RESUMO
Ghrelin is a metabolic signal regulating energy homeostasis. Circulating ghrelin levels rise during starvation and fall after a meal, and therefore, ghrelin may function as a signal of negative energy balance. Ghrelin may also act as a modulator of reproductive physiology, as acute ghrelin administration suppresses gonadotropin secretion and inhibits the neuroendocrine reproductive axis. Interestingly, ghrelin's effect in female metabolism varies according to the estrogen milieu predicting an interaction between ghrelin and estrogens, likely at the hypothalamic level. Here, we show that ghrelin receptor (GHSR) and estrogen receptor-α (ERα) are coexpressed in several hypothalamic sites. Higher levels of circulating estradiol increased the expression of GHSR mRNA and the coexpression of GHSR mRNA and ERα selectively in the arcuate nucleus (ARC). Subsets of preoptic and ARC Kiss1 neurons coexpressed GHSR. Increased colocalization was observed in ARC Kiss1 neurons of ovariectomized estradiol-treated (OVX + E2; 80%) compared with ovariectomized oil-treated (OVX; 25%) mice. Acute actions of ghrelin on ARC Kiss1 neurons were also modulated by estradiol; 75 and 22% of Kiss1 neurons of OVX + E2 and OVX mice, respectively, depolarized in response to ghrelin. Our findings indicate that ghrelin and estradiol may interact in several hypothalamic sites. In the ARC, high levels of E2 increase GHSR mRNA expression, modifying the colocalization rate with ERα and Kiss1 and the proportion of Kiss1 neurons acutely responding to ghrelin. Our findings indicate that E2 alters the responsiveness of kisspeptin neurons to metabolic signals, potentially acting as a critical player in the metabolic control of the reproductive physiology.
Assuntos
Estradiol/metabolismo , Receptor alfa de Estrogênio/agonistas , Grelina/metabolismo , Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Neurônios/metabolismo , Receptores de Grelina/agonistas , Acilação , Animais , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Estradiol/sangue , Estradiol/uso terapêutico , Receptor alfa de Estrogênio/metabolismo , Terapia de Reposição de Estrogênios , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Grelina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipotálamo/efeitos dos fármacos , Kisspeptinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Ovariectomia/efeitos adversos , Ratos , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The Jingle fallacy is the false assumption that instruments which share the same name measure the same underlying construct. In this experiment, we focus on the comprehension subtests of the Nelson Denny Reading Test (NDRT) and the Wechsler Individual Achievement Test (WIAT-II). 91 university students read passages for comprehension whilst their eye movements were recorded. Participants took part in two experimental blocks of which the order was counterbalanced, one with higher comprehension demands and one with lower comprehension demands. We assumed that tests measuring comprehension would be able to predict differences observed in eye movement patterns as a function of varying comprehension demands. Overall, readers were able to adapt their reading strategy to read more slowly, making more and longer fixations, coupled with shorter saccades when comprehension demands were higher. Within an experimental block, high scorers on the NDRT were able to consistently increase their pace of reading over time for both higher and lower comprehension demands, whereas low scorers approached a threshold where they could not continue to increase their reading speed or further reduce the number of fixations to read a text, even when comprehension demands were low. Individual differences based on the WIAT-II did not explain similar patterns. The NDRT comprehension test was therefore more predictive of differences in the reading patterns of skilled adult readers in response to comprehension demands than the WIAT-II (which also suffered from low reliability). Our results revealed that these different comprehension measures should not be used interchangeably, and researchers should be cautious when choosing reading comprehension tests for research.
