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Cells display complex intracellular organization by compartmentalization of metabolic processes into organelles, yet the resolution of these structures in the native tissue context and their functional consequences are not well understood. Here we resolved the three-dimensional structural organization of organelles in large (more than 2.8×105µm3) volumes of intact liver tissue (15 partial or full hepatocytes per condition) at high resolution (8nm isotropic pixel size) using enhanced focused ion beam scanning electron microscopy1,2 imaging followed by deep-learning-based automated image segmentation and 3D reconstruction. We also performed a comparative analysis of subcellular structures in liver tissue of lean and obese mice and found substantial alterations, particularly in hepatic endoplasmic reticulum (ER), which undergoes massive structural reorganization characterized by marked disorganization of stacks of ER sheets3 and predominance of ER tubules. Finally, we demonstrated the functional importance of these structural changes by monitoring the effects of experimental recovery of the subcellular organization on cellular and systemic metabolism. We conclude that the hepatic subcellular organization ofthe ER architecture are highly dynamic, integrated with the metabolic state and critical for adaptive homeostasis and tissue health.

Mitochondrial reactive oxygen species (mROS) are central to physiology. While excess mROS production has been associated with several disease states, its precise sources, regulation, and mechanism of generationin vivoremain unknown, limiting translational efforts. Here we show that in obesity, hepatic ubiquinone (Q) synthesis is impaired, which raises the QH2/Q ratio, driving excessive mROS production via reverse electron transport (RET) from site IQin complex I. Using multiple complementary genetic and pharmacological modelsin vivowe demonstrated that RET is critical for metabolic health. In patients with steatosis, the hepatic Q biosynthetic program is also suppressed, and the QH2/Q ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.

Three principal ER quality-control mechanisms, namely, the unfolded protein response, ER-associated degradation (ERAD), and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the 3 pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β cells. SEL1L-HRD1 ERAD deficiency in β cells triggers activation of autophagy, at least in part, via IRE1α (an endogenous ERAD substrate). In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β cells leads to diabetes in mice shortly after weaning, with premature death by approximately 11 weeks of age, associated with marked ER retention of proinsulin and β cell loss. Using focused ion beam scanning electron microscopy powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the 3 ER quality-control mechanisms in the dynamic regulation of organellar architecture and β cell function.

The endoplasmic reticulum (ER) is a key organelle involved in the regulation of lipid and glucose metabolism, proteostasis, Ca

O objetivo desta pesquisa foi determinar a incidência e analisar o perfil dos portadores de lesão por pressão, enfocando fatores de risco, caracterí­sticas clí­nicas e demográficas dos pacientes internados em Unidade de Terapia Intensiva (UTI) do hospital IMIP, assim como estádio e localização das lesões no corpo. Trata-se de um estudo descritivo, documental, com abordagem quantitativa. Realizado na UTI Cirúrgica do complexo hospitalar do Instituto de Medicina Integral Professor Fernando Figueira (IMIP), localizado em Recife no estado de Pernambuco, através da coleta de dados dos censos diários e do uso da escala de Braden com um formulário especí­fico durante o perí­odo de outubro de 2018 a dezembro de 2018. Percebe-se a importância de um trabalho em equipe, com planejamento estratégico, buscando sempre solucionar as falhas não de forma isolada, mas em conjunto, seja na área médica, da enfermagem, nutricional, fisioterapia, e dos outros profissionais envolvimentos no cuidado do paciente internado nesta unidade.

Inhibiting IP 3 Rs in adipocytes may prevent the development of obesity-induced metabolic complications.

Chronic metabolic inflammation is a key feature of obesity, insulin resistance, and diabetes. Here, we showed that altered regulation of the Ca(2+) channel inositol trisphosphate receptor (IP3R) was an adipocyte-intrinsic event involved in the emergence and propagation of inflammatory signaling and the resulting insulin resistance. Inflammation induced by cytokine exposure in vitro or by obesity in vivo led to increases in the abundance and activity of IP3Rs and in the phosphorylation of the Ca(2+)-dependent kinase CaMKII in adipocytes in a manner dependent on the kinase JNK. In mice, adipocyte-specific loss of IP3R1/2 protected against adipose tissue inflammation and insulin resistance, despite the mice exhibiting substantial diet-induced weight gain. Thus, this work suggests that increased IP3R activity is a key link between obesity, inflammation, and insulin resistance. These data also suggest that approaches to target IP3R-mediated Ca(2+) homeostasis in adipocytes may offer new therapeutic opportunities against metabolic diseases, especially because GWAS studies also implicate this locus in human obesity.

Cells display complex intracellular organization through compartmentalization of metabolic processes into organelles, yet neither the resolution of these structures in the native tissue context nor its functional consequences are well understood. Here, we resolved the 3-dimensional organelle structural organization in large (>2.8×105μm3) volumes of intact liver tissue (15 partial or full hepatocytes per condition) in high resolution (8nm isotropic pixel size) by utilizing enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging, followed by deep-learning-based image segmentation and 3D reconstruction. We also performed a comparative analysis of subcellular structures in liver tissue of lean and obese animals and found marked alterations particularly in hepatic endoplasmic reticulum (ER), which undergoes massive structural re-organization in obesity characterized by marked disorganization of stacks of ER sheets and predominance of ER tubules. Finally, we demonstrated the functional importance of these structural changes upon experimental recovery of the subcellular organization and its marked impact on cellular and systemic metabolism. We conclude that hepatic subcellular organization and ER’s architecture is highly dynamic, integrated with the metabolic state, and critical for adaptive homeostasis and tissue health.

