RESUMO
Ghrelin represents a key hormone regulating energy balance. Upon activation of the growth hormone secretagogue receptor (GHSR), ghrelin increases blood glucose levels, food intake, and promotes weight gain. The liver-expressed antimicrobial peptide 2 (LEAP2) acts as an endogenous antagonist of the GHSR. While the regulation of LEAP2 and its effect on the GHSR likely occur in an opposite pattern to that of ghrelin, the dietary regulation of LEAP2 remains to be described. We, therefore, examined the regulation of LEAP2 by different acute meal challenges (glucose, mixed meal, olive, lard, and fish oil) and diets (chow vs. high-fat) in C57BL/6 male mice. In addition, the effect of specific fatty acids (oleic, docosahexaenoic, and linoleic acid) on LEAP2 was assessed in murine intestinal organoids. While only mixed meal increased liver Leap2 expression, all meal challenges except fish oil increased jejunal Leap2 expression compared to water. Leap2 expression correlated with levels of hepatic glycogen and jejunal lipids. Lipid versus water dosing increased LEAP2 levels in the systemic circulation and portal vein where fish oil was associated with the smallest increase. In line with this, oleic acid, but not docosahexaenoic acid increased Leap2 expression in intestinal organoids. Feeding mice with high-fat versus chow diet not only increased plasma LEAP2 levels, but also the increment in plasma LEAP2 upon dosing with olive oil versus water. Taken together, these results show that LEAP2 is regulated by meal ingestion in both the small intestine and the liver according to the meal/diet of interest and local energy stores.
Assuntos
Dieta , Grelina , Animais , Masculino , Camundongos , Ácidos Graxos , Grelina/metabolismo , Camundongos Endogâmicos C57BL , Aumento de PesoRESUMO
To examine whether fasting plasma liver-expressed antimicrobial peptide 2 (FP-LEAP2) is associated with markers of cardiometabolic disease susceptibility in a cohort with prediabetes and overweight/obesity and whether antidiabetic interventions affect FP-LEAP2 levels. The analysis included 115 individuals with prediabetes [hemoglobin A1c (HbA1c) 39-47 mmol/mol, 5.7%-6.4%] and overweight/obesity [body mass index (BMI) ≥ 25 kg/m2] from a randomized controlled trial. Changes in FP-LEAP2 levels were assessed in relation to treatment with dapagliflozin (10 mg once daily), metformin (1,700 mg daily), or interval-based exercise (5 days/wk, 30 min/session) compared with control (habitual lifestyle) after 6 and 13 wk of treatment. FP-LEAP2 levels were positively associated with [standardized beta coefficient (95% CI)]: BMI 0.22 (0.03:0.41), P = 0.027; body weight 0.27 (0.06:0.48), P = 0.013; fat mass 0.2 (0.00:0.4), P = 0.048; lean mass 0.47 (0.13:0.8), P = 0.008; HbA1c 0.35 (0.17:0.53), P < 0.001; fasting plasma glucose (FPG) 0.32 (0.12:0.51), P = 0.001; fasting serum insulin 0.28 (0.09:0.47), P = 0.005; total cholesterol 0.19 (0.01:0.38), P = 0.043; triglycerides 0.31 (0.13:0.5), P < 0.001; and transaminases and fatty liver index (standardized beta coefficients 0.23-0.32), all P < 0.020. FP-LEAP2 levels were inversely associated with insulin sensitivity [-0.22 (-0.41: -0.03), P = 0.022] and kidney function [estimated glomerular filtration rate (eGFR) -0.34 (-0.56: -0.12), P = 0.003]. FP-LEAP2 levels were not associated with fat distribution or body fat percentage, fasting glucagon, postload glucose, ß-cell function, or low-density lipoprotein. The interventions were not associated with changes in FP-LEAP2. FP-LEAP2 is associated with body mass, impaired insulin sensitivity, liver-specific enzymes, and kidney function. The findings highlight the importance of studying LEAP2 in obesity, type 2 diabetes, and nonalcoholic fatty liver disease. FP-LEAP2 was not affected by metformin, dapaglifloxin, or exercise in this population.NEW & NOTEWORTHY LEAP2, primarily secreted by the liver, increases with greater body mass, insulin resistance, and liver-specific enzymes in individuals with prediabetes and overweight or obesity. Fasting glucose, body mass, and alanine aminotransferase independently predict LEAP2 levels. LEAP2 is inversely linked to impaired kidney function. Elevated LEAP2 levels might indicate an increased metabolic risk, warranting further investigation into its potential involvement in glucose and body weight control.
Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Resistência à Insulina , Metformina , Estado Pré-Diabético , Humanos , Estado Pré-Diabético/complicações , Hipoglicemiantes/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Hemoglobinas Glicadas , Sobrepeso , Glicemia/metabolismo , Obesidade/complicações , Metformina/uso terapêutico , Peso Corporal , Doenças Cardiovasculares/epidemiologiaRESUMO
Preclinical and clinical studies have identified the ghrelin receptor [growth hormone secretagogue receptor (GHSR)1a] as a potential target for treating alcohol use disorder. A recent phase 1a clinical trial of a GHSR1a antagonist/inverse agonist, PF-5190457, in individuals with heavy alcohol drinking identified a previously undetected major hydroxy metabolite of PF-5190457, namely PF-6870961. Here, we further characterized PF-6870961 by screening for off-target interactions in a high-throughput screen and determined its in vitro pharmacodynamic profile at GHSR1a through binding and concentration-response assays. Moreover, we determined whether the metabolite demonstrated an in vivo effect by assessing effects on food intake in male and female rats. We found that PF-6870961 had no off-target interactions and demonstrated both binding affinity and inverse agonist activity at GHSR1a. In comparison with its parent compound, PF-5190457, the metabolite PF-6870961 had lower binding affinity and potency at inhibiting GHSR1a-induced inositol phosphate accumulation. However, PF-6870961 had increased inhibitory potency at GHSR1a-induced ß-arrestin recruitment relative to its parent compound. Intraperitoneal injection of PF-6870961 suppressed food intake under conditions of both food restriction and with ad libitum access to food in male and female rats, demonstrating in vivo activity. The effects of PF-6870961 on food intake were abolished in male and female rats knockout for GHSR, thus demonstrating that its effects on food intake are in fact mediated by the GHSR receptor. Our findings indicate that the newly discovered major hydroxy metabolite of PF-5190457 may contribute to the overall activity of PF-5190457 by demonstrating inhibitory activity at GHSR1a. SIGNIFICANCE STATEMENT: Antagonists or inverse agonists of the growth hormone secretagogue receptor (GHSR)1a have demonstrated substantial potential as therapeutics for alcohol use disorder. We here expand understanding of the pharmacology of one such GHSR1a inverse agonist, PF-5190457, by studying the safety and pharmacodynamics of its major hydroxy metabolite, PF-6870961. Our data demonstrate biased inverse agonism of PF-6870961 at GHSR1a and provide new structure-activity relationship insight into GHSR1a inverse agonism.