Assuntos
Compreensão , Movimentos Oculares , Leitura , Humanos , Compreensão/fisiologia , Feminino , Masculino , Adulto Jovem , Movimentos Oculares/fisiologia , Adulto , AdolescenteRESUMO
When a preview contains substituted letters (SL; markey) word identification is more disrupted for a target word (monkey), compared to when the preview contains transposed letters (TL; mnokey). The transposed letter effect demonstrates that letter positions are encoded more flexibly than letter identities, and is a robust finding in adults. However, letter position encoding has been shown to gradually become more flexible as reading skills develop. It is unclear whether letter position encoding flexibility reaches maturation in skilled adult readers, or whether some differences in the magnitude of the TL effect remain in relation to individual differences in cognitive skills. We examined 100 skilled adult readers who read sentences containing a correct, TL or SL preview. Previews were replaced by the correct target word when the reader's gaze triggered an invisible boundary. Cognitive skills were assessed and grouped based on overlapping variance via Principal Components Analysis (PCA) and subsequently used to predict eye movement measures for each condition. Consistent with previous literature, adult readers were found to generally encode letter position more flexibly than letter identity. Very few differences were found in the magnitude of TL effects between adults based on individual differences in cognitive skills. The flexibility of letter position encoding appears to reach maturation (or near maturation) in skilled adult readers.
Assuntos
Individualidade , Leitura , Reconhecimento Visual de Modelos , Movimentos Oculares , IdiomaRESUMO
BACKGROUND: Microbial functioning on marine plastic surfaces has been poorly documented, especially within cold climates where temperature likely impacts microbial activity and the presence of hydrocarbonoclastic microorganisms. To date, only two studies have used metaproteomics to unravel microbial genotype-phenotype linkages in the marine 'plastisphere', and these have revealed the dominance of photosynthetic microorganisms within warm climates. Advancing the functional representation of the marine plastisphere is vital for the development of specific databases cataloging the functional diversity of the associated microorganisms and their peptide and protein sequences, to fuel biotechnological discoveries. Here, we provide a comprehensive assessment for plastisphere metaproteomics, using multi-omics and data mining on thin plastic biofilms to provide unique insights into plastisphere metabolism. Our robust experimental design assessed DNA/protein co-extraction and cell lysis strategies, proteomics workflows, and diverse protein search databases, to resolve the active plastisphere taxa and their expressed functions from an understudied cold environment. RESULTS: For the first time, we demonstrate the predominance and activity of hydrocarbonoclastic genera (Psychrobacter, Flavobacterium, Pseudomonas) within a primarily heterotrophic plastisphere. Correspondingly, oxidative phosphorylation, the citrate cycle, and carbohydrate metabolism were the dominant pathways expressed. Quorum sensing and toxin-associated proteins of Streptomyces were indicative of inter-community interactions. Stress response proteins expressed by Psychrobacter, Planococcus, and Pseudoalteromonas and proteins mediating xenobiotics degradation in Psychrobacter and Pseudoalteromonas suggested phenotypic adaptations to the toxic chemical microenvironment of the plastisphere. Interestingly, a targeted search strategy identified plastic biodegradation enzymes, including polyamidase, hydrolase, and depolymerase, expressed by rare taxa. The expression of virulence factors and mechanisms of antimicrobial resistance suggested pathogenic genera were active, despite representing a minor component of the plastisphere community. CONCLUSION: Our study addresses a critical gap in understanding the functioning of the marine plastisphere, contributing new insights into the function and ecology of an emerging and important microbial niche. Our comprehensive multi-omics and comparative metaproteomics experimental design enhances biological interpretations to provide new perspectives on microorganisms of potential biotechnological significance beyond biodegradation and to improve the assessment of the risks associated with microorganisms colonizing marine plastic pollution. Video Abstract.
Assuntos
Microbiota , Plásticos , Bactérias/genética , Multiômica , Biofilmes , Biodegradação Ambiental , Microbiota/genéticaRESUMO
A subset of neurons in the brain, known as 'glucose-excited' neurons, depolarize and increase their firing rate in response to increases in extracellular glucose. Similar to insulin secretion by pancreatic beta-cells, glucose excitation of neurons is driven by ATP-mediated closure of ATP-sensitive potassium (K(ATP)) channels. Although beta-cell-like glucose sensing in neurons is well established, its physiological relevance and contribution to disease states such as type 2 diabetes remain unknown. To address these issues, we disrupted glucose sensing in glucose-excited pro-opiomelanocortin (POMC) neurons via transgenic expression of a mutant Kir6.2 subunit (encoded by the Kcnj11 gene) that prevents ATP-mediated closure of K(ATP) channels. Here we show that this genetic manipulation impaired the whole-body response to a systemic glucose load, demonstrating a role for glucose sensing by POMC neurons in the overall physiological control of blood glucose. We also found that glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, suggesting that loss of glucose sensing by neurons has a role in the development of type 2 diabetes. The mechanism for obesity-induced loss of glucose sensing in POMC neurons involves uncoupling protein 2 (UCP2), a mitochondrial protein that impairs glucose-stimulated ATP production. UCP2 negatively regulates glucose sensing in POMC neurons. We found that genetic deletion of Ucp2 prevents obesity-induced loss of glucose sensing, and that acute pharmacological inhibition of UCP2 reverses loss of glucose sensing. We conclude that obesity-induced, UCP2-mediated loss of glucose sensing in glucose-excited neurons might have a pathogenic role in the development of type 2 diabetes.