SummaryChronic metabolic inflammation is a key feature of obesity, insulin resistance and diabetes, although the initiation and propagation mechanisms of metaflammation are not fully established, particularly in the adipose tissue. Here we show that in adipocytes, altered regulation of the Ca2+ channel inositol triphosphate receptor (IP3Rs) is a key, adipocyte-intrinsic, event involved in the emergence and propagation of inflammatory signaling and the resulting insulin resistance. Inflammation, either induced by cytokine exposure in vitro or by obesity in vivo lead to increased expression and activity of IP3Rs in adipocytes in a JNK-dependent manner. This results in increased cytosolic Ca2+ and impaired insulin action. In mice, adipocyte-specific loss of IP3R1/2 protected against adipose tissue inflammation and insulin resistance despite significant diet-induced weight gain. Thus, this work reveals that IP3R over-activation and the resulting increase in cytosolic Ca2+ is a key link between obesity, inflammation and insulin resistance, and suggests that approaches to target adipocyte Ca2+ homeostasis may offer new therapeutic opportunities against metabolic diseases, especially since GWAS studies also implicate this locus in human obesity.

Adipocytes possess remarkable adaptive capacity to respond to nutrient excess, fasting or cold exposure, and they are thus an important cell type for the maintenance of proper metabolic health. Although the endoplasmic reticulum (ER) is a critical organelle for cellular homeostasis, the mechanisms that mediate adaptation of the ER to metabolic challenges in adipocytes are unclear. Here we show that brown adipose tissue (BAT) thermogenic function requires an adaptive increase in proteasomal activity to secure cellular protein quality control, and we identify the ER-localized transcription factor nuclear factor erythroid 2-like 1 (Nfe2l1, also known as Nrf1) as a critical driver of this process. We show that cold adaptation induces Nrf1 in BAT to increase proteasomal activity and that this is crucial for maintaining ER homeostasis and cellular integrity, specifically when the cells are in a state of high thermogenic activity. In mice, under thermogenic conditions, brown-adipocyte-specific deletion of Nfe2l1 (Nrf1) resulted in ER stress, tissue inflammation, markedly diminished mitochondrial function and whitening of the BAT. In mouse models of both genetic and dietary obesity, stimulation of proteasomal activity by exogenously expressing Nrf1 or by treatment with the proteasome activator PA28α in BAT resulted in improved insulin sensitivity. In conclusion, Nrf1 emerges as a novel guardian of brown adipocyte function, providing increased proteometabolic quality control for adapting to cold or to obesity.

Objective: to compare the electrical activity of masseter muscles, bilaterally, according to the presence or absence of Temporomandibular Disorder (TMD) in college students with a high degree of anxiety. Methods: the study was conducted with a randomized sample of 31 Speech Therapy students aged between 17 and 32 years; 61.3% (n = 19) were females and 38.7% (n = 12) were males. They were divided into two groups, Group 1 (G1), comprising 11 students with TMD, and Group 2 (G2), composed of 20 students without TMD. The college students answered the State-Trait Anxiety Inventory (STAI) for anxiety investigation, and were evaluated by the protocol Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) for TMD diagnosis. The evaluation of muscular electrical activity took into account the records in the conditions of rest, Sustained Maximum Voluntary Activity (SMVA) and habitual chewing (HC). The data were analyzed using the version 22 IBM Statistical Package for Social Sciences (SPSS) software. The statistical analysis was performed using Student t test to compare means between groups, considering < 0,05 as the significant p-value. Results: college students, of both groups, presented high levels of anxiety traits. Significant statistical differences were observed on the percentage of electrical activity of right masseter muscle in chewing function, as well as muscle fibers recruitment during chewing, which were higher on the group without TMD. Conclusion: college students with TMD and a high degree of anxiety presented lower means of masseter muscle electromyografic activity during chewing, in most conditions assessed, as compared to volunteers without TMD, except for the left masseter muscle in rest and chewing. RESUMO Objetivo: comparar a atividade elétrica dos músculos masseteres bilateralmente de acordo com a presença ou ausência de Disfunção Temporomandibular (TMD) em estudantes universitários com alto grau de ansiedade. Métodos: o estudo foi realizado com 31 acadêmicos de Fonoaudiologia, selecionados aleatoriamente, com idades entre 17 e 32 anos, sendo 61,3% (n=19) do sexo feminino e 38,7% (n=12) do sexo masculino, divididos em dois grupos, sendo o Grupo 1 (G1) composto por 11 estudantes com DTM e o Grupo 2 (G2) formado por 20 estudantes sem DTM. Para investigação da ansiedade os estudantes universitários responderam o Inventário de Ansiedade Traço-Estado (IDATE), e para o diagnóstico de DTM, foram avaliados por meio do protocolo Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD). A avaliação da atividade elétrica muscular considerou os registros nas condições de repouso, Máxima Atividade Voluntária Sustentada (MAVS) e mastigação habitual (MH). Os dados foram analisados por meio do software IBM Statistical Package for Social Sciences (SPSS), versão 22. A estatística analítica foi realizada por meio do teste T de Student para comparação das médias entre os grupos. Os dados foram considerados significantes, quando o p-valor fosse inferior a 5%. Resultados: os graduandos, de ambos os grupos, apresentaram alto nível de ansiedade traço. Foram verificadas diferenças estatisticamente significantes para a porcentagem da atividade elétrica do músculo masseter direito na mastigação, bem como no recrutamento de fibras musculares na mastigação, as quais foram superiores no grupo sem DTM. Conclusões: universitários com DTM e alto grau de ansiedade apresentaram médias de atividade eletromiográfica do músculo masseter inferiores as do grupo sem DTM, na maioria das condições avaliadas, exceto para o masseter esquerdo em repouso e na mastigação.

