RESUMO
Mitochondrial reactive oxygen species (ROS) are important cellular signaling molecules, but can cause oxidative damage if not kept within tolerable limits. An important proximal form of ROS in mitochondria is superoxide. Its production is thought to occur in regulated stochastic bursts, but current methods using mitochondrial targeted cpYFP to assess superoxide flashes are confounded by changes in pH. Accordingly, these flashes are generally referred to as 'mitoflashes'. Here we provide regulatory insights into mitoflashes and pH fluctuations in skeletal muscle, and the role of uncoupling protein-3 (UCP3). Using quantitative confocal microscopy of mitoflashes in intact muscle fibers, we show that the mitoflash magnitude significantly correlates with the degree of mitochondrial inner membrane depolarization and ablation of UCP3 did not affect this correlation. We assessed the effects of the absence of UCP3 on mitoflash activity in intact skeletal muscle fibers, and found no effects on mitoflash frequency, amplitude or duration, with a slight reduction in the average size of mitoflashes. We further investigated the regulation of pH flashes (pHlashes, presumably a component of mitoflash) by UCP3 using mitochondrial targeted SypHer (mt-SypHer) in skeletal muscle fibers. The frequency of pHlashes was significantly reduced in the absence of UCP3, without changes in other flash properties. ROS scavenger, tiron, did not alter pHlash frequency in either WT or UCP3KO mice. High resolution respirometry revealed that in the absence of UCP3 there is impaired proton leak and Complex I-driven respiration and maximal coupled respiration. Total cellular production of hydrogen peroxide (H2O2) as detected by Amplex-UltraRed was unaffected. Altogether, we demonstrate a correlation between mitochondrial membrane potential and mitoflash magnitude in skeletal muscle fibers that is independent of UCP3, and a role for UCP3 in the control of pHlash frequency and of proton leak- and Complex I coupled-respiration in skeletal muscle fibers. The differential regulation of mitoflashes and pHlashes by UCP3 and tiron also indicate that the two events, though may be related, are not identical events.
Assuntos
Concentração de Íons de Hidrogênio , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteína Desacopladora 3/fisiologia , Animais , Metabolismo Energético , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Consumo de Oxigênio , Proteína Desacopladora 3/genéticaRESUMO
BACKGROUND: IL-15 is believed to play a role in the beneficial impact of exercise on muscle energy metabolism. However, previous studies have generally used supraphysiological levels of IL-15 that do not represent contraction-induced IL-15 secretion. METHODS: L6 myotubes were treated acutely (3â¯h) and chronically (48â¯h) with concentrations of IL-15 mimicking circulating (1-10â¯pg/ml) and muscle interstitial (100â¯pg/ml -20â¯ng/ml) IL-15 levels with the aim to better understand its autocrine/paracrine role on muscle glucose uptake and mitochondrial function. RESULTS: Acute exposure to IL-15 levels representing muscle interstitial IL-15 increased basal glucose uptake without affecting insulin sensitivity. This was accompanied by increased mitochondrial oxidative functions in association with increased AMPK pathway and formation of complex III-containing supercomplexes. Conversely, chronic IL-15 exposure resulted in a biphasic effect on mitochondrial oxidative functions and ETC supercomplex formation was increased with low IL-15 levels but decreased with higher IL-15 concentrations. The AMPK pathway was activated only by high levels of chronic IL-15 treatment. Similar results were obtained in skeletal muscle from muscle-specific IL-15 overexpressing mice that show very high circulating IL-15 levels. CONCLUSIONS: Acute IL-15 treatment that mimics local IL-15 concentrations enhances muscle glucose uptake and mitochondrial oxidative functions. That mitochondria respond differently to different levels of IL-15 during chronic treatments indicates that IL-15 might activate two different pathways in muscle depending on IL-15 concentrations. GENERAL SIGNIFICANCE: Our results suggest that IL-15 may act in an autocrine/paracrine fashion and be, at least in part, involved in the positive effect of exercise on muscle energy metabolism.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Respiração Celular/efeitos dos fármacos , Glucose/metabolismo , Interleucina-15/farmacologia , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Transporte de Elétrons/efeitos dos fármacos , Interleucina-15/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Oxirredução , RatosRESUMO
BACKGROUND/OBJECTIVES: Inter-individual variability in weight loss during obesity treatment is complex and poorly understood. Here we use whole body and tissue approaches to investigate fuel oxidation characteristics in skeletal muscle fibers, cells and distinct circulating protein biomarkers before and after a high fat meal (HFM) challenge in those who lost the most (obese diet-sensitive; ODS) vs the least (obese diet-resistant; ODR) amount of weight in a highly controlled weight management program. SUBJECTS/METHODS: In 20 weight stable-matched ODS and ODR women who previously completed a standardized clinical weight loss program, we analyzed whole-body energetics and metabolic parameters in vastus lateralis biopsies and plasma samples that were obtained in the fasting state and 6 h after a defined HFM, equivalent to 35% of total daily energy requirements. RESULTS: At baseline (fasting) and post-HFM, muscle fatty acid oxidation and maximal oxidative phosphorylation were significantly greater in ODS vs ODR, as was reactive oxygen species emission. Plasma proteomics of 1130 proteins pre and 1, 2, 5 and 6 h after the HFM demonstrated distinct group and interaction differences. Group differences identified S-formyl glutathione hydratase, heat shock 70 kDA protein 1A/B (HSP72), and eukaryotic translation initiation factor 5 (eIF5) to be higher in ODS vs ODR. Group-time differences included aryl hydrocarbon interacting protein (AIP), peptidylpropyl isomerase D (PPID) and tyrosine protein-kinase Fgr, which increased in ODR vs ODS over time. HSP72 levels correlated with muscle oxidation and citrate synthase activity. These proteins circulate in exosomes; exosomes isolated from ODS plasma increased resting, leak and maximal respiration rates in C2C12 myotubes by 58%, 21% and 51%, respectively, vs those isolated from ODR plasma. CONCLUSIONS: Findings demonstrate distinct muscle metabolism and plasma proteomics in fasting and post-HFM states corresponding in diet-sensitive vs diet-resistant obese women.