Assuntos
Glucose/metabolismo , Homeostase , Neurônios/metabolismo , Obesidade/fisiopatologia , Pró-Opiomelanocortina/metabolismo , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Humanos , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Canais Iônicos/metabolismo , Glicosídeos Iridoides , Iridoides/farmacologia , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Obesidade/induzido quimicamente , Obesidade/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteína Desacopladora 2RESUMO
Evidence suggests that the role played by the adipocyte-derived hormone leptin in female reproductive physiology is mediated in part by neurons located within the ventral premammillary nucleus (PMV). Leptin activates PMV neurons; however, the intracellular signaling pathway and channel(s) involved remain undefined. Notably, leptin's excitatory and inhibitory effects within hypothalamic and brainstem nuclei share the intracellular signaling cascade phosphoinositide 3 kinase (PI3K). Therefore, we assessed whether PI3K signaling is required for the acute effect of leptin to alter cellular activity of PMV neurons that express leptin receptors (LepR PMV neurons). Leptin caused a rapid depolarization in the majority of LepR PMV neurons in patch-clamp recordings of hypothalamic slices, while a subset of LepR PMV neurons were hyperpolarized in response to leptin. Data were obtained from both male and female mice and results demonstrate that the acute effect of leptin on LepR PMV neurons was identical for both sexes. Pharmacological inhibition of PI3K prevented the acute leptin-induced change in neuronal activity of LepR PMV neurons, indicating a PI3K-dependent mechanism of leptin action. Similarly, mice with genetically disrupted PI3K signaling in LepR PMV neurons failed to alter cellular activity in response to leptin. Moreover, the leptin-induced depolarization was dependent on a putative TRPC channel. In contrast, the leptin-induced-hyperpolarization required the activation of a putative Katp channel. Collectively, these results suggest that PI3K signaling in LepR PMV neurons is essential for leptin-induced alteration in cellular activity, and these data may suggest a cellular correlate in which leptin contributes to the initiation of reproductive development.
Assuntos
Hipotálamo/fisiologia , Leptina/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais/fisiologia , Androstadienos/farmacologia , Animais , Compostos de Boro/farmacologia , Cromonas/farmacologia , Feminino , Genes Reporter/genética , Hipotálamo/efeitos dos fármacos , Hipotálamo/enzimologia , Imidazóis/farmacologia , Canais KATP/fisiologia , Leptina/administração & dosagem , Leptina/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfolinas/farmacologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Fosfatidilinositol 3-Quinases/biossíntese , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Subunidades Proteicas/biossíntese , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/fisiologia , WortmaninaRESUMO
The importance of neuropeptides in the hypothalamus has been experimentally established. Due to difficulties in assessing function in vivo, the roles of the fast-acting neurotransmitters glutamate and GABA are largely unknown. Synaptic vesicular transporters (VGLUTs for glutamate and VGAT for GABA) are required for vesicular uptake and, consequently, synaptic release of neurotransmitters. Ventromedial hypothalamic (VMH) neurons are predominantly glutamatergic and express VGLUT2. To evaluate the role of glutamate release from VMH neurons, we generated mice lacking VGLUT2 selectively in SF1 neurons (a major subset of VMH neurons). These mice have hypoglycemia during fasting secondary to impaired fasting-induced increases in the glucose-raising pancreatic hormone glucagon and impaired induction in liver of mRNAs encoding PGC-1alpha and the gluconeogenic enzymes PEPCK and G6Pase. Similarly, these mice have defective counterregulatory responses to insulin-induced hypoglycemia and 2-deoxyglucose (an antimetabolite). Thus, glutamate release from VMH neurons is an important component of the neurocircuitry that functions to prevent hypoglycemia.