Assuntos
Alcoolismo , Ratos , Masculino , Feminino , Animais , Receptores de Grelina/metabolismo , Agonismo Inverso de DrogasRESUMO
Agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus regulates food intake and whole-body metabolism. NAD+ regulates multiple cellular processes controlling energy metabolism. Yet, its role in hypothalamic AgRP neurons to control food intake is poorly understood. Here, we aimed to assess whether genetic deletion of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in NAD+ production, affects AgRP neuronal function to impact whole-body metabolism and food intake. Metabolic parameters during fed and fasted states, and upon systemic ghrelin and leptin administration were studied in AgRP-specific Nampt knockout (ARNKO) mice. We monitored neuropeptide expression levels and density of AgRP neurons in ARNKO mice from embryonic to adult age. NPY cells were used to determine effects of NAMPT inhibition on neuronal viability, energy status, and oxidative stress in vitro. In these cells, NAD+ depletion reduced ATP levels, increased oxidative stress, and promoted cell death. Agrp expression in the hypothalamus of ARNKO mice gradually decreased after weaning due to progressive AgRP neuron degeneration. Adult ARNKO mice had normal glucose and insulin tolerance, but exhibited an elevated respiratory exchange ratio (RER) when fasted. Remarkably, fasting-induced food intake was unaffected in ARNKO mice when evaluated in metabolic cages, but fasting- and ghrelin-induced feeding and body weight gain decreased in ARNKO mice when evaluated outside metabolic cages. Collectively, deletion of Nampt in AgRP neurons causes progressive neurodegeneration and impairs fasting and ghrelin responses in a context-dependent manner. Our data highlight an essential role of Nampt in AgRP neuron function and viability.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Citocinas/fisiologia , Ingestão de Alimentos , Jejum , Grelina/farmacologia , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Nicotinamida Fosforribosiltransferase/fisiologia , Proteína Relacionada com Agouti/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismoRESUMO
INTRODUCTION: Food intake varies during the ovarian hormone/estrous cycle in humans and rodents, an effect mediated mainly by estradiol. A potential mediator of the central anorectic effects of estradiol is the neuropeptide relaxin-3 (RLN3) synthetized in the nucleus incertus (NI) and acting via the relaxin family peptide-3 receptor (RXFP3). METHODS: We investigated the relationship between RLN3/RXFP3 signaling and feeding behavior across the female rat estrous cycle. We used in situ hybridization to investigate expression patterns of Rln3 mRNA in NI and Rxfp3 mRNA in the hypothalamic paraventricular nucleus (PVN), lateral hypothalamic area (LHA), medial preoptic area (MPA), and bed nucleus of the stria terminalis (BNST), across the estrous cycle. We identified expression of estrogen receptors (ERs) in the NI using droplet digital PCR and assessed the electrophysiological responsiveness of NI neurons to estradiol in brain slices. RESULTS: Rln3 mRNA reached the lowest levels in the NI pars compacta during proestrus. Rxfp3 mRNA levels varied across the estrous cycle in a region-specific manner, with changes observed in the perifornical LHA, magnocellular PVN, dorsal BNST, and MPA, but not in the parvocellular PVN or lateral LHA. G protein-coupled estrogen receptor 1 (Gper1) mRNA was the most abundant ER transcript in the NI. Estradiol inhibited 33% of type 1 NI neurons, including RLN3-positive cells. CONCLUSION: These findings demonstrate that the RLN3/RXFP3 system is modulated by the estrous cycle, and although further studies are required to better elucidate the cellular and molecular mechanisms of estradiol signaling, current results implicate the involvement of the RLN3/RXFP3 system in food intake fluctuations observed across the estrous cycle in female rats.
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Estradiol/metabolismo , Ciclo Estral/metabolismo , Região Hipotalâmica Lateral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Área Pré-Óptica/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Relaxina/metabolismo , Núcleos Septais/metabolismo , Animais , Feminino , RNA Mensageiro/metabolismo , RatosRESUMO
Biased signaling has been suggested as a means of selectively modulating a limited fraction of the signaling pathways for G-protein-coupled receptor family members. Hence, biased ligands may allow modulation of only the desired physiological functions and not elicit undesired effects associated with pharmacological treatments. The ghrelin receptor is a highly sought antiobesity target, since the gut hormone ghrelin in humans has been shown to increase both food intake and fat accumulation. However, it also modulates mood, behavior, growth hormone secretion, and gastric motility. Thus, blocking all pathways of this receptor may give rise to potential side effects. In the present study, we describe a highly promiscuous signaling capacity for the ghrelin receptor. We tested selected ligands for their ability to regulate the various pathways engaged by the receptor. Among those, a biased ligand, YIL781, was found to activate the Gαq/11 and Gα12 pathways selectively without affecting the engagement of ß-arrestin or other G proteins. YIL781 was further characterized for its in vivo physiological functions. In combination with the use of mice in which Gαq/11 was selectively deleted in the appetite-regulating AgRP neurons, this biased ligand allowed us to demonstrate that selective blockade of Gαq/11, without antagonism at ß-arrestin or other G-protein coupling is sufficient to decrease food intake.
