RESUMO
From a forward mutagenetic screen to discover mutations associated with obesity, we identified mutations in the Spag7 gene linked to metabolic dysfunction in mice. Here, we show that SPAG7 KO mice are born smaller and develop obesity and glucose intolerance in adulthood. This obesity does not stem from hyperphagia, but a decrease in energy expenditure. The KO animals also display reduced exercise tolerance and muscle function due to impaired mitochondrial function. Furthermore, SPAG7-deficiency in developing embryos leads to intrauterine growth restriction, brought on by placental insufficiency, likely due to abnormal development of the placental junctional zone. This insufficiency leads to loss of SPAG7-deficient fetuses in utero and reduced birth weights of those that survive. We hypothesize that a 'thrifty phenotype' is ingrained in SPAG7 KO animals during development that leads to adult obesity. Collectively, these results indicate that SPAG7 is essential for embryonic development and energy homeostasis later in life.
Obesity rates are climbing worldwide, leading to an increase in associated conditions such as type 2 diabetes. While new pharmaceutical approaches are available to help individuals manage their weight, many patients do not respond to them or experience prohibitive side effects. Identifying alternative treatments will likely require pinpointing the genes and molecular actors involved in the biological processes that control weight regulation. Previous research suggests that a protein known as SPAG7 could help shape how mice use and store the energy they extract from food. Flaherty et al. therefore set out to investigate the role this protein plays in the body. To do so, they created a line of mice born without SPAG7, which they monitored closely throughout life. These animals were underweight at birth and did not eat more than other mice, yet they were obese as adults. Their ability to exercise was reduced, their muscles were weaker and contained fibers with functional defects. The mice also exhibited biological changes associated with the onset of diabetes. Yet deleting SPAG7 during adulthood led to no such changes; these mice maintained normal muscle function and body weight. Closely examining how SPAG7-deficient mice developed in the womb revealed placental defects which likely caused these animals to receive fewer nutrients from their mother. Such early-life deprivation is known to be associated with the body shifting towards maximizing its use of resources and privileging fat storage, even into and throughout adulthood. By shedding light on the biological role of SPAG7, the work by Flaherty et al. helps to better understand how developmental events can increase the likelihood of obesity later in life. Further investigations are now needed to explore whether this knowledge could help design interventions relevant to human health.
Assuntos
Retardo do Crescimento Fetal , Camundongos Knockout , Obesidade , Animais , Obesidade/genética , Obesidade/metabolismo , Retardo do Crescimento Fetal/genética , Camundongos , Feminino , Metabolismo Energético/genética , Deleção de Genes , Gravidez , Intolerância à Glucose/genéticaRESUMO
OBJECTIVE: In the small intestine, the products of digestion of dietary triacylglycerol (TAG), fatty acids (FA) and monoacylglycerol, are taken up by absorptive cells, enterocytes, for systemic energy delivery. These digestion products can also bind receptors on endocrine cells to stimulate the release of hormones capable of influencing systemic energy metabolism. The initial phase of intestinal FA absorption involves the acylation of FAs to acyl-CoA by the acyl-CoA long chain synthetase (ACSL) enzymes. ACSL5 is abundantly expressed in the small intestinal epithelium where it is the major ACSL isoform, contributing approximately 80% of total ACSL activity. In mice with whole body deficiency of ACSL5, the rate of dietary fat absorption is reduced and energy expenditure is increased. However, the mechanisms by which intestinal ACSL5 contributes to intestinal FA metabolism, enteroendocrine signaling, and regulation of energy expenditure remain undefined. Here, we