Endoplasmic reticulum stress (ERS) has a pathophysiological role in obesity-associated insulin resistance. Yet, the coordinated tissue response to ERS remains unclear. Increased connexin 43 (Cx43)-mediated intercellular communication has been implicated in tissue-adaptive and -maladaptive response to various chronic stresses. Here, we demonstrate that in hepatocytes, ERS results in increased Cx43 expression and cell-cell coupling. Co-culture of ER-stressed "donor" cells resulted in intercellular transmission of ERS and dysfunction to ERS-naive "recipient" cells ("bystander response"), which could be prevented by genetic or pharmacologic suppression of Cx43. Hepatocytes from obese mice were able to transmit ERS to hepatocytes from lean mice, and mice lacking liver Cx43 were protected from diet-induced ERS, insulin resistance, and hepatosteatosis. Taken together, our results indicate that in obesity, the increased Cx43-mediated cell-cell coupling allows intercellular propagation of ERS. This novel maladaptive response to over-nutrition exacerbates the tissue ERS burden, promoting hepatosteatosis and impairing whole-body glucose metabolism.

ObjectiveTo summarise the effects of herbal medications for the prevention of anxiety, depression, pain, and postoperative nausea and vomiting (PONV) in patients undergoing laparoscopic, obstetrical/gynaecological or cardiovascular surgical procedures.MethodsSearches of MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and LILACS up until January 2018 were performed to identify randomised controlled trials (RCTs). We included RCTs or quasi-RCTs evaluating any herbal medication among adults undergoing laparoscopic, obstetrical/gynaecological or cardiovascular surgeries. The primary outcomes were anxiety, depression, pain and PONV. We used the Grading of Recommendations Assessment, Development and Evaluation approach to rate overall certainty of the evidence for each outcome.ResultsEleven trials including 693 patients were eligible. Results from three RCTs suggested a statistically significant reduction in vomiting (relative risk/risk ratio (RR) 0.57; 95% CI 0.38 to 0.86) and nausea (RR 0.69; 95% CI 0.50 to 0.96) with the use ofZingiber officinale(ginger) compared with placebo in both laparoscopic and obstetrical/gynaecological surgeries. Results suggested a non-statistically significantly reduction in the need for rescue medication for pain (RR 0.52; 95% CI 0.13 to 2.13) withRosa damascena(damask rose) and ginger compared with placebo in laparoscopic and obstetrical/gynaecological surgery. None of the included studies reported on adverse events (AEs).ConclusionsThere is very low-certainty evidence regarding the efficacy of bothZingiber officinaleandRosa damascenain reducing vomiting (200 fewer cases per 1000; 288 fewer to 205 fewer), nausea (207 fewer cases per 1000; 333 fewer to 27 fewer) and the need for rescue medication for pain (666 fewer cases per 1000; 580 fewer to 752 more) in patients undergoing either laparoscopic or obstetrical/gynaecological surgeries. Among our eligible studies, there was no reported evidence on AEs.PROSPERO registration numberCRD42016042838