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Grelina/metabolismo , Receptores de Grelina/metabolismo , Animais , Ingestão de Alimentos/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismoRESUMO
The G-protein coupled receptor GPR39 is abundantly expressed in various tissues and can be activated by changes in extracellular Zn2+ in physiological concentrations. Previously, genetically modified rodent models have been able to shed some light on the physiological functions of GPR39, and more recently the utilization of novel synthetic agonists has led to the unraveling of several new functions in the variety of tissues GPR39 is expressed. Indeed, GPR39 seems to be involved in many important metabolic and endocrine functions, but also to play a part in inflammation, cardiovascular diseases, saliva secretion, bone formation, male fertility, addictive and depression disorders and cancer. These new discoveries offer opportunities for the development of novel therapeutic approaches against many diseases where efficient therapeutics are still lacking. This review focuses on Zn2+ as an endogenous ligand as well as on the novel synthetic agonists of GPR39, placing special emphasis on the recently discovered physiological functions and discusses their pharmacological potential.
Assuntos
Biomarcadores , Descoberta de Drogas , Receptores Acoplados a Proteínas G/fisiologia , Animais , Suscetibilidade a Doenças , Descoberta de Drogas/métodos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Especificidade de Órgãos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Zinco/metabolismoRESUMO
AIMS/HYPOTHESIS: A genetic risk score (GRS) consisting of 53 insulin resistance variants (GRS53) was recently demonstrated to associate with insulin resistance in adults. We speculated that the GRS53 might already associate with insulin resistance during childhood, and we therefore aimed to investigate this in populations of Danish children and adolescents. Furthermore, we aimed to address whether the GRS associates with components of the metabolic syndrome and altered body composition in children and adolescents. METHODS: We examined a total of 689 children and adolescents who were overweight or obese and 675 children and adolescents from a population-based study. Anthropometric data, dual-energy x-ray absorptiometry scans, BP, fasting plasma glucose, fasting serum insulin and fasting plasma lipid measurements were obtained, and HOMA-IR was calculated. The GRS53 was examined for association with metabolic traits in children by linear regressions using an additive genetic model. RESULTS: In overweight/obese children and adolescents, the GRS53 associated with higher HOMA-IR (ß = 0.109 ± 0.050 (SE); p = 2.73 × 10-2), fasting plasma glucose (ß = 0.010 ± 0.005 mmol/l; p = 2.51 × 10-2) and systolic BP SD score (ß = 0.026 ± 0.012; p = 3.32 × 10-2) as well as lower HDL-cholesterol (ß = -0.008 ± 0.003 mmol/l; p = 1.23 × 10-3), total fat-mass percentage (ß = -0.143 ± 0.054%; p = 9.15 × 10-3) and fat-mass percentage in the legs (ß = -0.197 ± 0.055%; p = 4.09 × 10-4). In the population-based sample of children, the GRS53 only associated with lower HDL-cholesterol concentrations (ß = -0.007 ± 0.003 mmol/l; p = 1.79 × 10-2). CONCLUSIONS/INTERPRETATION: An adult-based GRS comprising 53 insulin resistance susceptibility SNPs associates with insulin resistance, markers of the metabolic syndrome and altered fat distribution in a sample of Danish children and adolescents who were overweight or obese.