test the hypothesis that intestinal ACSL5 regulates energy metabolism by influencing dietary fat absorption and enteroendocrine signaling. METHODS: To explore the role of intestinal ACSL5 in energy balance and intestinal dietary fat absorption, a novel mouse model of intestine specific ACSL5 deficiency (ACSL5IKO) was generated by breeding ACSL5 floxed (ACSL5loxP/loxP) to mice harboring the tamoxifen inducible, villin-Cre recombinase. ACSL5IKO and control, ACSL5loxP/loxP mice were fed chow (low in fat) or a 60% high fat diet (HFD), and metabolic phenotyping was performed including, body weight, body composition, insulin and glucose tolerance tests, energy expenditure, physical activity, and food intake studies. Pair-feeding studies were performed to determine the role of food intake in regulating development of obesity. Studies of dietary fat absorption, fecal lipid excretion, intestinal mucosal FA content, and circulating levels of glucagon like peptide 1 (GLP-1) and peptide YY (PYY) in response to a TAG challenge were performed. Treatment with a GLP-1 receptor antagonist was performed to determine the contribution of GLP-1 to acute regulation of food intake. RESULTS: We found that ACSL5IKO mice experienced rapid and sustained protection from body weight and fat mass accumulation during HFD feeding. While intestine specific deficiency of ACSL5 delayed gastric emptying and reduced dietary fat secretion, it did not result in increased excretion of dietary lipid in feces. Energy expenditure and physical activity were not increased in ACSL5IKO mice. Mice deficient in intestinal ACSL5 display significantly reduced energy intake during HFD, but not chow feeding. When HFD intake of control mice was matched to ACSL5IKO during pair-feeding studies, no differences in body weight or fat mass gain were observed between groups. Postprandial GLP-1 and PYY were significantly elevated in ACSL5IKO mice secondary to increased FA content in the distal small intestine. Blockade of GLP-1 signaling by administration of a long-acting GLP-1 receptor antagonist partially restored HFD intake of ACSL5IKO. CONCLUSIONS: These data indicate that intestinal ACSL5 serves as a critical regulator of energy balance, protecting mice from diet-induced obesity exclusively by increasing satiety and reducing food intake during HFD feeding. The reduction in food intake observed in ACSL5IKO mice is driven, in part, by increased postprandial GLP-1 and PYY secretion. These effects are only observed during HFD feeding, suggesting that altered processing of dietary fat following intestinal ACSL5 ablation contributes to GLP-1 and PYY mediated increases in satiety.
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Coenzima A Ligases , Dieta Hiperlipídica , Peptídeo 1 Semelhante ao Glucagon , Obesidade , Peptídeo YY , Animais , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Camundongos , Obesidade/metabolismo , Masculino , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo YY/metabolismo , Camundongos Endogâmicos C57BL , Ingestão de Alimentos , Período Pós-Prandial , Metabolismo Energético , Camundongos KnockoutRESUMO
Urocortin 2 (UCN2) acts as a ligand for the G protein-coupled receptor corticotropin-releasing hormone receptor 2 (CRHR2). UCN2 has been reported to improve or worsen insulin sensitivity and glucose tolerance in vivo. Here we show that acute dosing of UCN2 induces systemic insulin resistance in male mice and skeletal muscle. Inversely, chronic elevation of UCN2 by injection with adenovirus encoding UCN2 resolves metabolic complications, improving glucose tolerance. CRHR2 recruits Gs in response to low concentrations of UCN2, as well as Gi and ß-Arrestin at high concentrations of UCN2. Pre-treating cells and skeletal muscle ex vivo with UCN2 leads to internalization of CRHR2, dampened ligand-dependent increases in cAMP, and blunted reductions in insulin signaling. These results provide mechanistic insights into how UCN2 regulates insulin sensitivity and glucose metabolism in skeletal muscle and in vivo. Importantly, a working model was derived from these results that unifies the contradictory metabolic effects of UCN2.