Defective Ca2+ handling is a key mechanism underlying hepatic endoplasmic reticulum (ER) dysfunction in obesity. ER Ca2+ level is in part monitored by the store-operated Ca2+ entry (SOCE) system, an adaptive mechanism that senses ER luminal Ca2+ concentrations through the STIM proteins and facilitates import of the ion from the extracellular space. Here, we show that hepatocytes from obese mice displayed significantly diminished SOCE as a result of impaired STIM1 translocation, which was associated with aberrant STIM1 O-GlycNAcylation. Primary hepatocytes deficient in STIM1 exhibited elevated cellular stress as well as impaired insulin action, increased glucose production and lipid droplet accumulation. Additionally, mice with acute liver deletion of STIM1 displayed systemic glucose intolerance. Conversely, over-expression of STIM1 in obese mice led to increased SOCE, which was sufficient to improve systemic glucose tolerance. These findings demonstrate that SOCE is an important mechanism for healthy hepatic Ca2+ balance and systemic metabolic control., eLife digest Obesity is a chronic metabolic disorder. Some people’s genetics make them more vulnerable to the condition, and it is generally caused by eating too much and moving too little. The resulting surplus of nutrients affects the cells and organs of the body in several adverse ways. For example, excessive nutrients impair a compartment within cells called the endoplasmic reticulum. This compartment is where many proteins and fats are made and transported. It is also the site for a lot of metabolic processes, and the main place in the cell where calcium ions are stored. Many proteins need calcium ions to work properly, including metabolic enzymes. In obesity, the endoplasmic reticulum becomes less able to store calcium ions. A protein called STIM1 senses and regulates the levels of calcium ions in the endoplasmic reticulum. When calcium levels drop, STIM1 moves along the endoplasmic reticulum membrane towards the part that is next to the cell surface. Here, STIM1 joins up with a calcium channel called Orai1. The STIM1-Orai1 complex allows calcium ions to enter the cell and replenish its levels in the endoplasmic reticulum. Arruda, Pers et al. have now asked if STIM1 is altered in obesity and, if so, whether it contributes to the endoplasmic reticulum’s inability to maintain proper calcium levels. High-resolution microscopy and biochemical techniques confirmed that STIM1 is indeed compromised in liver cells from obese mice. In these cells, STIM1 was found in unusual small clusters. It also could not move along the endoplasmic reticulum membrane when calcium levels dropped. As a result of these navigational errors, STIM1 failed to couple with Orai1, meaning less calcium could enter the cell. Further work identified that a small sugar molecule that is added onto STIM1 in obesity is behind its reduced ability to move accurately. Arruda, Pers et al. next created mice that lacked STIM1 in their liver. These mice showed signs of metabolic abnormalities. Notably, when STIM1 levels were experimentally increased in obese mice, it restored calcium levels in the endoplasmic reticulum closer to normal, and improved metabolism too. Thus, regulating calcium levels in the endoplasmic reticulum via proteins such as STIM1 is essential for maintaining a healthy metabolism. Interventions to correct calcium levels may have therapeutic promise to combat metabolic diseases.

Proper function of the endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site has been linked to pathophysiological states, including metabolic diseases. Although the ER and mitochondria play distinct cellular roles, these organelles also form physical interactions with each other at sites defined as mitochondria-associated ER membranes (MAMs), which are essential for calcium, lipid and metabolite exchange. Here we show that in the liver, obesity leads to a marked reorganization of MAMs resulting in mitochondrial calcium overload, compromised mitochondrial oxidative capacity and augmented oxidative stress. Experimental induction of ER-mitochondria interactions results in oxidative stress and impaired metabolic homeostasis, whereas downregulation of PACS-2 or IP3R1, proteins important for ER-mitochondria tethering or calcium transport, respectively, improves mitochondrial oxidative capacity and glucose metabolism in obese animals. These findings establish excessive ER-mitochondrial coupling as an essential component of organelle dysfunction in obesity that may contribute to the development of metabolic pathologies such as insulin resistance and diabetes.

TNFalpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNFalpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNFalpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNFalpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNFalpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNFalpha action to be important mediator of the wastage syndrome in cachexia.

Short-term response to cold promotes a small but significant rise in serum T3 in euthyroid rabbits, where the heart is an important target of T3 action. In this work, we measured changes in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and phospholamban (PLB) in hearts of hypo- and hyperthyroid rabbits and compared them with modifications induced by short- and long-term cold exposure. Short-term cold exposure promotes a small increase in T3 and, similar to hyperthyroidism, induces an increase of heart SERCA2a expression. The total PLB content does not change in hyperthyroidism, but short-term cold exposure promotes a significant decrease in total PLB and an increase in the ratio between phosphorylated and total PLB. The temperature of a given tissue depends on the balance between the heat provided by blood circulation and the rate of heat production by the tissue. In an attempt to evaluate the heat contribution of cardiac tissue, we measured mitochondrial respiration in permeabilized cardiac muscle and heat produced by cardiac sarcoplasmic reticulum (SR) during Ca2+transport. We observed that there was an increase in oxygen consumption and heat production during Ca2+transport by cardiac SR in both hyperthyroidism and short-term cold exposure. In contrast, both the mitochondrial respiration rate and heat derived from Ca2+transport were decreased in hypothyroid rabbits. The heart changes in oxygen consumption, SERCA2a-PLB ratio, and Ca2+-ATPase activity detected during short-term cold exposure were abolished after cold adaptation. We hypothesize that the transient rise in serum T3 contributes to the short-term response to cold exposure.

Brown adipose tissue (BAT) is involved in rat and mice thermoregulation, and heat produced by BAT depends on the concerted action of thyroid hormones and catecholamines. Little is known about cold-induced thermogenesis in mammals that have little or no BAT, such as rabbits. In these animals, thermogenesis primarily occurs in skeletal muscle. In this work, we have studied the effect of cold acclimation (4 C for 10 d) in normal and hypothyroid rabbits. It is known that hypothyroid rats die after a few hours of cold exposure. We now show that, different from rats, hypothyroid rabbits sustain their body temperature and survive after 10 d cold exposure. When compared with rabbits kept at room temperature, the muscles of cold-exposed rabbits showed a dark red color characteristic of oxidative muscle fibers. According to this pattern, we observed that in both normal and hypothyroid rabbits, cold exposure promotes an increase in oxygen consumption by skeletal muscle mitochondria. Moreover, in red muscle, cold acclimation induces an increase in the expression and activity of sarcoplasmic reticulum Ca2+ ATPase isoform 1 (SERCA1), one of the muscle enzymes involved in heat production. We conclude that rabbit cold tolerance is probably related to increased muscle oxidative metabolism and heat production by SERCA1 and that these changes are not completely dependent on normal thyroid function.