Assuntos
Predisposição Genética para Doença , Resistência à Insulina , Sobrepeso/genética , Obesidade Infantil/genética , Adolescente , Adulto , Antropometria , Composição Corporal , Criança , HDL-Colesterol/metabolismo , Dinamarca , Diabetes Mellitus Tipo 2 , Genótipo , Humanos , Modelos Lineares , Síndrome Metabólica/metabolismo , Pessoa de Meia-Idade , Fenótipo , RiscoRESUMO
BACKGROUND: p.R270H (rs116454156) in the long chain fatty acid 7TM receptor FFAR4 (GPR120) which results in impaired Gαq (Gq) coupled signalling, has been associated with obesity. We aimed to extend the functional in vitro analyses of p.R270H and to investigate the association with obesity and glucose-related traits in the Danish population. METHODS: Surface expression, Gq and Gi coupled signalling as well as ß-arrestin recruitment were examined in vitro. p.R270H was genotyped using the exome chip array in 11 479 Danish adult individuals. Of these 4391 were obese and 4415 were normal weight. Association with quantitative metabolic traits comprised 8720 non-diabetic individuals. RESULTS: p.R270H showed reduced surface expression of FFAR4. Ligand-independent activity was eliminated and strongly impaired through the Gq and Gi signalling pathways, respectively. The ligand-induced maximal signalling efficacy of p.R270H was reduced only through the Gq pathway. The p.R270H variant did not affect ß-arrestin recruitment. p.R270H was not associated with increased risk of obesity nor increased fasting plasma glucose levels in the Danish study populations. Nor was it associated with these two traits in the European Network for Genetic and Genomic Epidemiology consortium data (N=34â 901-71â 175; p>0.70). It was also not associated with waist-hip ratio, glucose metabolism during an oral glucose tolerance test, lipid levels or with markers of adiposity (leptin, adiponectin), inflammation (high-sensitive C reactive protein; hs-CRP) and liver function (alanine aminotransferase) in the Danish population (p>0.05). CONCLUSIONS: We demonstrate that p.R270H of FFAR4 impairs Gq and Gi signalling of FFAR4 in vitro; however, this impaired signalling for p.R270H does not translate into associations with human metabolic phenotypes in the investigated populations.
Assuntos
Variação Genética/genética , Receptores Acoplados a Proteínas G/genética , Adulto , Biomarcadores/metabolismo , Glicemia/genética , Estudos de Casos e Controles , Linhagem Celular , Feminino , Glucose/genética , Teste de Tolerância a Glucose/métodos , Células HEK293 , Humanos , Inflamação/genética , Ligantes , Lipídeos/genética , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade/genética , Transdução de Sinais/genética , beta-Arrestinas/genéticaRESUMO
Ghrelin receptor (Ghr-R) signaling in neurons of the ventral tegmental area (VTA) can modulate dopaminergic function and the reward-related effects of both palatable foods and drugs of abuse. In this study, we re-introduced the Ghr-R in VTA neurons in Ghr-R knockout mice (Ghr-RVTA mice) to specifically study the importance of the constitutively active Ghr-R for VTA neuronal signaling. Our results showed that re-introduction of the Ghr-R in the VTA had no impact on body weight or food intake under basal conditions. However, during novel environment stress Ghr-RVTA mice showed increased food intake and energy expenditure compared to Ghr-R knockout mice, demonstrating the significance of Ghr-R signaling in the response to stress. Ghr-RVTA mice also showed increased cocaine-induced locomotor activity compared to Ghr-R knockout mice, highlighting the importance of ghrelin signaling for the reward-related effects of activation of VTA neurons. Overall, our data suggest that re-introduction of the Ghr-R in the mesolimbic reward system of Ghr-R knockout mice increases the level of activation induced by both cocaine and novelty stress.
Assuntos
Comportamento Animal/fisiologia , Receptores de Grelina/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Peso Corporal , Dependovirus/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Ingestão de Alimentos , Metabolismo Energético , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Consumo de Oxigênio , Receptores de Dopamina D2/metabolismo , Receptores de Grelina/deficiência , Receptores de Grelina/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
AIMS/HYPOTHESIS: Normal glucose metabolism depends on pancreatic secretion of insulin and glucagon. The bihormonal hypothesis states that while lack of insulin leads to glucose underutilisation, glucagon excess is the principal factor in diabetic glucose overproduction. A recent study reported that streptozotocin-treated glucagon receptor knockout mice have normal glucose tolerance. We investigated the impact of acute disruption of glucagon secretin or action in a mouse model of severe diabetes by three different approaches: (1) alpha cell elimination; (2) glucagon immunoneutralisation; and (3) glucagon receptor antagonism, in order to evaluate the effect of these on glucose tolerance. METHODS: Severe diabetes was induced in transgenic and wild-type mice by streptozotocin. Glucose metabolism was investigated using OGTT in transgenic mice with the human diphtheria toxin receptor expressed in proglucagon producing cells allowing for diphtheria toxin (DT)-induced alpha cell ablation and in mice treated with either a specific high affinity glucagon antibody or a specific glucagon receptor antagonist. RESULTS: Near-total alpha cell elimination was induced in transgenic mice upon DT administration and resulted in a massive decrease in pancreatic glucagon content. Oral glucose tolerance in diabetic mice was neither affected by glucagon immunoneutralisation, glucagon receptor antagonism, nor alpha cell removal, but did not deteriorate further compared with mice with intact alpha cell mass. CONCLUSIONS/INTERPRETATION: Disruption of glucagon action/secretion did not improve glucose tolerance in diabetic mice. Near-total alpha cell elimination may have prevented further deterioration. Our findings support insulin lack as the major factor underlying hyperglycaemia in beta cell-deficient diabetes.
