Ghrelin Facilitates GLUT2-, SGLT1- and SGLT2-mediated Intestinal Glucose Transport in Goldfish (Carassius auratus)

Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish

Characterization of Ghrelin O-Acyltransferase (GOAT) in goldfish (Carassius auratus)

Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin’s physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish

Cyberbullying and Gambling Disorder: Associations with Emotion Regulation and Coping Strategies

The presence of unsuitable coping and emotion regulation strategies in young populations with gambling disorder (GD) and in those who have experienced cyberbullying victimization has been suggested. However, this association has not been explored in depth. In this study, our aim was to analyze individual differences in emotion regulation, coping strategies, and substance abuse in a clinical sample of adolescents and young adult patients with GD (n = 31) and in a community sample (n = 250). Furthermore, we aimed to examine the association between cyberbullying and GD. Participants were evaluated using the Cyberbullying Questionnaire-Victimization, the Canadian Adolescent Gambling Inventory, the Coping Strategies Inventory, the Difficulties in Emotion Regulation Scale, the Alcohol Use Disorders Identification Test and the Drug Use Disorders Identification Test. Structural Equation Modeling was used to explore associations between these factors in a community sample and in a clinical group. In both groups, exposure to cyberbullying behaviors was positively associated with higher emotion dysregulation and the use of maladaptative coping styles. Our findings uphold that adolescents and young adults who were victims of cyberbullying show difficulties in emotion regulation and maladaptive coping strategies when trying to solve problems. The specific contribution of sex, age, gambling severity, emotion regulation, and coping strategies on cyberbullying severity is also discussed. Populations at vulnerable ages could potentially benefit from public prevention policies that target these risk factors

COVID-19 Severity and Survival over Time in Patients with Hematologic Malignancies: A Population-Based Registry Study

Mortality rates for COVID-19 have declined over time in the general population, but data in patients with hematologic malignancies are contradictory. We identified independent prognostic factors for COVID-19 severity and survival in unvaccinated patients with hematologic malignancies, compared mortality rates over time and versus non-cancer inpatients, and investigated post COVID-19 condition. Data were analyzed from 1166 consecutive, eligible patients with hematologic malignancies from the population-based HEMATO-MADRID registry, Spain, with COVID-19 prior to vaccination roll-out, stratified into early (February–June 2020; n = 769 (66%)) and later (July 2020–February 2021; n = 397 (34%)) cohorts. Propensity-score matched non-cancer patients were identified from the SEMI-COVID registry. A lower proportion of patients were hospitalized in the later waves (54.2%) compared to the earlier (88.6%), OR 0.15, 95%CI 0.11–0.20. The proportion of hospitalized patients admitted to the ICU was higher in the later cohort (103/215, 47.9%) compared with the early cohort (170/681, 25.0%, 2.77; 2.01–3.82). The reduced 30-day mortality between early and later cohorts of non-cancer inpatients (29.6% vs. 12.6%, OR 0.34; 0.22–0.53) was not paralleled in inpatients with hematologic malignancies (32.3% vs. 34.8%, OR 1.12; 0.81–1.5). Among evaluable patients, 27.3% had post COVID-19 condition. These findings will help inform evidence-based preventive and therapeutic strategies for patients with hematologic malignancies and COVID-19 diagnosis.Depto. de MedicinaFac. de MedicinaTRUEFundación Madrileña de Hematología y HemoterapiaFundación Leucemia y LinfomaAsociación Madrileña de Hematología y Hemoterapiapu

Triptófano hidroxilasa en los fotorreceptores de la retina de Xenopus laevis : regulación de la expresión de la actividad enzimática por el fotoperiodo y mensajeros intracelulares y extracelulares

La inyección ocular de ácido Kaínico es un método adecuado para la obtención de retinas de Xenopus laevis enriquecidas en fotorreceptores, siendo estas preparaciones un buen modelo para el estudio de la síntesis de melatonina en la retina. En los fotorreceptores de Xenopus laevis, la actividad TPH muestra un ritmo diario paralelo a los ritmos de actividad NAT y melatonina ocular. El ciclo luz-oscuridad dirige los principales sistemas de comunicación intracelular de los fotorreceptores de Xenopus laevis. La entrada de calcio a través de los canales tipo L desencadena una serie de procesos dependientes del complejo calcio-calmodulina, como una estimulación de la CaMKII y de la adenilato ciclasa tipo I. CaMKII fosforina directamente la TPH, activándola. La activación de la adenilato ciclasa aumenta el contenido de cAMP e induce una fosforilación de la TPH por la PKA. La dopamina a través de receptores D2 regula negativamente la actividad TPH de los fotorreceptores, mimetizando el efecto de la luz. Los niveles extracelulares de la adenosina regulan la síntesis de melatonina activando a través de receptores A2 las actividades TPH y NAT y mimetizando el efecto de la oscurida