Assuntos
Ácido Glutâmico/metabolismo , Hipoglicemia/metabolismo , Hipotálamo/citologia , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Eletrofisiologia , Glucagon/metabolismo , Glucose-6-Fosfatase/metabolismo , Hibridização In Situ , Insulina , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transativadores/metabolismo , Fatores de Transcrição , Proteína Vesicular 2 de Transporte de Glutamato/genéticaRESUMO
Acute leptin administration results in a depolarization and concomitant increase in the firing rate of a subpopulation of arcuate proopiomelanocortin (POMC) cells. This rapid activation of POMC cells has been implicated as a cellular correlate of leptin effects on energy balance. In contrast to leptin, insulin inhibits the activity of some POMC neurons. Several studies have described a "cross talk" between leptin and insulin within the mediobasal hypothalamus via the intracellular enzyme, phosphoinositol-3-kinase (PI3K). Interestingly, both insulin and leptin regulate POMC cellular activity by activation of PI3K; however, it is unclear whether leptin and insulin effects are observed in similar or distinct populations of POMC cells. We therefore used dual label immunohistochemistry/in situ hybridization and whole-cell patch-clamp electrophysiology to map insulin and leptin responsive arcuate POMC neurons. Leptin-induced Fos activity within arcuate POMC neurons was localized separate from POMC neurons that express insulin receptor. Moreover, acute responses to leptin and insulin were largely segregated in distinct subpopulations of POMC cells. Collectively, these data suggest that cross talk between leptin and insulin occurs within a network of cells rather than within individual POMC neurons.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Leptina/farmacologia , Neurônios/classificação , Neurônios/efeitos dos fármacos , Pró-Opiomelanocortina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Proteínas de Fluorescência Verde/genética , Hipotálamo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Oncogênicas v-fos/genética , Proteínas Oncogênicas v-fos/metabolismo , Técnicas de Patch-Clamp/métodos , Pró-Opiomelanocortina/genética , RNA Mensageiro/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
Suppressor of cytokine signaling-3 (Socs-3) negatively regulates the action of various cytokines, as well as the metabolic hormones leptin and insulin. Mice with haploinsufficiency of Socs-3, or those with neuronal deletion of Socs-3, are lean and more leptin and insulin sensitive. To examine the role of Socs-3 within specific neurons critical to energy balance, we created mice with selective deletion of Socs-3 within pro-opiomelanocortin (POMC)-expressing cells. These mice had enhanced leptin sensitivity, measured by weight loss and food intake after leptin infusion. On chow diet, glucose homeostasis was improved despite normal weight gain. On a high-fat diet, the rate of weight gain was reduced, due to increased energy expenditure rather than decreased food intake; glucose homeostasis and insulin sensitivity were substantially improved. These studies demonstrate that Socs-3 within POMC neurons regulates leptin sensitivity and glucose homeostasis, and plays a key role in linking high-fat diet to disordered metabolism.