Assuntos
Resistência à Insulina , Animais , Masculino , Camundongos , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/metabolismo , Glucose/metabolismo , Insulina , Ligantes , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Urocortinas/genética , Urocortinas/metabolismoRESUMO
OBJECTIVE: The liver is a central regulator of energy metabolism exerting its influence both through intrinsic processing of substrates such as glucose and fatty acid as well as by secreting endocrine factors, known as hepatokines, which influence metabolism in peripheral tissues. Human genome wide association studies indicate that a predicted loss-of-function variant in the Inhibin ßE gene (INHBE), encoding the putative hepatokine Activin E, is associated with reduced abdominal fat mass and cardiometabolic disease risk. However, the regulation of hepatic Activin E and the influence of Activin E on adiposity and metabolic disease are not well understood. Here, we examine the relationship between hepatic Activin E and adipose metabolism, testing the hypothesis that Activin E functions as part of a liver-adipose, inter-organ feedback loop to suppress adipose tissue lipolysis in response to elevated serum fatty acids and hepatic fatty acid exposure. METHODS: The relationship between hepatic Activin E and non-esterified fatty acids (NEFA) released from adipose lipolysis was assessed in vivo using fasted CL 316,243 treated mice and in vitro using Huh7 hepatocytes treated with fatty acids. The influence of Activin E on adipose lipolysis was examined using a combination of Inhbe knockout mice, a mouse model of hepatocyte-specific overexpression of Activin E, and mouse brown adipocytes treated with Activin E enriched media. RESULTS: Increasing hepatocyte NEFA exposure in vivo by inducing adipose lipolysis through fasting or CL 316,243 treatment increased hepatic Inhbe expression. Similarly, incubation of Huh7 human hepatocytes with fatty acids increased expression of INHBE. Genetic ablation of Inhbe in mice increased fasting circulating NEFA and hepatic triglyceride accumulation. Treatment of mouse brown adipocytes with Activin E conditioned media and overexpression of Activin E in mice suppressed adipose lipolysis and reduced serum FFA levels, respectively. The suppressive effects of Activin E on lipolysis were lost in CRISPR-mediated ALK7 deficient cells and ALK7 kinase deficient mice. Disruption of the Activin E-ALK7 signaling axis in Inhbe KO mice reduced adiposity upon HFD feeding, but caused hepatic steatosis and insulin resistance. CONCLUSIONS: Taken together, our data suggest that Activin E functions as part of a liver-adipose feedback loop, such that in response to increased serum free fatty acids and elevated hepatic triglyceride, Activin E is released from hepatocytes and signals in adipose through ALK7 to suppress lipolysis, thereby reducing free fatty acid efflux to the liver and preventing excessive hepatic lipid accumulation. We find that disrupting this Activin E-ALK7 inter-organ communication network by ablation of Inhbe in mice increases lipolysis and reduces adiposity, but results in elevated hepatic triglyceride and impaired insulin sensitivity. These results highlight the liver-adipose, Activin E-ALK7 signaling axis as a critical regulator of metabolic homeostasis.
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Ativinas , Tecido Adiposo , Ácidos Graxos , Subunidades beta de Inibinas , Lipólise , Fígado , Animais , Camundongos , Fígado/metabolismo , Tecido Adiposo/metabolismo , Humanos , Masculino , Subunidades beta de Inibinas/metabolismo , Subunidades beta de Inibinas/genética , Ácidos Graxos/metabolismo , Ativinas/metabolismo , Camundongos Endogâmicos C57BL , Hepatócitos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos não Esterificados/sangue , Camundongos Knockout , AdiposidadeRESUMO
CONTEXT: Biological and translational insights from large-scale, array-based genetic studies of fat distribution, a key determinant of metabolic health, have been limited by the difficulty in linking predominantly noncoding variants to specific gene targets. Rare coding variant analyses provide greater confidence that a specific gene is involved, but do not necessarily indicate whether gain or loss of function (LoF) would be of most therapeutic benefit. OBJECTIVE: This work aimed to identify genes/proteins involved in determining fat distribution. METHODS: We combined the power of genome-wide analysis of array-based rare, nonsynonymous variants in 450â 562 individuals in the UK Biobank with exome-sequence-based rare LoF gene burden testing in 184â 246 individuals. RESULTS: The data indicate that the LoF of 4 genes (PLIN1 [LoF variants, Pâ =â 5.86â ×â 10-7], INSR [LoF variants, Pâ =â 6.21â ×â 10-7], ACVR1C [LoFâ +â moderate impact variants, Pâ =â 1.68â ×â 10-7; moderate impact variants, Pâ =â 4.57â ×â 10-7], and PDE3B [LoF variants, Pâ =â 1.41â ×â 10-6]) is associated with a beneficial effect on body mass index-adjusted waist-to-hip ratio and increased gluteofemoral fat mass, whereas LoF of PLIN4 (LoF variants, Pâ =â 5.86â ×â 10-7 adversely affects these parameters. Phenotypic follow-up suggests that LoF of PLIN1, PDE3B, and ACVR1C favorably affects metabolic phenotypes (eg, triglycerides [TGs] and high-density lipoprotein [HDL] cholesterol concentrations) and reduces the risk of cardiovascular disease, whereas PLIN4 LoF has adverse health consequences. INSR LoF is associated with lower TG and HDL levels but may increase the risk of type 2 diabetes. CONCLUSION: This study robustly implicates these genes in the regulation of fat distribution, providing new and in some cases somewhat counterintuitive insight into the potential consequences of targeting these molecules therapeutically.