The sarcoplasmic reticulum Ca(2+) ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca(2+) transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca(2+) channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (DeltaH(cal)) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg(2+) or ruthenium red, conditions that close the ryanodine Ca(2+) channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the DeltaH(cal) values of ATP hydrolysis increased significantly. Neither Mg(2+) nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the DeltaH(cal) of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca(2+) channel is opened or closed.

A number of chronic metabolic pathologies, including obesity, diabetes, cardiovascular disease, asthma, and cancer cluster together to present the greatest threat to human health. As research in this field has advanced, it has become clear that unresolved metabolic inflammation, organelle dysfunction, and other cellular and metabolic stresses underlie the development of these chronic metabolic diseases. However, the relationship between these systems and pathological mechanisms is poorly understood. Here, we will discuss the role of cellular Ca2+ homeostasis as a critical mechanism integrating the myriad of cellular and subcellular dysfunctional networks found in metabolic tissues such as liver and adipose tissue in the context of metabolic disease particularly in obesity and diabetes.

The endoplasmic reticulum (ER) plays a critical role in protein, lipid, and glucose metabolism as well as cellular calcium signaling and homeostasis. Perturbation of ER function and chronic ER stress are associated with many pathologies ranging from diabetes and neurodegenerative diseases to cancer and inflammation. Although ER targeting shows therapeutic promise in preclinical models of obesity and other pathologies, the available chemical entities generally lack the specificity and other pharmacological properties required for effective clinical translation. To overcome these challenges and identify new potential therapeutic candidates, we first designed and chemically and genetically validated two high-throughput functional screening systems that independently measure the free chaperone content and protein-folding capacity of the ER. With these quantitative platforms, we characterized a small-molecule compound, azoramide, that improves ER protein-folding ability and activates ER chaperone capacity to protect cells against ER stress in multiple systems. This compound also exhibited potent antidiabetic efficacy in two independent mouse models of obesity by improving insulin sensitivity and pancreatic β cell function. Together, these results demonstrate the utility of this functional, phenotypic assay platform for ER-targeted drug discovery and provide proof of principle for the notion that specific ER modulators can be potential drug candidates for type 2 diabetes.

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca2+ concentration from 0.05 up to 0.70 μm. It is not known how the rise of Ca2+ concentration activates BAT thermogenesis. In this report we compared the effects of Ca2+ in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca2+-ATPase bound to the inner membrane of BAT mitochondria. A Ca2+-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca2+ enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca2+ concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 μm. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca2+-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca2+ did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca2+-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca2+ concentrations similar to those noted in the cell during adrenergic stimulation.

Enzymes are able to handle the energy derived from the hydrolysis of phosphate compounds in such a way as to determine the parcel that is used for work and the fraction that is converted into heat. The sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) is a family of membrane-bound ATPases that are able to transport Ca2+ ion across the membrane using the chemical energy derived from ATP hydrolysis. The heat released during ATP hydrolysis by SERCA may vary from 10 up to 30 kcal/mol depending on the SERCA isoform used and on whether or not a Ca2+ gradient is formed across the membrane. Drugs such as heparin, dimethyl sulfoxide and the platelet-activating factor (PAF) are able to modify the fraction of the chemical energy released during ATP hydrolysis that is used for Ca2+ transport and the fraction that is dissipated in the surrounding medium as heat. The thyroid hormone 3,5,3′-triiodo l-thyronine (T3) regulates the expression and function of the thermogenic SERCA isoforms. Modulation of heat production by SERCA might be one of the mechanisms involved in the increased thermogenesis found in hyperthyroidism.

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca2+ release, and the light fraction enriched in Ca(2+)-ATPase (SERCA), an enzyme responsible for Ca2+ transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca(2+)- ATPase activity and Ca(2+)-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca(2+)-uptake and Ca(2+)-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.

The sarcoplasmic reticulum Ca2+-ATPase is able to modulate the distribution of energy released during ATP hydrolysis, so that a portion of energy is used for Ca2+ transport (coupled ATPase activity) and a portion is converted into heat (uncoupled ATPase activity). In this report it is shown that T4 administration to rabbits promotes an increase in the rates of both the uncoupled ATPase activity and heat production in sarcoplasmic reticulum vesicles, and that the degree of activation varies depending on the muscle type used. In white muscles hyperthyroidism promotes a 0.8-fold increase of the uncoupled ATPase activity and in red muscle a 4-fold increase. The yield of vesicles from hyperthyroid muscles is 3–4-fold larger than that obtained from normal muscles; thus the rate of heat production by the Ca2+-ATPase expressed in terms of g of muscle in hyperthyroidism is increased by a factor of 3.6 in white muscles and 12.0 in red muscles. The data presented suggest that the Ca2+-ATPase uncoupled activity may represent one of the heat sources that contributes to the enhanced thermogenesis noted in hyperthyroidism.

Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol.

In both human and experimental obesity, inflammatory damage to the hypothalamus plays an important role in the loss of the coordinated control of food intake and energy expenditure. Upon prolonged maintenance of increased body mass, the brain changes the defended set point of adiposity, and returning to normal weight becomes extremely difficult. Here we show that in prolonged but not in short-term obesity, the ubiquitin/proteasome system in the hypothalamus fails to maintain an adequate rate of protein recycling, leading to the accumulation of ubiquitinated proteins. This is accompanied by an increased colocalization of ubiquitin and p62 in the arcuate nucleus and reduced expression of autophagy markers in the hypothalamus. Genetic protection from obesity is accompanied by the normal regulation of the ubiquitin/proteasome system in the hypothalamus, whereas the inhibition of proteasome or p62 results in the acceleration of body mass gain in mice exposed for a short period to a high-fat diet. Thus, the defective regulation of the ubiquitin/proteasome system in the hypothalamus may be an important mechanism involved in the progression and autoperpetuation of obesity.

O presente estudo avaliou a influência de três variáveis, catalisador (C) NaOH e KOH, temperatura de 60 e 80°C e razão molar álcool/óleo (R) de 6:1 e 12:1, na transesterificação do óleo de pequi por meio de um planejamento fatorial 23. Todas as variáveis foram avaliadas em seus níveis máximos e mínimos. Os resultados demonstraram que o maior rendimento em ésteres correspondeu a 73%, utilizando catalisador (C) KOH, razão molar (R) de 12:1 (etanol:óleo) e temperatura de 60 °C, sendo que rendimentos ligeiramente menores 37,25% sem significância foram obtidos com o emprego NaOH, razão molar 12:1 (etanol:óleo) e a 80°C. Separadamente a variável de maior importância para a transesterificação óleo de pequi foi a temperatura (T) em nível menor (60 ºC) e o catalisador não apresentou efeito significativo no rendimento da reação. A partir dos cálculos de efeito da combinação entre as três variáveis, observou-se que os melhores rendimentos são encontrados quando se utiliza o catalisador em seu maior nível (KOH), combinado com níveis contrários de razão molar e temperatura. Ou seja, os melhores rendimentos são encontrados quando a transesterificação é realizada utilizando como catalisador o KOH, uma razão molar de 12:1 e uma temperatura de 60ºC, ou como catalisador o KOH, uma razão molar de 6:1 e uma temperatura de 80ºC.

Defective liver gluconeogenesis is the main mechanism leading to fasting hyperglycemia in type 2 diabetes, and, in concert with steatosis, it is the hallmark of hepatic insulin resistance. Experimental obesity results, at least in part, from hypothalamic inflammation, which leads to leptin resistance and defective regulation of energy homeostasis. Pharmacological or genetic disruption of hypothalamic inflammation restores leptin sensitivity and reduces adiposity. Here, we evaluate the effect of a hypothalamic anti-inflammatory approach to regulating hepatic responsiveness to insulin. Obese rodents were treated by intracerebroventricular injections, with immunoneutralizing antibodies against Toll-like receptor (TLR)4 or tumor necrosis factor (TNF)α, and insulin signal transduction, hepatic steatosis, and gluconeogenesis were evaluated. The inhibition of either TLR4 or TNFα reduced hypothalamic inflammation, which was accompanied by the reduction of hypothalamic resistance to leptin and improved insulin signal transduction in the liver. This was accompanied by reduced liver steatosis and reduced hepatic expression of markers of steatosis. Furthermore, the inhibition of hypothalamic inflammation restored defective liver glucose production. All these beneficial effects were abrogated by vagotomy. Thus, the inhibition of hypothalamic inflammation in obesity results in improved hepatic insulin signal transduction, leading to reduced steatosis and reduced gluconeogenesis. All these effects are mediated by parasympathetic signals delivered by the vagus nerve.

Inflammation plays an important pathogenic role in a number of metabolic diseases such as obesity, type 2 diabetes, and atherosclerosis. The activation of inflammation in these diseases depends at least in part on the combined actions of TLR4 signaling and endoplasmic reticulum stress, which by acting in concert can boost the inflammatory response. Defining the mechanisms involved in this phenomenon may unveil potential targets for the treatment of metabolic/inflammatory diseases. Here we used LPS to induce endoplasmic reticulum stress in the human monocyte cell-line, THP-1. The unfolded protein response, produced after LPS, was dependent on CD14 activity but not on RNA-dependent protein kinase and could be inhibited by an exogenous chemical chaperone. The induction of the endoplasmic reticulum resident chaperones, GRP94 and GRP78, by LPS was of a much lower magnitude than the effect of LPS on TLR4 and MD-2 expression. In face of this apparent insufficiency of chaperone expression, we induced the expression of GRP94 and GRP78 by glucose deprivation. This approach completely reverted endoplasmic reticulum stress. The inhibition of either GRP94 or GRP78 with siRNA was sufficient to rescue the protective effect of glucose deprivation on LPS-induced endoplasmic reticulum stress. Thus, insufficient LPS-induced chaperone expression links TLR4 signaling to endoplasmic reticulum stress.