Assuntos
Gorduras na Dieta/farmacologia , Glucose/metabolismo , Homeostase , Leptina/farmacologia , Pró-Opiomelanocortina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Animais , Células Cultivadas , Fígado Gorduroso/prevenção & controle , Camundongos , Camundongos Knockout , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Redução de PesoRESUMO
The anorexigenic effect of serotonergic compounds has largely been attributed to activation of serotonin 2C receptors (Htr2cs). Using mouse genetic models in which Htr2c can be selectively deleted or restored (in Htr2c-null mice), we investigate the role of Htr2c in forebrain Sim1 neurons. Unexpectedly, we find that Htr2c acts in these neurons to promote food intake and counteract the anorectic effect of serotonergic appetite suppressants. Furthermore, Htr2c marks a subset of Sim1 neurons in the paraventricular nucleus of the hypothalamus (PVH). Chemogenetic activation of these neurons in adult mice suppresses hunger, whereas their silencing promotes feeding. In support of an orexigenic role of PVH Htr2c, whole-cell patch-clamp experiments demonstrate that activation of Htr2c inhibits PVH neurons. Intriguingly, this inhibition is due to Gαi/o-dependent activation of ATP-sensitive K+ conductance, a mechanism of action not identified previously in the mammalian nervous system.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Animais , Anorexia , Depressores do Apetite/metabolismo , Depressores do Apetite/farmacologia , Metabolismo Energético/fisiologia , Comportamento Alimentar/fisiologia , Fome/fisiologia , Hipotálamo/metabolismo , Hipotálamo/fisiologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Neurônios/fisiologia , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/fisiologia , Potássio/metabolismo , Receptor 5-HT2C de Serotonina/genética , Serotonina/metabolismo , Serotonina/farmacologia , SerotoninérgicosRESUMO
Leptin, an adipocyte-derived hormone, acts directly on the brain to control food intake and energy expenditure. An important question is the identity of first-order neurons initiating leptin's anti-obesity effects. A widely held view is that most, if not all, of leptin's effects are mediated by neurons located in the arcuate nucleus of the hypothalamus. However, leptin receptors (LEPRs) are expressed in other sites as well, including the ventromedial hypothalamus (VMH). The possible role of leptin acting in "nonarcuate" sites has largely been ignored. In the present study, we show that leptin depolarizes and increases the firing rate of steroidogenic factor-1 (SF1)-positive neurons in the VMH. We also show, by generating mice that lack LEPRs on SF1-positive neurons, that leptin action at this site plays an important role in reducing body weight and, of note, in resisting diet-induced obesity. These results reveal a critical role for leptin action on VMH neurons.
Assuntos
Peso Corporal/fisiologia , Proteínas de Homeodomínio/fisiologia , Homeostase/fisiologia , Leptina/farmacologia , Neurônios/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/fisiologia , Animais , Composição Corporal/efeitos dos fármacos , Composição Corporal/genética , Composição Corporal/fisiologia , Dieta , Eletrofisiologia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Homeostase/efeitos dos fármacos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/fisiopatologia , Técnicas de Patch-Clamp , Fenótipo , Sondas RNA , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores para Leptina , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator Esteroidogênico 1 , Fatores de Transcrição/genética , Núcleo Hipotalâmico Ventromedial/citologiaRESUMO
The neural pathways through which central serotonergic systems regulate food intake and body weight remain to be fully elucidated. We report that serotonin, via action at serotonin1B receptors (5-HT1BRs), modulates the endogenous release of both agonists and antagonists of the melanocortin receptors, which are a core component of the central circuitry controlling body weight homeostasis. We also show that serotonin-induced hypophagia requires downstream activation of melanocortin 4, but not melanocortin 3, receptors. These results identify a primary mechanism underlying the serotonergic regulation of energy balance and provide an example of a centrally derived signal that reciprocally regulates melanocortin receptor agonists and antagonists in a similar manner to peripheral adiposity signals.
Assuntos
Ingestão de Alimentos/fisiologia , Neurônios/fisiologia , Receptor Tipo 3 de Melanocortina/fisiologia , Receptor 5-HT1B de Serotonina/fisiologia , Receptores de Melanocortina/fisiologia , Serotonina/fisiologia , Animais , Ingestão de Alimentos/efeitos dos fármacos , Estimulação Elétrica , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Camundongos Transgênicos , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Piridinas/farmacologia , Receptor Tipo 4 de Melanocortina/agonistas , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Receptor Tipo 4 de Melanocortina/fisiologia , Receptores de Melanocortina/agonistas , Receptores de Melanocortina/antagonistas & inibidores , Serotonina/farmacologia , Agonistas do Receptor 5-HT1 de SerotoninaRESUMO
