RESUMO
The endocannabinoid system (ECS) is composed of a group of Gi-coupled protein receptors and enzymes, producing and degrading the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoyl-ethanolamine (AEA). Endocannabinoid-mediated signaling modulates brain functions, such as pain, mood, memory, and feeding behavior. The activation of the ECS is associated with overeating and obesity; however, the expression of components of this system has been only partially studied in the hypothalamus, a critical region implicated in feeding behavior. Within this brain region, anorexigenic, and orexigenic neurons of the arcuate nucleus (ARC) are in close contact with tanycytes, glial radial-like cells that line the lateral walls and floor of the third ventricle (3V). The specific function of tanycytes and the effects of metabolic signals generated by them on adjacent neurons is starting to be elucidated. We have proposed that the ECS within tanycytes modulates ARC neurons, thus modifying food intake. Here, we evaluated the expression and the loss of function of the 2-AG-producing enzyme, diacylglycerol lipase-alpha (DAGLα). Using Western blot and immunohistochemistry analyses in basal hypothalamus sections of adult rats under several glycemic conditions, we confirm that DAGLα is strongly expressed at the basal hypothalamus in glial and neuronal cells, increasing further in response to greater extracellular glucose levels. Using a DAGLα-inhibiting adenovirus (shRNA), suppression of DAGLα expression in tanycytes altered the usual response to intracerebroventricular glucose in terms of neuropeptides produced by neurons of the ARC. Thus, these results strongly suggest that the tanycytes could generate 2-AG, which modulates the function of anorexigenic and orexigenic neurons.
RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Background: The beta-amyloid peptide (Aß) involved in Alzheimer's disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aß in cultured hippocampal neurons. Methods: To decrease or increase the levels of cholesterol in the membrane we used methyl-ß-cyclodextrin (MßCD) and MßCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively. Results: The results showed that cholesterol removal decreased the macroscopic association of Aß to neuronal membranes (fluorescent-puncta/20 µm: control = 18 ± 2 vs. MßCD = 10 ± 1, p < 0.05) and induced a facilitation of the membrane perforation by Aß with respect to control cells (half-time for maximal charge transferred: control = 7.2 vs. MßCD = 4.4). Under this condition, we found an increase in membrane fluidity (46 ± 3.3% decrease in GP value, p < 0.001). On the contrary, increasing cholesterol levels incremented membrane rigidity (38 ± 2.7% increase in GP value, p < 0.001) and enhanced the association and clustering of Aß (fluorescent-puncta/20 µm: control = 18 ± 2 vs. MßCD = 10 ± 1, p < 0.01), but inhibited membrane disruption. Conclusion: Our results strongly support the significance of plasma membrane organization in the toxic effects of Aß in hippocampal neurons, since fluidity can regulate distribution and insertion of the Aß peptide in the neuronal membrane.
RESUMO
Glucose is a key modulator of feeding behavior. By acting in peripheral tissues and in the central nervous system, it directly controls the secretion of hormones and neuropeptides and modulates the activity of the autonomic nervous system. GLUT2 is required for several glucoregulatory responses in the brain, including feeding behavior, and is localized in the hypothalamus and brainstem, which are the main centers that control this behavior. In the hypothalamus, GLUT2 has been detected in glial cells, known as tanycytes, which line the basal walls of the third ventricle (3V). This study aimed to clarify the role of GLUT2 expression in tanycytes in feeding behavior using 3V injections of an adenovirus encoding a shRNA against GLUT2 and the reporter EGFP (Ad-shGLUT2). Efficient in vivo GLUT2 knockdown in rat hypothalamic tissue was demonstrated by qPCR and Western blot analyses. Specificity of cell transduction in the hypothalamus and brainstem was evaluated by EGFP-fluorescence and immunohistochemistry, which showed EGFP expression specifically in ependymal cells, including tanycytes. The altered mRNA levels of both orexigenic and anorexigenic neuropeptides suggested a loss of response to increased glucose in the 3V. Feeding behavior analysis in the fasting-feeding transition revealed that GLUT2-knockdown rats had increased food intake and body weight, suggesting an inhibitory effect on satiety. Taken together, suppression of GLUT2 expression in tanycytes disrupted the hypothalamic glucosensing mechanism, which altered the feeding behavior.