Brain Mapping of Ghrelin O-Acyltransferase in Goldfish (Carassius Auratus): Novel Roles for the Ghrelinergic System in Fish?

Ghrelin O-acyltransferase (GOAT) is the enzyme responsible for acylation of ghrelin, a gut-brain hormone with important roles in many physiological functions in vertebrates. Many aspects of GOAT remain to be elucidated, especially in fish, and particularly its anatomical distribution within the different brain areas has never been reported to date. The present study aimed to characterize the brain mapping of GOAT using RT-qPCR and immunohistochemistry in a teleost, the goldfish (Carassius auratus). Results show that goat transcripts are expressed in different brain areas of the goldfish, with the highest levels in the vagal lobe. Using immunohistochemistry, we also report the presence of GOAT immunoreactive cells in different encephalic areas, including the telencephalon, some hypothalamic nuclei, pineal gland, optic tectum and cerebellum, although they are especially abundant in the hindbrain. Particularly, an important signal is observed in the vagal lobe and some fiber tracts of the brainstem, such as the medial longitudinal fasciculus, Mauthneri fasciculus, secondary gustatory tract and spinothalamic tract. Most of the forebrain areas where GOAT is detected, particularly the hypothalamic nuclei, also express the ghs-r1a ghrelin receptor and other appetite-regulating hormones (e.g., orexin and NPY), supporting the role of ghrelin as a modulator of food intake and energy balance in fish. Present results are the first report on the presence of GOAT in the brain using imaging techniques. The high presence of GOAT in the hindbrain is a novelty, and point to possible new functions for the ghrelinergic system in fish

Ghrelin suppresses cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) in the intestine, and attenuates the anorectic effects of CCK, PYY and GLP-1 in goldfish (Carassius auratus)

Ghrelin is an important gut-derived hormone with an appetite stimulatory role, while most of the intestinal hormones, including cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1), are appetite-inhibitors. Whether these important peptides with opposing roles on food intake interact to regulate energy balance in fish is currently unknown. The aim of this study was to characterize the putative crosstalk between ghrelin and CCK, PYY and GLP-1 in goldfish (Carassius auratus). We first determined the localization of CCK, PYY and GLP-1 in relation to ghrelin and its main receptor GHS-R1a (growth hormone secretagogue 1a) in the goldfish intestine by immunohistochemistry. Colocalization of ghrelin/GHS-R1a and CCK/PYY/GLP-1 was found primarily in the luminal border of the intestinal mucosa. In an intestinal explant culture, a significant decrease in prepro-cck, prepro-pyy and proglucagon transcript levels was observed after 60 min of incubation with ghrelin, which was abolished by preincubation with the GHS-R1a ghrelin receptor antagonist [D-Lys3]-GHRP-6 (except for proglucagon). The protein expression of PYY and GLP-1 was also downregulated by ghrelin. Finally, intraperitoneal co-administration of CCK, PYY or GLP-1 with ghrelin results in no modification of food intake in goldfish. Overall, results of the present study show for the first time in fish that ghrelin exerts repressive effects on enteric anorexigens. It is likely that these interactions mediate the stimulatory effects of ghrelin on feeding and metabolism in fish

Periprandial changes and effects of short- and long-term fasting on ghrelin, GOAT, and ghrelin receptors in goldfish (Carassius auratus)

