RESUMO
GPR139 is an orphan G-protein-coupled receptor that is expressed in restricted areas of the nervous system, including the hypothalamus. In this study, we hypothesized that GPR139 could be involved in the regulation of energy balance and metabolism. In the first part of the study, we confirmed that GPR139 is expressed in the hypothalamus and particularly in proopiomelanocortin and agouti-related peptide neurons of the mediobasal hypothalamus. Using a lentivirus with a short-hairpin RNA, we inhibited the expression of GPR139 bilaterally in the mediobasal hypothalamus of mice. The intervention promoted a 40% reduction in the hypothalamic expression of GPR139, which was accompanied by an increase in body mass, a reduction in fasting blood glucose levels, and an increase in insulin levels. In the hypothalamus, inhibition of GPR139 was accompanied by a reduction in the expression of orexin. As previous studies using a pharmacological antagonist of orexin showed a beneficial impact on type 2 diabetes and glucose metabolism, we propose that the inhibition of hypothalamic GPR139 could be acting indirectly through the orexin system to control systemic glucose and insulin. In conclusion, this study advances the characterization of GPR139 in the hypothalamus, demonstrating its involvement in the regulation of body mass, blood insulin, and glycemia.
Assuntos
Diabetes Mellitus Tipo 2 , Insulina , Camundongos , Animais , Orexinas/metabolismo , Insulina/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hipotálamo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas do Tecido Nervoso/metabolismoRESUMO
BACKGROUND: The hypothalamic proopiomelanocortin (Pomc) neurons act as first-order sensors of systemic energy stores, providing signals that regulate caloric intake and energy expenditure. In experimental obesity, dietary saturated fatty acids affect Pomc endopeptidases (PCs), resulting in the abnormal production of the neurotransmitters α-melanocyte-stimulating hormone (α-MSH) and ß-endorphin, thus impacting energy balance. The cAMP response element-binding protein (CREB) is one of the transcription factors that control the expression of Pomc endopeptidases; however, it was previously unknown if dietary fats could affect CREB and consequently the expression of Pomc endopeptidases. METHODS: Here, we used single-cell RNA sequencing analysis, PCR, immunoblot, ELISA and immunofluorescence histological assays to determine the impact of a high-fat diet (HFD) on the expression and function of hypothalamic CREB and its impact on the melanocortinergic system. RESULTS: The results indicate that CREB is expressed in arcuate nucleus Pomc neurons and is activated as early as nine hours after the introduction of a high-fat diet. The inhibition of hypothalamic CREB using a short-hairpin RNA lentiviral vector resulted in increased diet-induced body-mass gain and reduced energy expenditure. This was accompanied by reduced expression of the Pomc endopeptidases, protein convertase 2, which are encoded by Pcsk2, and by the loss of the high-fat-diet-induced effect to inhibit the production of α-MSH. CONCLUSIONS: This study provides the first evidence for the involvement of CREB in the abnormal regulation of the hypothalamic Pomc endopeptidase system in experimental obesity.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Pró-Opiomelanocortina , Dieta Hiperlipídica , Endopeptidases , Humanos , Obesidade/metabolismo , Pró-Opiomelanocortina/genética , Pró-Proteína Convertase 2 , alfa-MSH/farmacologiaRESUMO
Currently, up to 35% off all drugs approved for the treatment of human diseases belong to the G-protein-coupled receptor (GPCR) family. Out of the almost 800 existing GPCRs, 25% have no known endogenous ligands and are regarded as orphan receptors; many of these are currently under investigation as potential pharmacological targets. Here, we hypothesised that orphan GPCRs expressed in the hypothalamus could be targets for the treatment of obesity and other metabolic diseases. Using bioinformatic tools, we identified 78 class A orphan GPCRs that are expressed in the hypothalamus of mice. Initially, we selected two candidates and determined their responsivities to nutritional interventions: GPR162, the GPCR with highest expression in the hypothalamus, and GPR68, a GPCR with intermediate expression in the hypothalamus and that has never been explored for its potential involvement in metabolic regulation. GPR162 expression was not modified by fasting/feeding or by the consumption of a high-fat diet, and was therefore not subsequently evaluated. Conversely, GPR68 expression increased in response to the consumption of a high-fat diet and reduced under fasting conditions. Using immunofluorescence, GPR68 was identified in both proopiomelanocortin-expressing and agouti-related peptide-expressing neurons in the hypothalamic arcuate nucleus. Acute inhibition of GPR68 with an allosteric modulator promoted an increase in the expression of the orexigenic agouti-related peptide and neuropeptide Y, whereas 4- and 12-h inhibition of GPR68 resulted in increased caloric intake. Thus, GPR68 has emerged as an orphan GPCR that is expressed in the hypothalamus and is involved in the regulation of feeding.