Assuntos
Diabetes Mellitus Tipo 2 , Receptores de Ativinas Tipo I/genética , Distribuição da Gordura Corporal , Diabetes Mellitus Tipo 2/genética , Exoma , Variação Genética , Estudo de Associação Genômica Ampla , HumanosRESUMO
An imbalance in the storage and breakdown of hepatic lipid droplet (LD) triglyceride (TAG) leads to hepatic steatosis, a defining feature of non-alcoholic fatty liver disease (NAFLD). The two primary cellular pathways regulating hepatic TAG catabolism are lipolysis, initiated by adipose triglyceride lipase (ATGL), and lipophagy. Each of these processes requires access to the LD surface to initiate LD TAG catabolism. Ablation of perilipin 2 (PLIN2), the most abundant lipid droplet-associated protein in steatotic liver, protects mice from diet-induced NAFLD. However, the mechanisms underlaying this protection are unclear. We tested the contributions of ATGL and lipophagy mediated lipolysis to reduced hepatic TAG in mice with liver-specific PLIN2 deficiency (PLIN2LKO) fed a Western-type diet for 12 weeks. We observed enhanced autophagy in the absence of PLIN2, as determined by ex vivo p62 flux, as well as increased p62- and LC3-positive autophagic vesicles in PLIN2LKO livers and isolated primary hepatocytes. Increased levels of autophagy correlated with significant increases in cellular fatty acid (FA) oxidation in PLIN2LKO hepatocytes. We observed that inhibition of either autophagy or ATGL blunted the increased FA oxidation in PLIN2LKO hepatocytes. Additionally, combined inhibition of ATGL and autophagy reduced FA oxidation to the same extent as treatment with either inhibitor alone. In sum, these studies show that protection against NAFLD in the absence of hepatic PLIN2 is driven by the integrated actions of both ATGL and lipophagy.
Assuntos
Tecido Adiposo/enzimologia , Autofagia , Dieta/efeitos adversos , Lipase/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Perilipina-2/fisiologia , Animais , Lipase/genética , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Triglicerídeos/metabolismoRESUMO
OBJECTIVE: Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction. METHODS: We developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings. RESULTS: Ad-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro. CONCLUSION: These studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE).
Assuntos
Adipócitos/metabolismo , Coenzima A Ligases/genética , Obesidade/metabolismo , Células 3T3 , Adipócitos/patologia , Adiposidade , Animais , Células Cultivadas , Coenzima A Ligases/metabolismo , Dieta Hiperlipídica/efeitos adversos , Feminino , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/patologia , Consumo de Oxigênio , Fosfolipídeos/metabolismoRESUMO
OBJECTIVE: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ACSL isoforms. In vitro studies have suggested a role for ACSL5 in triglyceride synthesis; however, we have limited understanding of the in vivo actions of this ACSL isoform. METHODS: To elucidate the in vivo actions of ACSL5 we generated a line of mice in which ACSL5 expression was ablated in all tissues (ACSL5 (-/-) ). RESULTS: Ablation of ACSL5 reduced ACSL activity by â¼80% in jejunal mucosa, â¼50% in liver, and â¼37% in brown adipose tissue lysates. Body composition studies revealed that ACSL5 (-/-) , as compared to control ACSL5 (loxP/loxP) , mice had significantly reduced fat mass and adipose fat pad weights. Indirect calorimetry studies demonstrated that ACSL5 (-/-) had increased metabolic rates, and in the dark phase, increased respiratory quotient. In ACSL5 (-/-) mice, fasting glucose and serum triglyceride were reduced; and insulin sensitivity was improved during an insulin tolerance test. Both hepatic mRNA (â¼16-fold) and serum levels of fibroblast growth factor 21 (FGF21) (â¼13-fold) were increased in ACSL5 (-/-) as compared to ACSL5 (loxP/loxP) . Consistent with increased FGF21 serum levels, uncoupling protein-1 gene (Ucp1) and PPAR-gamma coactivator 1-alpha gene (Pgc1α) transcript levels were increased in gonadal adipose tissue. To further evaluate ACSL5 function in intestine, mice were gavaged with an olive oil bolus; and the rate of triglyceride appearance in serum was found to be delayed in ACSL5 (-/-) mice as compared to control mice. CONCLUSIONS: In summary, ACSL5 (-/-) mice have increased hepatic and serum FGF21 levels, reduced adiposity, improved insulin sensitivity, increased energy expenditure and delayed triglyceride absorption. These studies suggest that ACSL5 is an important regulator of whole-body energy metabolism and ablation of ACSL5 may antagonize the development of obesity and insulin resistance.