Hypothalamic inflammation and dysfunction are common features of experimental obesity. An imbalance between caloric intake and energy expenditure is generated as a consequence of this inflammation, leading to the progressive increase of body adiposity. Thermogenesis, is one of the main functions affected by obesity-linked hypothalamic dysfunction and the complete characterization of the mechanisms involved in this process may offer new therapeutic perspectives for obesity. The brown adipose tissue is an important target for hypothalamic action in thermogenesis. This tissue has been thoroughly studied in rodents and hibernating mammals; however, until recently, its advocated role in human thermogenesis was neglected due to the lack of substantial evidence of its presence in adult humans. The recent demonstration of the presence of functional brown adipose tissue in adult humans has renovated the interest in this tissue. Here, we review some of the work that shows how inflammation and dysfunction of the hypothalamus can control brown adipose tissue activity and how this can impact on whole body thermogenesis and energy expenditure.

Taurine is known to modulate a number of metabolic parameters such as insulin secretion and action and blood cholesterol levels. Recent data have suggested that taurine can also reduce body adiposity in C. elegans and in rodents. Since body adiposity is mostly regulated by insulin-responsive hypothalamic neurons involved in the control of feeding and thermogenesis, we hypothesized that some of the activity of taurine in the control of body fat would be exerted through a direct action in the hypothalamus. Here, we show that the intracerebroventricular injection of an acute dose of taurine reduces food intake and locomotor activity, and activates signal transduction through the Akt/FOXO1, JAK2/STAT3 and mTOR/AMPK/ACC signaling pathways. These effects are accompanied by the modulation of expression of NPY. In addition, taurine can enhance the anorexigenic action of insulin. Thus, the aminoacid, taurine, exerts a potent anorexigenic action in the hypothalamus and enhances the effect of insulin on the control of food intake.

In diet-induced obesity, hypothalamic inflammation is triggered as an outcome of prolonged exposure to dietary fats. Toll-like receptor 4 (TLR4) activation plays a central role in this process, inducing endoplasmic reticulum stress and activating inflammatory cytokine gene transcription. Although saturated fatty acids can induce endoplasmic reticulum stress in the hypothalamus, it is unknown whether inflammatory cytokines alone can activate this mechanism. Here, rats were treated with TNF-α or lyposaccharide (LPS) and endoplasmic reticulum stress and unfolded protein response were evaluated by immunoblot and polymerase chain reaction (PCR). Activation of TLR4 by LPS was capable of inducing a complete endoplasmic reticulum stress and unfolded protein response through the PERK/eIF2α and IRE1α/XBP1 pathways. Conversely, TNF-α, injected either locally or systemically, was unable to induce a complete program of unfolded protein response, although the activation of endoplasmic reticulum stress was achieved to a certain degree. Thus, in the hypothalamus, the isolated action of TNF-α is insufficient to produce the activation of a complete program of unfolded protein response.

Fatty-acid-induced endoplasmic reticulum stress has been recently described as a novel mechanism involved in the genesis of atherosclerosis. Here we show that statins, a class of drug widely employed in the clinical management of hypercholesterolemia, reduces lipid-induced macrophage endoplasmic reticulum stress in an isolated cell system and in LDL receptor knockout mice. Given the importance of endoplasmic reticulum stress as an inducer of inflammation, we suspect that the novel mechanism of action herein described for statins may play a major role on its beneficial effects in the prevention of cardiovascular disease.

In this work, we compared the effect of K+ on vesicles derived from the longitudinal (LSR) and terminal cisternae (HSR) of rabbit white muscle. In HSR, K+ was found to inhibit both the Ca2+ accumulation and the heat released during ATP hydrolysis by the Ca2+-ATPase (SERCA1). This was not observed in LSR. Valinomycin abolished the HSR Ca2+-uptake inhibition promoted by physiological K+ concentrations, but it did not modify the thermogenic activity of the Ca2+ pump. The results with HSR are difficult to interpret, assuming that a single K+ is binding to either the ryanodine channel or to the Ca2+-ATPase. It is suggested that an increase of K+ in the assay medium alters the interactions among the various proteins found in HSR, thus modifying the properties of both the ryanodine channel and SERCA1.

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca(2+) concentration from 0.05 up to 0.70 mum. It is not known how the rise of Ca(2+) concentration activates BAT thermogenesis. In this report we compared the effects of Ca(2+) in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca(2+)-ATPase bound to the inner membrane of BAT mitochondria. A Ca(2+)-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca(2+) enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca(2+) concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 mum. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca(2+) did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca(2+)-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca(2+) concentrations similar to those noted in the cell during adrenergic stimulation.