Leptin is required for normal energy and glucose homeostasis. The hypothalamic arcuate nucleus (ARH) has been proposed as an important site of leptin action. To assess the physiological significance of leptin signaling in the ARH, we used mice homozygous for a FLPe-reactivatable, leptin receptor null allele (Lepr(neo/neo) mice). Similar to Lepr(db/db) mice, these mice are obese, hyperglycemic, hyperinsulinemic, infertile, and hypoactive. To selectively restore leptin signaling in the ARH, we generated an adeno-associated virus expressing FLPe-recombinase, which was delivered unilaterally into the hypothalamus using stereotaxic injections. We found that unilateral restoration of leptin signaling in the ARH of Lepr(neo/neo) mice leads to a modest decrease in body weight and food intake. In contrast, unilateral reactivation markedly improved hyperinsulinemia and normalized blood glucose levels and locomotor activity. These data demonstrate that leptin signaling in the ARH is sufficient for mediating leptin's effects on glucose homeostasis and locomotor activity.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Glucose/metabolismo , Hipotálamo/metabolismo , Leptina/metabolismo , Alelos , Animais , Composição Corporal , Peso Corporal , Núcleo Celular/metabolismo , DNA Nucleotidiltransferases/metabolismo , Fertilidade , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Homozigoto , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Atividade Motora , Neurônios/metabolismo , Consumo de Oxigênio , Receptores de Superfície Celular/metabolismo , Receptores para Leptina , Transdução de Sinais , Fatores de TempoRESUMO
OBJECTIVE: Histaminergic neurons of the tuberomammillary nucleus (TMN) are wake-promoting and contribute to the regulation of energy homeostasis. Evidence indicates that melanocortin 4 receptors (MC4R) are expressed within the TMN. However, whether the melanocortin system influences the activity and function of TMN neurons expressing histidine decarboxylase (HDC), the enzyme required for histamine synthesis, remains undefined. METHODS: We utilized Hdc-Cre mice in combination with whole-cell patch-clamp electrophysiology and in vivo chemogenetic techniques to determine whether HDC neurons receive metabolically relevant information via the melanocortin system. RESULTS: We found that subsets of HDC-expressing neurons were excited by melanotan II (MTII), a non-selective melanocortin receptor agonist. Use of melanocortin receptor selective agonists (THIQ, [D-Trp8]-γ-MSH) and inhibitors of synaptic transmission (TTX, CNQX, AP5) indicated that the effect was mediated specifically by MC4Rs and involved a glutamatergic dependent presynaptic mechanism. MTII enhanced evoked excitatory post-synaptic currents (EPSCs) originating from electrical stimulation of the perifornical lateral hypothalamic area (PeFLH), supportive of melanocortin effects on the glutamatergic PeFLH projection to the TMN. Finally, in vivo chemogenetic inhibition of HDC neurons strikingly enhanced the anorexigenic effects of intracerebroventricular administration of MTII, suggesting that MC4R activation of histaminergic neurons may restrain the anorexigenic effects of melanocortin system activation. CONCLUSIONS: These experiments identify a functional interaction between the melanocortin and histaminergic systems and suggest that HDC neurons act naturally to restrain the anorexigenic effect of melanocortin system activation. These findings may have implications for the control of arousal and metabolic homeostasis, especially in the context of obesity, in which both processes are subjected to alterations.
Assuntos
Histamina/metabolismo , Histidina Descarboxilase/metabolismo , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/metabolismo , Melanocortinas/metabolismo , Neurônios/metabolismo , Receptor Tipo 4 de Melanocortina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Histidina Descarboxilase/genética , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Receptor Tipo 4 de Melanocortina/agonistas , Receptor Tipo 4 de Melanocortina/genética , alfa-MSH/análogos & derivados , alfa-MSH/farmacologiaRESUMO
Ghrelin administration increases food intake, body weight (BW), adiposity, and blood glucose. In contrast, although mouse models lacking ghrelin or its receptor (Growth Hormone Secretagogue Receptor (GHSR)) exhibit life-threatening hypoglycemia in starvation-like states, they do not exhibit appreciable reductions in food intake, BW, adiposity, blood glucose, or survival when food availability is unrestricted. This suggests the existence of a parallel neuromodulatory system that can compensate for disruptions in the ghrelin system in certain settings. Here, we hypothesized that the cannabinoid CB1 receptor (CB1R) may encode this putative redundancy, and as such, that genetic deletion of both GHSR and CB1R would exaggerate the metabolic deficits associated with deletion of GHSR alone. To test this hypothesis, we assessed food intake, BW, blood glucose, survival, and plasma acyl-ghrelin in ad libitum-fed male wild-type mice and those that genetically lack GHSR (GHSR-nulls), CB1R (CB1R-nulls), or both GHSR and CB1R (double-nulls). BW, fat mass, and lean mass were similar in GHSR-nulls and wild-types, lower in CB1R-nulls, but not further reduced in double-nulls. Food intake, plasma acyl-ghrelin, and blood glucose were similar among genotypes. Deletion of either GHSR or CB1R alone did not have a statistically-significant effect on survival, but double-nulls demonstrated a statistical trend towards decreased survival (pâ¯=â¯0.07). We conclude that CB1R is not responsible for the normal BW, adiposity, food intake, and blood glucose observed in GHSR-null mice in the setting of unrestricted food availability. Nor is CB1R required for plasma acyl-ghrelin secretion in that setting. However, GHSR may be protective against exaggerated mortality associated with CB1R deletion.