Assuntos
Núcleo Arqueado do Hipotálamo , Hipotálamo , Receptores Acoplados a Proteínas G , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Hipotálamo/metabolismo , Camundongos , Neuropeptídeo Y/metabolismo , Pró-Opiomelanocortina/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Nescient helix-loop-helix 2 (NHLH2) is a hypothalamic transcription factor that controls the expression of prohormone convertase 1/3, therefore having an impact on the processing of proopiomelanocortin and thus on energy homeostasis. Studies have shown that KO of Nhlh2 results in increased body mass, reduced physical activity, and hypogonadism. In humans, a polymorphism of the NHLH2 gene is associated with obesity; and in Prader-Willi syndrome, a condition characterized by obesity, hypogonadism and behavioral abnormalities, the expression of NHLH2 is reduced. Despite clinical and experimental evidence suggesting that NHLH2 could be a good target for the treatment of obesity, no previous study has evaluated the impact of NHLH2 overexpression in obesity. Here, in mice fed a high-fat diet introduced right after the arcuate nucleus intracerebroventricular injection of a lentivirus that promoted 40% increase in NHLH2, there was prevention of the development of obesity by a mechanism dependent on the reduction of caloric intake. When hypothalamic overexpression of NHLH2 was induced in previously obese mice, the beneficial impact on obesity-associated phenotype was even greater; thus, there was an 80% attenuation in body mass gain, reduced whole-body adiposity, increased brown adipose tissue temperature, reduced hypothalamic inflammation, and reduced liver steatosis. In this setting, the beneficial impact of hypothalamic overexpression of NHLH2 was a result of combined effects on caloric intake, energy expenditure, and physical activity. Moreover, the hypothalamic overexpression of NHLH2 reduced obesity-associated anxiety/depression behavior. Thus, we provide an experimental proof of concept supporting that hypothalamic NHLH2 is a good target for the treatment of obesity.SIGNIFICANCE STATEMENT Obesity is a highly prevalent medical condition that lacks an effective treatment. The main advance provided by this study is the demonstration of the beneficial metabolic and behavioral outcomes resulting from the overexpression of NHLH2 in the hypothalamus. When NHLH2 was overexpressed simultaneously with the introduction of a high-fat diet, there was prevention of obesity by a mechanism dependent on reduced caloric intake. Conversely, when NHLH2 was overexpressed in previously obese mice, there was reduction of the obese phenotype because of a combination of reduced caloric intake, increased physical activity, and increased thermogenesis. In addition, the overexpression of NHLH2 reduced anxiety/depression-like behavior. Thus, NHLH2 emerges as a potential target for the combined treatment of obesity and its associated anxiety/depression-like behavior.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Obesidade/metabolismo , Animais , Ansiedade/metabolismo , Índice de Massa Corporal , Depressão/metabolismo , Dieta Hiperlipídica/efeitos adversos , Feminino , Masculino , Camundongos , Obesidade/psicologiaRESUMO
Currently, experimental animals are widely used in biological and medical research. However, the scientific community has raised several bioethical concerns, such as the number of animals required to achieve reproducible and statistically relevant results. These concerns involve aspects related to pain, discomfort, and unwanted animal loss. Retrospectively, we compare two different approaches for anesthesia dosage: a mobile app for dose calculation and a standard dose calculation. A total of 939 C57BL/6J and Swiss mice were analyzed. We collected data on intraoperative and anesthesia-related mortality as described in electronic or physical handwritten records. Our results showed that the mobile app approach significantly reduces anesthetic-related deaths upon using doses of ketamine and xylazine. The results suggest that anesthesia-related mortality can be minimized even more using information technology approaches, helping to solve an old but transversal challenge for researchers working with experimental mice. The mobile app is a free and open code which could be implemented worldwide as an essential requirement for all anesthetic procedures in mice using xylazine and ketamine combination. As an open code app, the Labinsane initiative could also represent the starting point to unify and validate other anesthetic procedures in different species and strains.
RESUMO
Glutamic acid is the main excitatory neurotransmitter acting both in the brain and in peripheral tissues. Abnormal distribution of glutamic acid receptors occurs in skin hyperproliferative conditions such as psoriasis and skin regeneration; however, the biological function of glutamic acid in the skin remains unclear. Using ex vivo, in vivo and in silico approaches, we showed that exogenous glutamic acid promotes hair growth and keratinocyte proliferation. Topical application of glutamic acid decreased the expression of genes related to apoptosis in the skin, whereas glutamic acid increased cell viability and proliferation in human keratinocyte cultures. In addition, we identified the keratinocyte glutamic acid excitotoxic concentration, providing evidence for the existence of a novel skin signalling pathway mediated by a neurotransmitter that controls keratinocyte and hair follicle proliferation. Thus, glutamic acid emerges as a component of the peripheral nervous system that acts to control cell growth in the skin. These results raise the perspective of the pharmacological and nutritional use of glutamic acid to treat skin diseases.