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For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-clamp technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-clamp fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.
Assuntos
Canais Iônicos/metabolismo , Neurônios/fisiologia , Oócitos/fisiologia , Xenopus , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Feminino , Microscopia Eletrônica de Varredura , Neurônios/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Membrana Vitelina/ultraestrutura , Xenopus laevisRESUMO
BACKGROUND: In addition to lowering LDL-C, emerging data suggests that phytosterols (PS) may reduce blood triglycerides (TG), however, the underlying mechanisms are not known. METHODS: We examined the TG-lowering mechanisms of dietary PS in Syrian golden hamsters randomly assigned to a high fat (HF) diet or the HF diet supplemented with PS (2%) for 6 weeks (n = 12/group). An additional subset of animals (n = 12) was provided the HF diet supplemented with ezetimibe (EZ, 0.002%) as a positive control as it is a cholesterol-lowering agent with known TG-lowering properties. RESULTS: In confirmation of diet formulation and compound delivery, both the PS and EZ treatments lowered (p < 0.05) intestinal cholesterol absorption (24 and 31%, respectively), blood non-HDL cholesterol (61 and 66%, respectively), and hepatic cholesterol (45 and 55%, respectively) compared with the HF-fed animals. Blood TG concentrations were lower (p < 0.05) in the PS (49%) and EZ (68%)-treated animals compared with the HF group. The TG-lowering response in the PS-supplemented group was associated with reduced (p < 0.05) intestinal SREBP1c mRNA (0.45 fold of HF), hepatic PPARα mRNA (0.73 fold of HF), hepatic FAS protein abundance (0.68 fold of HD), and de novo lipogenesis (44%) compared with the HF group. Similarly, lipogenesis was lower in the EZ-treated animals, albeit through a reduction in the hepatic protein abundance of ACC (0.47 fold of HF). CONCLUSIONS: Study results suggest that dietary PS are protective against diet-induced hypertriglyceridemia, likely through multiple mechanisms that involve modulation of intestinal fatty acid metabolism and a reduction in hepatic lipogenesis.
Assuntos
Anticolesterolemiantes/farmacologia , Hipertrigliceridemia/tratamento farmacológico , Fitosteróis/farmacologia , Animais , Anticolesterolemiantes/uso terapêutico , Azetidinas/farmacologia , Azetidinas/uso terapêutico , HDL-Colesterol/sangue , Cricetinae , Dieta Hiperlipídica/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Ezetimiba , Ácidos Graxos/metabolismo , Expressão Gênica/efeitos dos fármacos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/etiologia , Absorção Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Lipogênese , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Mesocricetus , Fitosteróis/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Early work suggested that dietary cholesterol increases plasma total cholesterol concentrations in humans. Given the relationship between elevated plasma cholesterol concentrations and cardiovascular disease risk dietary guidelines have consistently recommended limiting food sources of cholesterol. Current intakes are approaching recommended levels. Recently there have been calls to reassess the importance of continuing to recommend limiting dietary cholesterol. Over the past 10 years there have been a limited number of studies addressing this issue. Striking among these studies is the high degree of variability in background diet, subject characteristics and study design. Within the context of current levels of dietary cholesterol intake, the effect on plasma lipids concentrations, with primary interest in LDL-C cholesterol concentrations, is modest and appears to be limited to population subgroups. In these cases, restrictions in dietary cholesterol intake are likely warranted. The biological determinants of inter-individual variability remain a relatively understudied area.
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Thermoregulatory neurons in the preoptic area of the anterior hypothalamus (POA) form synaptic networks, which affect responses that regulate body temperature. To characterize these pathways of activation, projections to effector control areas, like the dorsomedial hypothalamus (DMH), require labeling in live tissue slices. Traditional fluorescent dyes label axon terminals near an injection site, but unfortunately, also that of nearby fibers of passage. Here, we describe a novel methodology for retrograde labeling of neurons in vitro, which will allow for further electrophysiological recording. To determine if POA neurons project to the DMH, we have used nanometer-sized, gold nanoprobes, which provide for specific neuronal entry, via synapses in close proximity to the injection site. Upon neuronal entry, these nanoprobe complexes diffuse to the soma, where they are readily visualized and quantified. We found that conjugation of these gold nanoprobes with VGLUT-2 antibodies and polyethyleneimine (PEI) facilitates neuronal entry and a high level of labeling efficacy. This novel method, adapted from emerging cancer therapy technologies, is highly specific for determining axon terminal projections within particular neuronal populations, while maintaining neuronal viability for targeted live cell electrophysiological recording.