Assuntos
Canabinoides , Receptores de Grelina , Animais , Peso Corporal , Ingestão de Alimentos , Grelina/análogos & derivados , Masculino , Camundongos , Receptor CB1 de Canabinoide/genética , Receptores de Grelina/genéticaRESUMO
SIRT1 is a nicotinamide adenosine dinucleotide-dependent deacetylase that orchestrates key metabolic adaptations to nutrient deprivation in peripheral tissues. SIRT1 is induced also in the brain by reduced energy intake. However, very little is known about SIRT1 distribution and the biochemical phenotypes of SIRT1-expressing cells in the neuraxis. Unknown are also the brain sites in which SIRT1 is regulated by energy availability and whether these regulations are altered in a genetic model of obesity. To address these issues, we performed in situ hybridization histochemistry analyses and found that Sirt1 mRNA is highly expressed in metabolically relevant sites. These include, but are not limited to, the hypothalamic arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the area postrema and the nucleus of the solitary tract in the hindbrain. Of note, our single-cell reverse transcription-PCR analyses revealed that Sirt1 mRNA is expressed in pro-opiomelanocortin neurons that are critical for normal body weight and glucose homeostasis. We also found that SIRT1 protein levels are restrictedly increased in the hypothalamus in the fasted brain. Of note, we found that this hypothalamic-specific, fasting-induced SIRT1 regulation is altered in leptin-deficient, obese mice. Collectively, our findings establish the distribution of Sirt1 mRNA throughout the neuraxis and suggest a previously unrecognized role of brain SIRT1 in regulating energy homeostasis.
Assuntos
Química Encefálica/fisiologia , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Metabolismo Energético/fisiologia , Sirtuínas/metabolismo , Animais , Encéfalo/fisiologia , Homeostase/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Sirtuína 1 , Sirtuínas/fisiologiaRESUMO
The PI3K-Akt-FoxO1 pathway contributes to the actions of insulin and leptin in several cell types, including neurons in the CNS. However, identifying these actions in chemically identified neurons has proven difficult. To address this problem, we have developed a reporter mouse for monitoring PI3K-Akt signaling in specific populations of neurons, based on FoxO1 nucleocytoplasmic shuttling. The reporter, FoxO1 fused to green fluorescent protein (FoxO1GFP), is expressed under the control of a ubiquitous promoter that is silenced by a loxP flanked transcriptional blocker. Thus, the expression of the reporter in selected cells is dependent on the action of Cre recombinase. Using this model, we found that insulin treatment resulted in the nuclear exclusion of FoxO1GFP within POMC and AgRP neurons in a dose- and time-dependent manner. FoxO1GFP nuclear exclusion was also observed in POMC neurons following in vivo administration of insulin. In addition, leptin induced transient nuclear export of FoxO1GFP in POMC neurons in a dose dependent manner. Finally, insulin-induced nuclear export was impaired in POMC neurons by pretreatment with free fatty acids, a paradigm known to induce insulin resistance in peripheral insulin target tissues. Thus, our FoxO1GFP mouse provides a tool for monitoring the status of PI3K-Akt signaling in a cell-specific manner under physiological and pathophysiological conditions.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Animais , Ácidos Graxos não Esterificados/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Hipotálamo/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Insulina/metabolismo , Leptina/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Ghrelin is a peptide hormone with many known functions, including orexigenic, blood glucose-regulatory, and antidepressant actions, among others. Mature ghrelin is unique in that it is the only known naturally occurring peptide to be posttranslationally modified by O-acylation with octanoate. This acylation is required for many of ghrelin's actions, including its effects on promoting increases in food intake and body weight. GOAT (ghrelin O-acyltransferase), one of 16 members of the MBOAT family of membrane-bound O-acyltransferases, has recently been identified as the enzyme responsible for catalyzing the addition of the octanoyl group to ghrelin. Although the initial reports of GOAT have localized its encoding mRNA to tissues known to contain ghrelin, it is as yet unclear whether the octanoylation occurs within ghrelin-producing cells or in neighboring cells. Here, we have performed dual-label histochemical analysis on mouse stomach sections and quantitative PCR on mRNAs from highly enriched pools of mouse gastric ghrelin cells to demonstrate a high degree of GOAT mRNA expression within ghrelin-producing cells of the gastric oxyntic mucosa. We also demonstrate that GOAT is the only member of the MBOAT family whose expression is highly enriched within gastric ghrelin cells and whose whole body distribution mirrors that of ghrelin.