Assuntos
Ácido Glutâmico/farmacologia , Folículo Piloso/efeitos dos fármacos , Cabelo/efeitos dos fármacos , Fenômenos Fisiológicos da Pele , Pele/efeitos dos fármacos , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Simulação por Computador , Desenvolvimento de Medicamentos , Fibroblastos/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Queratinócitos/citologia , Masculino , Camundongos , Mapeamento de Interação de Proteínas , Regeneração , Transdução de Sinais , Pele/metabolismoRESUMO
Under high-fat feeding, the hypothalamus atypically undergoes pro-inflammatory signaling activation. Recent data from transcriptomic analysis of microglia from rodents and humans has allowed the identification of several microglial subpopulations throughout the brain. Numerous studies have clarified the roles of these cells in hypothalamic inflammation, but how each microglial subset plays its functions upon inflammatory stimuli remains unexplored. Fortunately, these data unveiling microglial heterogeneity have triggered the development of novel experimental models for studying the roles and characteristics of each microglial subtype. In this review, we explore microglial heterogeneity in the hypothalamus and their crosstalk with astrocytes under high fat diet-induced inflammation. We present novel currently available ex vivo and in vivo experimental models that can be useful when designing a new research project in this field of study. Last, we examine the transcriptomic data already published to identify how the hypothalamic microglial signature changes upon short-term and prolonged high-fat feeding.
Assuntos
Astrócitos/patologia , Dieta Hiperlipídica/efeitos adversos , Hipotálamo/patologia , Inflamação/patologia , Microglia/patologia , Transcriptoma , Animais , Astrócitos/metabolismo , Humanos , Hipotálamo/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Microglia/metabolismoRESUMO
Obesity in menopausal women occurs because of the systemic effects of loss of ovarian function, resulting in increased body weight and oxidative stress. Caloric restriction (CR) is essential for weight loss, since it provides benefits associated with metabolic normalization resulting from the action of sirtuins. The aim of this work was to evaluate the physiological effects of weight cycling in ovariectomized females. Females aged 2 mo (n = 8/group) were submitted to simulated surgery, ovariectomy (OVX group), and ovariectomy with weight fluctuation (WF group). In the WF group, weight cycling was performed two times, using 21 days of ad libitum commercial feed and 21 days of caloric restriction with 40% of the feed consumed by the OVX group. After 17 wk, the animals were evaluated experimentally. Weight fluctuations reduced triacylglycerol and the adipose tissue index of the WF animals, while increasing the expression of antioxidant proteins. In addition to causing fluctuations in the physiological parameters, the weight cycling led to increases of adipocyte number and serum fatty acids. These effects were reflected in increased expression of the sirtuin (SIRT) 1 and SIRT4 proteins, as well as protein complexes of the mitochondrial electron transport chain, especially in the liver and adipose tissues. The weight-cycling results suggested that mitochondrial and nuclear sirtuins were active in cellular signaling for the control of lipid metabolism, oxidative phosphorylation, and redox status. Weight cycling was able to restore the health characteristics of lean animals.
Assuntos
Ovariectomia , Sirtuína 1/fisiologia , Aumento de Peso/fisiologia , Redução de Peso/fisiologia , Adipócitos , Tecido Adiposo/metabolismo , Animais , Antioxidantes/metabolismo , Restrição Calórica , Ingestão de Alimentos , Ácidos Graxos não Esterificados/sangue , Feminino , Metabolismo dos Lipídeos/genética , Ratos , Ratos Wistar , Sirtuína 1/genética , Triglicerídeos/metabolismoRESUMO
Hypothalamic adult neurogenesis provides the basis for renewal of neurons involved in the regulation of whole-body energy status. In addition to hormones, cytokines and growth factors, components of the diet, particularly fatty acids, have been shown to stimulate hypothalamic neurogenesis; however, the mechanisms behind this action are unknown. Here, we hypothesized that GPR40 (FFAR1), the receptor for medium and long chain unsaturated fatty acids, could mediate at least part of the neurogenic activity in the hypothalamus. We show that a GPR40 ligand increased hypothalamic cell proliferation and survival in adult mice. In postnatal generated neurospheres, acting in synergy with brain-derived neurotrophic factor (BDNF) and interleukin 6, GPR40 activation increased the expression of doublecortin during the early differentiation phase and of the mature neuronal marker, microtubule-associated protein 2 (MAP2), during the late differentiation phase. In Neuro-2a proliferative cell-line GPR40 activation increased BDNF expression and p38 activation. The chemical inhibition of p38 abolished GPR40 effect in inducing neurogenesis markers in neurospheres, whereas BDNF immunoneutralization inhibited GPR40-induced cell proliferation in the hypothalamus of adult mice. Thus, GPR40 acts through p38 and BDNF to induce hypothalamic neurogenesis. This study provides mechanistic advance in the understating of how a fatty acid receptor regulates adult hypothalamic neurogenesis.