Assuntos
Compostos de Ouro , Hipotálamo/fisiologia , Nanopartículas Metálicas , Neurônios/fisiologia , Área Pré-Óptica/fisiologia , Animais , Anticorpos/metabolismo , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Hipotálamo/citologia , Técnicas In Vitro , Masculino , Nanotecnologia , Vias Neurais/citologia , Vias Neurais/fisiologia , Marcadores do Trato Nervoso , Neurônios/citologia , Polietilenoimina/metabolismo , Área Pré-Óptica/citologia , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologia , Proteína Vesicular 2 de Transporte de Glutamato/imunologia , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
BACKGROUND: Transient hyperthermic shifts in body temperature have been linked to the endogenous hormone calcitonin gene-related peptide (CGRP), which can increase sympathetic activation and metabolic heat production. Recent studies have demonstrated that these centrally mediated responses may result from CGRP dependent changes in the activity of thermoregulatory neurons in the preoptic and anterior regions of the hypothalamus (POAH). RESULTS: Using a tissue slice preparation, we recorded the single-unit activity of POAH neurons from the adult male rat, in response to temperature and CGRP (10 muM). Based on the slope of firing rate as a function of temperature, neurons were classified as either warm sensitive or temperature insensitive. All warm sensitive neurons responded to CGRP with a significant decrease in firing rate. While CGRP did not alter the firing rates of some temperature insensitive neurons, responsive neurons showed an increase in firing rate. CONCLUSION: With respect to current models of thermoregulatory control, these CGRP dependent changes in firing rate would result in hyperthermia. This suggests that both warm sensitive and temperature insensitive neurons in the POAH may play a role in producing this hyperthermic shift in temperature.
Assuntos
Núcleo Hipotalâmico Anterior/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Hipotálamo Anterior/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Núcleo Hipotalâmico Anterior/citologia , Núcleo Hipotalâmico Anterior/fisiologia , Regulação da Temperatura Corporal/efeitos dos fármacos , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Hipotálamo Anterior/citologia , Hipotálamo Anterior/fisiologia , Masculino , Microeletrodos , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Área Pré-Óptica/citologia , Área Pré-Óptica/efeitos dos fármacos , Área Pré-Óptica/fisiologia , Ratos , Ratos Sprague-Dawley , Análise de Regressão , Temperatura , Técnicas de Cultura de TecidosRESUMO
Peripheral exposure to LPS induces a biphasic fever thought to be initiated via vagal afferents to the preoptic area of the anterior hypothalamus (POAH), an important thermoregulatory control center in the brain. Previous studies have shown that norepinephrine synaptically mediates this Prostaglandin E(2) (PGE(2))-dependent change in temperature through the selective activation of alpha-2 adrenoreceptors (AR). However, there is clear evidence that alpha-1 AR activation of thermoregulatory hypothalamic neurons will result in a rapid hyperthermia that is not dependent on PGE(2). This direct action of norepinephrine in the POAH was tested in the present study by recording the single-unit activity of POAH neurons in a tissue slice preparation from the adult male rat, in response to temperature and the selective alpha-1 AR agonist Cirazoline (1-100 microM). Neurons were classified as either warm sensitive or temperature insensitive. Warm sensitive neurons responded to Cirazoline with a decrease in firing rate, while temperature insensitive neurons showed a firing rate increase. These responses are similar to those reported for PGE(2) and suggest that both warm sensitive and temperature insensitive neurons in the POAH are important in mediating this alpha-1 AR-dependent hyperthermic shift in body temperature.