Assuntos
Aciltransferases/metabolismo , Mucosa Gástrica/metabolismo , Grelina/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Aciltransferases/genética , Animais , Células Cultivadas , Duodeno/metabolismo , Grelina/genética , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Distribuição TecidualRESUMO
OBJECTIVE: The sympathetic nervous system (SNS) is a key regulator of the metabolic and endocrine functions of adipose tissue. Increased SNS outflow promotes fat mobilization, stimulates non-shivering thermogenesis, promotes browning, and inhibits leptin production. Most of these effects are attributed to norepinephrine activation of the Gs-coupled beta adrenergic receptors located on the surface of the adipocytes. Evidence suggests that other adrenergic receptor subtypes, including the Gi-coupled alpha 2 adrenergic receptors might also mediate the SNS effects on adipose tissue. However, the impact of acute stimulation of adipocyte Gs and Gi has never been reported. METHODS: We harness the power of chemogenetics to develop unique mouse models allowing the specific and spatiotemporal stimulation of adipose tissue Gi and Gs signaling. We evaluated the impact of chemogenetic stimulation of these pathways on glucose homeostasis, lipolysis, leptin production, and gene expression. RESULTS: Stimulation of Gs signaling in adipocytes induced rapid and sustained hypoglycemia. These hypoglycemic effects were secondary to increased insulin release, likely consequent to increased lipolysis. Notably, we also observed differences in gene regulation and ex vivo lipolysis in different adipose depots. In contrast, acute stimulation of Gi signaling in adipose tissue did not affect glucose metabolism or lipolysis, but regulated leptin production. CONCLUSION: Our data highlight the significance of adipose Gs signaling in regulating systemic glucose homeostasis. We also found previously unappreciated heterogeneity across adipose depots following acute stimulation. Together, these results highlight the complex interactions of GPCR signaling in adipose tissue and demonstrate the usefulness of chemogenetic technology to better understand adipocyte function.
Assuntos
Adipócitos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Glucose/metabolismo , Hipoglicemia/metabolismo , Animais , Homeostase , Insulina/metabolismo , Leptina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Phosphoinositide 3-kinase (PI3K) signaling in hypothalamic neurons integrates peripheral metabolic cues, including leptin and insulin, to coordinate systemic glucose and energy homeostasis. PI3K is composed of different subunits, each of which has several unique isoforms. However, the role of the PI3K subunits and isoforms in the ventromedial hypothalamus (VMH), a prominent site for the regulation of glucose and energy homeostasis, is unclear. Here we investigated the role of subunit p110ß in steroidogenic factor-1 (SF-1) neurons of the VMH in the regulation of metabolism. Our data demonstrate that the deletion of p110ß in SF-1 neurons disrupts glucose metabolism, rendering the mice insulin resistant. In addition, the deletion of p110ß in SF-1 neurons leads to the whitening of brown adipose tissues and increased susceptibility to diet-induced obesity due to blunted energy expenditure. These results highlight a critical role for p110ß in the regulation of glucose and energy homeostasis via VMH neurons.