Assuntos
Comportamento Alimentar/fisiologia , Transportador de Glucose Tipo 2/metabolismo , Hipotálamo/metabolismo , Neuroglia/metabolismo , Saciação/fisiologia , Animais , Peso Corporal , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Células Cultivadas , Jejum/metabolismo , Técnicas de Silenciamento de Genes , Transportador de Glucose Tipo 2/genética , Hipotálamo/citologia , Masculino , Neuroglia/citologia , Neuropeptídeos/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
Glucokinase (GK), the hexokinase involved in glucosensing in pancreatic ß-cells, is also expressed in arcuate nucleus (AN) neurons and hypothalamic tanycytes, the cells that surround the basal third ventricle (3V). Several lines of evidence suggest that tanycytes may be involved in the regulation of energy homeostasis. Tanycytes have extended cell processes that contact the feeding-regulating neurons in the AN, particularly, agouti-related protein (AgRP), neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC) neurons. In this study, we developed an adenovirus expressing GK shRNA to inhibit GK expression in vivo. When injected into the 3V of rats, this adenovirus preferentially transduced tanycytes. qRT-PCR and Western blot assays confirmed GK mRNA and protein levels were lower in GK knockdown animals compared to the controls. In response to an intracerebroventricular glucose injection, the mRNA levels of anorexigenic POMC and CART and orexigenic AgRP and NPY neuropeptides were altered in GK knockdown animals. Similarly, food intake, meal duration, frequency of eating events and the cumulative eating time were increased, whereas the intervals between meals were decreased in GK knockdown rats, suggesting a decrease in satiety. Thus, GK expression in the ventricular cells appears to play an important role in feeding behavior.
Assuntos
Adenoviridae/fisiologia , Comportamento Alimentar , Glucoquinase/metabolismo , Hipotálamo/metabolismo , Hipotálamo/fisiopatologia , Infecções por Adenoviridae , Animais , Encefalite/etiologia , Encefalite/metabolismo , Encefalite/patologia , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Hipotálamo/patologia , Hipotálamo/virologia , Masculino , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Seabird excrements (guano) have been preserved in the arid climate of Northern Chile since at least the Pliocene. The deposits of marine organic material in coastal areas potentially open a window into the present and past composition of the coastal ocean and its food web. We use the stable isotope composition of nitrogen and carbon as well as element contents to compare the principal prey of the birds, the Peruvian anchovy, with the composition of modern guano. We also investigate the impact of diagenetic changes on the isotopic composition and elemental contents of the pure ornithogenic sediments, starting with modern stratified deposits and extending to fossil guano. Where possible, 14C systematics is used for age information. The nitrogen and carbon isotopic composition of the marine prey (Peruvian anchovy) of the birds is complex as it shows strong systematic variations with latitude. The detailed study of a modern profile that represents a few years of guano deposition up to present reveals systematic changes in nitrogen and carbon isotopic composition towards heavier values that increase with age, i.e. depth. Only the uppermost, youngest layers of modern guano show compositional affinity to the prey of the birds. In the profile, the simultaneous loss of nitrogen and carbon occurs by degassing, and non-volatile elements like phosphorous and calcium are passively enriched in the residual guano. Fossil guano deposits are very low in nitrogen and low in carbon contents, and show very heavy nitrogen isotopic compositions. One result of the study is that the use of guano for tracing nitrogen and carbon isotopic and elemental composition in the marine food web of the birds is restricted to fresh material. Despite systematic changes during diagenesis, there is little promise to retrieve reliable values of marine nitrogen and carbon signatures from older guano. However, the changes in isotopic composition from primary marine nitrogen isotopic signatures towards very heavy values generate a compositionally unique material. These compositions trace the presence of guano in natural ecosystems and its use as fertilizer in present and past agriculture.
Assuntos
Aves/fisiologia , Carbono/análise , Fósseis , Sedimentos Geológicos , Marcação por Isótopo/métodos , Nitrogênio/análise , Envelhecimento , Animais , Isótopos de Carbono , Chile , Geografia , Isótopos de Nitrogênio , VolatilizaçãoRESUMO
Anterior cruciate ligament tears are common and affect young individuals who participate in jumping and pivoting sports. After injury many individuals undergo ligament reconstruction (ACLR) but do not return to play, suffer recurrent injury and osteoarthritis. Outcome studies show that after ACLR, 81% of individuals return to sports, 65% return to their preinjury level and 55% return to competitive sports. Systematic reviews place the risk of ipsilateral retears at 5.8% and contralateral injuries at 11.8%, with recent reports of over 20% failure rate. Approximately 20% to 50% of patients will have evidence of OA within 10 to 20 yr. Factors important in reducing complications include timing of surgery, individualized return to play protocols, and prevention programs for injury. Further understanding of the factors that increase return to play percentages, reduce the risk of recurrent injury and improve long-term outcomes after ACL injury is needed to reduce the burden of these injuries on society.