Assuntos
Diabetes Mellitus Experimental , Glucagon , Intolerância à Glucose , Insulina/deficiência , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Toxina Diftérica , Glucagon/antagonistas & inibidores , Glucagon/metabolismo , Glucagon/fisiologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/patologia , Intolerância à Glucose/sangue , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/genética , Teste de Tolerância a Glucose , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/genética , EstreptozocinaRESUMO
BACKGROUND: Besides the well-known effects of ghrelin on adiposity and food intake regulation, the ghrelin system has been shown to regulate aspects of behavior including anxiety and stress. However, the effect of virus-mediated overexpression of the ghrelin receptor in the amygdala has not previously been addressed directly. METHODS: First, we examined the acute effect of peripheral ghrelin administration on anxiety- and depression-like behavior using the open field, elevated plus maze, forced swim, and tail suspension tests. Next, we examined the effect of peripheral ghrelin administration and ghrelin receptor deficiency on stress in a familiar and social environment using the Intellicage system. Importantly, we also used a novel approach to study ghrelin receptor signaling in the brain by overexpressing the ghrelin receptor in the amygdala. We examined the effect of ghrelin receptor overexpression on anxiety-related behavior before and after acute stress and measured the modulation of serotonin receptor expression. RESULTS: We found that ghrelin caused an anxiolytic-like effect in both the open field and elevated plus maze tests. Additionally, it attenuated air-puff-induced stress in the social environment, while the opposite was shown in ghrelin receptor deficient mice. Finally, we found that overexpression of the ghrelin receptor in the basolateral division of the amygdala caused an anxiolytic-like effect and decreased the 5HT1a receptor expression. CONCLUSIONS: Ghrelin administration and overexpression of the ghrelin receptor in the amygdala induces anxiolytic-like behavior. Since the ghrelin receptor has high constitutive activity, ligand-independent signaling in vivo may be important for the observed anxiolytic-like effects. The anxiolytic effects seem to be mediated independently from the HPA axis, potentially engaging the central serotonin system.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Ansiolíticos/farmacologia , Ansiedade/prevenção & controle , Comportamento Animal/efeitos dos fármacos , Grelina/farmacologia , Receptores de Grelina/agonistas , Transdução de Sinais/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiopatologia , Animais , Ansiedade/genética , Ansiedade/metabolismo , Ansiedade/psicologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Elevação dos Membros Posteriores , Humanos , Locomoção/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Comportamento Social , Estresse Psicológico/complicações , Estresse Psicológico/metabolismo , Estresse Psicológico/psicologia , Natação , Fatores de TempoRESUMO
Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.
