal1 receptor heteromers mediate the dopaminergic effects of opioids that may lead to a lower addictive liability of opioids with selective low potency for the µ-opioid-Gal1 receptor heteromer, exemplified by methadone.