The Ca2+-ATPase (SERCA) found in vesicles derived from the sarco/endoplasmic reticulum vesicles of rats brown adipose tissue and rabbit white muscle were identified by gel electrophoresis, Western blot, electron microscopy and immunolabeling with gold particles. In both tissues, the isoform found was SERCA 1. The Ca2+ affinity of the fat SERCA 1 was different from the muscle isoform. The degree of uncoupling is estimated measuring the ratio between Ca2+ transport and ATP cleaved. In brown fat vesicles the degree of uncoupling varied depending on the Ca2+ concentration of the medium. This was not observed in vesicles derived from muscle. At all Ca2+ concentrations tested, the uncoupling was not related to Ca2+ leakage from the membrane and was far more pronounced in fat than in muscle vesicle. When a Ca2+ gradient was formed across the vesicles membrane the heat released during ATP hydrolysis varied between 22 and 26 Kcal/mol in both fat and muscle vesicles but in the absence of a gradient the heat released was 17 Kcal/mol in fat and 12 Kcal/mol in muscle. The data reported indicate that the SERCA 1 of brown adipocytes is far more thermogenic than the white muscle SERCA 1, and suggest that, in addition to storing Ca2+ inside the endoplasmic reticulum, the SERCA 1 may represent a source of heat production contributing to the thermogenic function of brown adipose tissue.

After formation of a Ca(2+) gradient, the amount of heat released during the hydrolysis of each mol of ATP cleaved (DeltaH(cal)) varies depending on the Ca(2+)-ATPase isoform expressed by the muscle cell. In vesicles derived from the sarcoplasmic reticulum of white muscle (SERCA 1) most of the ATP cleaved is not coupled to Ca(2+) transport, and the DeltaH(cal) varies between -20 and -22 kcal/mol. In contrast, in vesicles derived from red muscle (SERCA 2a) the hydrolysis of ATP is coupled with Ca(2+) transport, and the DeltaH(cal) varies between -12 and -14 kcal/mol. Hyperthyroidism increases the rate of heat production by the Ca(2+)-ATPase fourfold in white muscle and 40-fold in red muscle. In hyperthyroid rabbits, the amount of sarcoplasmic reticulum protein recovered from white and red muscle is four- to fivefold greater than that obtained from control rabbits. Hyperthyroid red muscle expresses SERCA 1, and the vesicles derived from these muscle hydrolyze ATP through a catalytic route that is not coupled to Ca(2+) transport, thus increasing the amount of heat released during ATP hydrolysis, the DeltaH(cal) varying between -20 and -22 kcal/mol.

Iniciativas no sentido de mensurar a espiritualidade e a religiosidade vêm sendo desenvolvidas, com alguns instrumentos construídos e validados para a utilização em diferentes contextos. Esses temas têm sido estudados em vários âmbitos, como nas inter-relações com a saúde física, saúde mental, como fator de risco/proteção e coping. As associações encontradas podem estar relacionadas aos vários construtos utilizados e aos instrumentos delineados com base nesses conceitos. Geralmente a construção de escalas e instrumentos de mensuração parte de reflexões teóricas e pesquisas. Alguns estudos preferem partir de observações de populações para construir os instrumentos. Atualmente várias medidas estão disponíveis, principalmente na língua inglesa, para que se possa investigar e aprofundar a extensão da influência da religiosidade e da espiritualidade na vida de indivíduos e grupos. No Brasil, são poucos os instrumentos desenvolvidos e/ou validados que mensurem diferentes dimensões da religiosidade, bem como construtos correlatos. Este é um trabalho de cunho teórico, cujo objetivo é explorar a variedade de instrumentos de mensuração da espiritualidade e religiosidade analisando os construtos presentes.#]

Hypothalamic inflammation is present in animal models of obesity, and the intracerebroventricular injection of TNFα can reproduce a number of features of the hypothalamus of obese animals. Because obesity is a risk factor for type 2 diabetes (DM2) we hypothesized that, by inducing hypothalamic inflammation, we could reproduce some clinical features of DM2. Lean Wistar rats and TNF receptor 1-knockout mice were employed to determine the effects of hypothalamic actions of TNFα on thermogenesis and metabolic parameters. Signal transduction and protein expression were evaluated by immunoblot and real-time PCR. Thermogenesis was evaluated in living rats, and respirometry was determined in isolated muscle fiber. In Wistar rats, hypothalamic TNFα blunts the anorexigenic effect of leptin, which is accompanied by reduced leptin signaling and increased expression of suppressor of cytokine signaling 3. In addition, hypothalamic TNFα reduces O2 consumption and the expression of thermogenic proteins in brown adipose tissue and skeletal muscle. Furthermore, hypothalamic inflammation increases base-line plasma insulin and insulin secretion by isolated pancreatic islets, which is accompanied by an impaired insulin signal transduction in liver and skeletal muscle. Hypothalamic inflammation induced by stearic acid also reduces O2 consumption and blunts peripheral insulin signal transduction. The use of intracerebroventricular infliximab restores O2 consumption in obese rats, whereas TNF receptor 1-knockout mice are protected from diet-induced reduced thermogenesis and defective insulin signal transduction. Thus, low-grade inflammation of the hypothalamus is sufficient to induce changes in a number of parameters commonly impaired in obesity and DM2, and TNFα is an important mediator of this process.