Assuntos
Intolerância à Glucose/metabolismo , Transtornos do Crescimento/metabolismo , Proteínas Nucleares/deficiência , Vesículas Secretórias/metabolismo , Animais , Autoantígenos/fisiologia , Células COS , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Drosophila melanogaster , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Glucose/metabolismo , Intolerância à Glucose/genética , Transtornos do Crescimento/genética , Hormônio do Crescimento/deficiência , Hormônio do Crescimento/metabolismo , Homeostase , Insulina/metabolismo , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Hipófise/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Imagem com Lapso de Tempo , Rede trans-Golgi/metabolismoRESUMO
Dietary advanced glycation end products (AGE) formed during heating of food have gained interest as potential nutritional toxins with adverse effects on inflammation and glucose metabolism. In the present study, we investigated the short-term effects of high and low molecular weight (HMW and LMW) dietary AGE on insulin sensitivity, expression of the receptor for AGE (RAGE), the AGE receptor 1 (AGER1) and TNF-α, F2-isoprostaglandins, body composition and food intake. For 2 weeks, thirty-six Sprague-Dawley rats were fed a diet containing 20% milk powder with different proportions of this being given as heated milk powder (0, 40 or 100%), either native (HMW) or hydrolysed (LMW). Gene expression of RAGE and AGER1 in whole blood increased in the group receiving a high AGE LMW diet, which also had the highest urinary excretion of the AGE, methylglyoxal-derived hydroimidazolone 1 (MG-H1). Urinary excretion of N ε-carboxymethyl-lysine increased with increasing proportion of heat-treated milk powder in the HMW and LMW diets but was unrelated to gene expression. There was no difference in insulin sensitivity, F2-isoprostaglandins, food intake, water intake, body weight or body composition between the groups. In conclusion, RAGE and AGER1 expression can be influenced by a high AGE diet after only 2 weeks in proportion to MG-H1 excretion. No other short-term effects were observed.
Assuntos
Dieta/efeitos adversos , Produtos Finais de Glicação Avançada/efeitos adversos , Hexosiltransferases/metabolismo , Receptor para Produtos Finais de Glicação Avançada/agonistas , Regulação para Cima , Animais , Biomarcadores/sangue , Biomarcadores/urina , Ingestão de Energia , Produtos Finais de Glicação Avançada/administração & dosagem , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/urina , Hexosiltransferases/sangue , Hexosiltransferases/química , Hexosiltransferases/genética , Temperatura Alta/efeitos adversos , Imidazóis/urina , Imidazolinas/urina , Lisina/análogos & derivados , Lisina/urina , Masculino , Proteínas do Leite/administração & dosagem , Proteínas do Leite/efeitos adversos , Proteínas do Leite/química , Peso Molecular , Proteólise , Distribuição Aleatória , Ratos Sprague-Dawley , Receptor para Produtos Finais de Glicação Avançada/sangue , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Eliminação Renal , Testes de Toxicidade Subaguda , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
To characterize mechanisms responsible for fat accumulation we used a selectively bred obesity-prone (OP) and obesity-resistant (OR) rat model where the rats were fed a Western diet for 76 days. Body composition was assessed by magnetic resonance imaging scans, and as expected, the OP rats developed a higher degree of fat accumulation compared with OR rats. Indirect calorimetry showed that the OP rats had higher respiratory exchange ratio (RER) compared with OR rats, indicating an impaired ability to oxidize fat. The OP rats had lower expression of carnitine palmitoyltransferase 1b in intra-abdominal fat, and higher expression of stearoyl-CoA desaturase 1 in subcutaneous fat compared with OR rats, which could explain the higher fat accumulation and RER values. Basal metabolic parameters were also examined in juvenile OP and OR rats before and during the introduction of the Western diet. Juvenile OP rats likewise had higher RER values, indicating that this trait may be a primary and contributing factor to their obese phenotype. When the adult obese rats were exposed to the orexigenic and adipogenic hormone ghrelin, we observed increased RER values in both OP and OR rats, while OR rats were more sensitive to the orexigenic effects of ghrelin as well as ghrelin-induced attenuation of activity and energy expenditure. Thus increased fat accumulation characterizing obesity may be caused by impaired oxidative capacity due to decreased carnitine palmitoyltransferase 1b levels in the white adipose tissue, whereas ghrelin sensitivity did not seem to be a contributing factor.