Assuntos
Lesões do Ligamento Cruzado Anterior/epidemiologia , Lesões do Ligamento Cruzado Anterior/terapia , Reconstrução do Ligamento Cruzado Anterior/estatística & dados numéricos , Traumatismos em Atletas/epidemiologia , Traumatismos em Atletas/terapia , Desempenho Atlético/estatística & dados numéricos , Volta ao Esporte/estatística & dados numéricos , Adolescente , Adulto , Criança , Tomada de Decisão Clínica , Medicina Baseada em Evidências , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Recuperação de Função Fisiológica , Fatores de Risco , Resultado do Tratamento , Adulto JovemRESUMO
Alzheimer's disease is a neurodegenerative disorder that affects mostly the elderly. The main histopathological markers are the senile plaques formed by amyloid-ß peptide (Aß) aggregates that can perforate the plasma membrane of cells, increasing the intracellular calcium levels and releasing synaptic vesicles that finally lead to a delayed synaptic failure. Several membrane proteins and lipids interact with Aß affecting its toxicity in neurons. Here, we focus on NMDA receptors (NMDARs) as proteins that could be modulating the association and neurotoxic perforation induced by Aß on the plasma membrane. In fact, our results showed that decreasing NMDARs, using enzymatic or siRNA approaches, increased the association of Aß to the neurons. Furthermore, overexpression of NMDARs also resulted in an enhanced association between NMDA and Aß. Functionally, the reduction in membrane NMDARs augmented the process of membrane perforation. On the other hand, overexpressing NMDARs had a protective effect because Aß was now unable to cause membrane perforation, suggesting a complex relationship between Aß and NMDARs. Because previous studies have recognized that Aß oligomers are able to increase membrane permeability and produce amyloid pores, the present study supports the conclusion that NMDARs play a critical protective role on Aß actions in hippocampal neurons. These results could explain the lack of correlation between brain Aß burden and clinically observed dementia.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Membrana Celular/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Células HEK293 , Hipocampo/citologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Proteínas Associadas aos Microtúbulos/metabolismo , N-Metilaspartato/farmacologia , Gravidez , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Historically, the foundation of physical medicine and rehabilitation training has provided the capabilities to optimize nonoperative treatments of a variety of musculoskeletal conditions, including acute and chronic muscle, tendon, ligament, and cartilage disorders. Such treatments include the use of nonsteroidal anti-inflammatory drugs (NSAIDs), therapeutic modalities (eg, thermal and manual therapies), and corticosteroid injections in conjunction with specific rehabilitation exercises. Although NSAIDs, modalities, and corticosteroids may be helpful for short-term pain reduction and early recovery of function, they do not typically reverse the structural changes associated with degenerative conditions and may contribute to worse long-term outcomes by potentially interfering with tissue healing. Regenerative interventions, including platelet-rich plasma and mesenchymal stem cells, recently have been used to treat refractory painful conditions such as chronic tendinopathies because of the potential of these interventions to facilitate tissue healing. The future development of these regenerative techniques will require a variety of conditions to be met, including determining the most appropriate procedures based on the disease being treated; establishing the optimal preparations of these regenerative techniques; and providing clinicians, patients, and regulatory agencies with high-quality evidence demonstrating the safety, effectiveness, and long-term results of these treatments. Clarification of current regulatory uncertainty, improved access for all patients, proper training for clinicians who incorporate these techniques into their practice, and determination of the most appropriate postinjection protocols will allow physical medicine and rehabilitation specialists to play a unique role in the long-term management of patients with musculoskeletal and sports injuries. This article will also address the role physiatrists should have in the inevitable growth of regenerative medicine applications.
Assuntos
Doenças Musculoesqueléticas/terapia , Medicina Física e Reabilitação/métodos , Medicina Regenerativa/métodos , HumanosRESUMO
The mechanism by which amyloid-ß (Aß) produces brain dysfunction in patients with Alzheimer's disease is largely unknown. According to previous studies, Aß might share perforating properties with gramicidin, a well-accepted membrane-disrupting peptide. Therefore, we hypothesize that the key steps leading to synaptotoxicity by Aß and gramicidin involve peptide aggregation, pore formation, and calcium dysregulation. Here, we show that Aß and gramicidin form aggregates enriched in ß-sheet structures using electron microscopy, and Thioflavin and Congo Red staining techniques. Also, we found that Aß and gramicidin display fairly similar actions in hippocampal cell membranes, i.e. inducing Ca(2+) entry and synaptoxicity characterized by the loss of synaptic proteins and a decrease in neuronal viability. These effects were not observed in a Ca(2+) free solution, indicating that both Aß and gramicidin induce neurotoxicity by a Ca(2+)-dependent mechanism. Using combined perforated patch clamp and imaging recordings, we found that only Aß produced a perforation that progressed from a small (Cl(-)-selective pore) to a larger perforation that allowed the entry of fluorescent molecules. Therefore, based on these results, we propose that the perforation at the plasma membrane by Aß is a dynamic process that is critical in producing neurotoxicity similar to that found in the brains of AD patients.