The periprandial profile and effects of short- (7 days) and long-term (30 days) fasting on the ghrelinergic system were studied in goldfish (Carassius auratus). Plasma levels of acyl-ghrelin, desacyl-ghrelin, and ghrelin O-acyl transferase (GOAT) were analyzed by enzymoimmunoassays, and expression of preproghrelin, goat and growth hormone secretagogue receptors (ghs-r) was quantified by real-time PCR. Circulating levels of acyl-ghrelin and GOAT rise preprandially, supporting the role of acyl-ghrelin as a meal initiator in this teleost. Consistently, preproghrelin and ghs-r1a1 expression increases 1 h before feeding time in intestinal bulb, suggesting that this receptor subtype might be involved in the preprandial action of ghrelin in this tissue. Significant postfeeding variations are detected for preproghrelin in telencephalon, goat in telencephalon and hypothalamus, ghs-r1a1 in vagal lobe, ghs-r1a2 and ghs-r2a1 in hypothalamus and ghs-r2a2 in telencephalon and vagal lobe, especially in unfed fish. Short- and long-term fasting significantly increase preproghrelin expression in telencephalon and gut. Goat expression is upregulated by short-term fasting in telencephalon and hypothalamus, and by both short- and long-term fasting in gut. Expression of ghs-r increases by fasting in telencephalon, while an upregulation of type 2, but not type 1, receptors is observed in vagal lobe. In intestinal bulb, ghs-r1a2 transcripts increase after both short- and long-term fasting. These results show a high dependence of the ghrelinergic system on feeding and nutritional status in fish, and demonstrate for the first time a differential implication of the various components of this system suggesting different roles for the four ghrelinergic receptor subtypes

Tissue-specific expression of ghrelinergic and NUCB2/nesfatin-1 systems in goldfish (Carassius auratus) is modulated by macronutrient composition of diets

The macronutrient composition of diets is a very important factor in the regulation of body weight and metabolism. Several lines of research in mammals have shown that macronutrients differentially regulate metabolic hormones, including ghrelin and nesfatin-1 that have opposing effects on energy balance. This study aimed to determine whether macronutrients modulate the expression of ghrelin and the nucleobindin-2 (NUCB2) encoded nesfatin-1 in goldfish (Carassius auratus). Fish were fed once daily on control, high-carbohydrate, high-protein, high-fat and very high-fat diets for 7 (short-term) or 28 (long-term) days. The expression of preproghrelin, ghrelin O-acyl transferase (goat), growth hormone secretagogue receptor 1 (ghs-r1) and nucb2/nesfatin-1 mRNAs was quantified in the hypothalamus, pituitary, gut and liver. Short-term feeding with fat-enriched diets significantly increased nucb2 mRNA levels in hypothalamus and liver, preproghrelin, goat and ghs-r1 expression in pituitary, and ghs-r1 expression in gut. Fish fed on a high-protein diet exhibited a significant reduction in preproghrelin and ghs-r1 mRNAs in the liver. After long-term feeding, fish fed on high-carbohydrate and very high-fat diets had significantly increased preproghrelin, goat and ghs-r1 expression in pituitary. Feeding on a high-carbohydrate diet also upregulated goat and ghs-r1 transcripts in gut, while feeding on a high-fat diet elicited the same effect only for ghs-r1 in liver. Nucb2 expression increased in pituitary, while it decreased in gut after long-term feeding of a high-protein diet. Collectively, these results show for the first time in fish that macronutrients differentially regulate the expression of ghrelinergic and NUCB2/nesfatin-1 systems in central and peripheral tissues of goldfish

Interplay between the endocrine and circadian systems in fishes

The circadian system is responsible for the temporal organisation of physiological functions which, in part, involves daily cycles of hormonal activity. In this review, we analyse the interplay between the circadian and endocrine systems in fishes. We first describe the current model of fish circadian system organisation and the basis of the molecular clockwork that enables different tissues to act as internal pacemakers. This system consists of a net of central and peripherally located oscillators and can be synchronised by the light–darkness and feeding–fasting cycles. We then focus on two central neuroendocrine transducers (melatonin and orexin) and three peripheral hormones (leptin, ghrelin and cortisol), which are involved in the synchronisation of the circadian system in mammals and/or energy status signalling. We review the role of each of these as overt rhythms (i.e. outputs of the circadian system) and, for the first time, as key internal temporal messengers that act as inputs for other endogenous oscillators. Based on acute changes in clock gene expression, we describe the currently accepted model of endogenous oscillator entrainment by the light–darkness cycle and propose a new model for non-photic (endocrine) entrainment, highlighting the importance of the bidirectional cross-talking between the endocrine and circadian systems in fishes. The flexibility of the fish circadian system combined with the absence of a master clock makes these vertebrates a very attractive model for studying communication among oscillators to drive functionally coordinated outputs