Assuntos
Adiposidade , Carnitina O-Palmitoiltransferase/metabolismo , Metabolismo Energético , Gordura Intra-Abdominal/enzimologia , Obesidade/enzimologia , Gordura Subcutânea/enzimologia , Adiposidade/efeitos dos fármacos , Animais , Calorimetria Indireta , Carnitina O-Palmitoiltransferase/genética , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação para Baixo , Ingestão de Alimentos , Metabolismo Energético/efeitos dos fármacos , Regulação da Expressão Gênica , Grelina/administração & dosagem , Hipotálamo/enzimologia , Insulina/sangue , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/fisiopatologia , Leptina/sangue , Imageamento por Ressonância Magnética , Masculino , Músculo Esquelético/enzimologia , Obesidade/sangue , Obesidade/etiologia , Obesidade/genética , Obesidade/fisiopatologia , Oxirredução , Ratos , Gordura Subcutânea/efeitos dos fármacos , Gordura Subcutânea/fisiopatologia , Fatores de Tempo , Aumento de PesoRESUMO
Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti-diabetic and anti-obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC-MS. Native NMU was found to be rapidly cleaved at the C-terminus between Arg(24) and Asn(25) , followed by cleavage between Arg(16) and Gly(17) . Lipidated NMU analogs were generated using solid-phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293-based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor-interacting C-terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose-dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Neuropeptídeos/síntese química , Neuropeptídeos/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Células HEK293 , Humanos , Masculino , Camundongos , Neuropeptídeos/sangue , Neuropeptídeos/química , Estabilidade ProteicaRESUMO
BACKGROUND: Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. METHODS: In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail suspension test, and light/dark test. We also investigated whether lack of GPR39 would change levels of cAMP response element-binding protein (CREB),brain-derived neurotrophic factor (BDNF) and tropomyosin related kinase B (TrkB) protein in the hippocampus and frontal cortex of GPR39 knockout mice subjected to the forced swim test, as measured by Western-blot analysis. RESULTS: In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB and BDNF levels in the hippocampus, but not in the frontal cortex, which indicates region specificity for the impaired CREB/BDNF pathway (which is important in antidepressant response) in the absence of GPR39. There were no changes in TrkB protein in either structure. In the present study, we also investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. CONCLUSIONS: There were no changes in the GPR39 knockout mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn(2+)-sensing receptor in the pathophysiology of depression with component of anxiety.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação a CREB/metabolismo , Depressão , Regulação para Baixo/genética , Hipocampo/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Animais , Corticosterona/sangue , Adaptação à Escuridão/genética , Depressão/genética , Depressão/metabolismo , Depressão/patologia , Modelos Animais de Doenças , Elevação dos Membros Posteriores , Resposta de Imobilidade Tônica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Receptor trkB/metabolismo , Receptores Acoplados a Proteínas G/genética , Natação/psicologia , Fatores de Tempo , Zinco/metabolismoRESUMO
Midbrain dopaminergic neurons (DANs) are subject to extensive metabotropic regulation, but the repertoire of G protein-coupled receptors (GPCRs) present in these neurons has not been mapped. Here, we isolate DANs from Dat-eGFP mice to generate a GPCR atlas by unbiased qPCR array expression analysis of 377 GPCRs. Combined with data mining of scRNA-seq databases, we identify multiple receptors in DAN subpopulations with 38 of these receptors representing the majority of transcripts. We identify 41 receptors expressed in midbrain DANs but not in non-DAN midbrain cells, including the free fatty acid receptor 4 (FFAR4). Functional expression of FFAR4 is validated by ex vivo Ca2+ imaging, and in vivo experiments support that FFAR4 negatively regulates food and water intake and bodyweight. In addition to providing a critical framework for understanding metabotropic DAN regulation, our data suggest fatty acid sensing by FFAR4 as a mechanism linking high-energy intake to the dopamine-reward pathway.
Assuntos
Neurônios Dopaminérgicos , Receptores Acoplados a Proteínas G , Animais , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Neurônios Dopaminérgicos/metabolismo , Camundongos , Ingestão de Alimentos , Ingestão de Líquidos , Camundongos Endogâmicos C57BL , Masculino , Mesencéfalo/metabolismoRESUMO
In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the ß(2)-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides â¼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the ß(2)-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.