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Potenciais de Ação/efeitos dos fármacos , Agonistas alfa-Adrenérgicos/farmacologia , Núcleo Hipotalâmico Anterior/citologia , Temperatura Alta , Imidazóis/farmacologia , Neurônios/efeitos dos fármacos , Animais , Masculino , Neurônios/classificação , Neurônios/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Physiological and morphological evidence suggests that activation of the ventromedial preoptic area of the hypothalamus (VMPO) is an essential component of an intravenous LPS-dependent fever. In response to the endogenous pyrogen prostaglandin E2 (PGE2), the majority of temperature insensitive neurons in the VMPO show an increase in firing rate, while warm sensitive neurons are inhibited. We have hypothesized that these PGE2 dependent effects on firing rate are due to changes in the inherent electrical properties of VMPO neurons, which are regulated by the activity of specific ionic currents. RESULTS: To characterize the electrical properties of VMPO neurons, whole-cell recordings were made in tissue slices from male Sprague-Dawley rats. Our results indicate that PGE2 dependent firing rate responses were not the result of changes in resting membrane potential, action potential amplitude and duration, or local synaptic input. However, PGE2 reduced the input resistance of all VMPO neurons, while increasing the excitability of temperature insensitive neurons and decreasing the excitability of warm sensitive neurons. In addition, the majority of temperature insensitive neurons responded to PGE2 with an increase in the rate of rise of the depolarizing prepotential that precedes each action potential. This response to PGE2 was reversed for warm sensitive neurons, in which the prepotential rate of rise decreased. CONCLUSION: We would therefore suggest that PGE2 is having an effect on the ionic currents that regulate firing rate by controlling how fast membrane potential rises to threshold during the prepotential phase of the action potential.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Dinoprostona/farmacologia , Neurônios/efeitos dos fármacos , Área Pré-Óptica/efeitos dos fármacos , Temperatura , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Temperatura Alta , Hipotálamo/efeitos dos fármacos , Hipotálamo/fisiologia , Técnicas In Vitro , Masculino , Neurônios/classificação , Neurônios/fisiologia , Área Pré-Óptica/fisiologia , Ratos , Ratos Sprague-Dawley , Núcleo Hipotalâmico Ventromedial/fisiologiaRESUMO
The primary motivation for integrating any form of education technology into a particular course or curriculum should always be to enhance student learning. However, it can be difficult to determine which technologies will be the most appropriate and effective teaching tools. Through the alignment of technology-enhanced learning experiences with a clear set of learning objectives, teaching becomes more efficient and effective and learning is truly enhanced. In this article, I describe how I have made extensive use of technology in two neuroscience courses that differ in structure and content. Course websites function as resource centers and provide a forum for student interaction. PowerPoint presentations enhance formal lectures and provide an organized outline of presented material. Some lectures are also supplemented with interactive CD-ROMs, used in the presentation of difficult physiological concepts. In addition, a computer-based physiological recording system is used in laboratory sessions, improving the hands-on experience of group learning while reinforcing the concepts of the research method. Although technology can provide powerful teaching tools, the enhancement of the learning environment is still dependent on the instructor. It is the skill and enthusiasm of the instructor that determines whether technology will be used effectively.
Assuntos
Aprendizagem , Neurociências/educação , Estudantes , Materiais de Ensino , Ensino , Tecnologia , CD-ROM , Instrução por Computador , Avaliação Educacional/métodos , Humanos , Internet , Tecnologia/normas , Interface Usuário-ComputadorRESUMO
In response to an immune system challenge with lipopolysaccharide (LPS), recent work has shown that Fos immunoreactivity is displayed by neurons in the ventromedial preoptic area of the hypothalamus (VMPO). In addition, neurons in this region show distinct axonal projections to the anterior perifornical area (APFx) and the paraventricular nucleus (PVN). It has been hypothesized that neurons within the VMPO integrate their local responses to temperature with changes in firing activity that result from LPS induced production of prostaglandin E(2) (PGE(2)). This may be an important mechanism by which the set-point regulation of thermoeffector neurons in the APFx and PVN is altered, resulting in hyperthermia. To characterize the firing rate activity of VMPO neurons, single-unit recordings were made of neuronal extracellular activity in rat hypothalamic tissue slices. Based on the slope of firing rate as a function of tissue temperature, neurons were classified as either warm sensitive or temperature insensitive. Neurons were then treated with PGE(2) (200 nM) while tissue temperature was held at a constant level ( approximately 36 degrees C). The majority of temperature insensitive neurons responded to PGE(2) with an increase in firing rate activity, while warm sensitive neurons showed a reduction in firing rate. This suggests that both warm sensitive and temperature insensitive neurons in the VMPO may play critical and contrasting roles in the production of a fever during an acute phase response to infection.