Assuntos
Proteínas Sanguíneas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Obesidade , Proteoma/metabolismo , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Estudos de Casos e Controles , Dieta , Exossomos/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Obesidade/sangue , Obesidade/dietoterapia , Obesidade/epidemiologia , Obesidade/metabolismo , Proteoma/análise , Falha de TratamentoRESUMO
BACKGROUND: In utero undernutrition is associated with obesity and insulin resistance, although its effects on skeletal muscle remain poorly defined. Therefore, in the current study we explored the effects of in utero food restriction on muscle energy metabolism in mice. METHODS: We used an experimental mouse model system of maternal undernutrition during late pregnancy to examine offspring from undernourished dams (U) and control offspring from ad libitum-fed dams (C). Weight loss of 10-week-old offspring on a 4-week 40% calorie-restricted diet was also followed. Experimental approaches included bioenergetic analyses in isolated mitochondria, intact (permeabilized) muscle and at the whole body level. RESULTS: U have increased adiposity and decreased glucose tolerance compared to C. Strikingly, when U are put on a 40% calorie-restricted diet they lose half as much weight as calorie-restricted controls. Mitochondria from muscle overall from U had decreased coupled (state 3) and uncoupled (state 4) respiration and increased maximal respiration compared to C. Mitochondrial yield was lower in U than C. In permeabilized fiber preparations from mixed fiber-type muscle, U had decreased mitochondrial content and decreased adenylate-free leak respiration, fatty acid oxidative capacity and state 3 respiratory capacity through complex I. Fiber maximal oxidative phosphorylation capacity did not differ between U and C but was decreased with calorie restriction. CONCLUSIONS: Our results reveal that in utero undernutrition alters metabolic physiology through a profound effect on skeletal muscle energetics and blunts response to a hypocaloric diet in adulthood. We propose that mitochondrial dysfunction links undernutrition in utero with metabolic disease in adulthood.
Assuntos
Glicemia/metabolismo , Restrição Calórica/efeitos adversos , Recém-Nascido de Baixo Peso/metabolismo , Desnutrição/patologia , Músculo Esquelético/patologia , Adiposidade , Animais , Modelos Animais de Doenças , Camundongos , Redução de PesoRESUMO
Growing evidence highlights a role for mitochondrial dysfunction and oxidative stress as underlying contributors to Parkinson's disease (PD) pathogenesis. DJ-1 (PARK7) is a recently identified recessive familial PD gene. Its loss leads to increased susceptibility of neurons to oxidative stress and death. However, its mechanism of action is not fully understood. Presently, we report that DJ-1 deficiency in cell lines, cultured neurons, mouse brain and lymphoblast cells derived from DJ-1 patients display aberrant mitochondrial morphology. We also show that these DJ-1-dependent mitochondrial defects contribute to oxidative stress-induced sensitivity to cell death since reversal of this fragmented mitochondrial phenotype abrogates neuronal cell death. Reactive oxygen species (ROS) appear to play a critical role in the observed defects, as ROS scavengers rescue the phenotype and mitochondria isolated from DJ-1 deficient animals produce more ROS compared with control. Importantly, the aberrant mitochondrial phenotype can be rescued by the expression of Pink1 and Parkin, two PD-linked genes involved in regulating mitochondrial dynamics and quality control. Finally, we show that DJ-1 deficiency leads to altered autophagy in murine and human cells. Our findings define a mechanism by which the DJ-1-dependent mitochondrial defects contribute to the increased sensitivity to oxidative stress-induced cell death that has been previously reported.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Doença de Parkinson/genética , Acetilcisteína/farmacologia , Animais , Autofagia/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Proteínas Mutantes/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Neostriado/patologia , Neostriado/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Neurônios/ultraestrutura , Doença de Parkinson/patologia , Peroxirredoxinas , Fenótipo , Proteína Desglicase DJ-1 , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
AIMS/HYPOTHESIS: AMP-activated protein kinase (AMPK) has a broad role in the regulation of glucose and lipid metabolism making it a promising target in the treatment of type 2 diabetes mellitus. We therefore sought to characterise for the first time the effects of chronic AMPK activation on skeletal muscle carbohydrate metabolism in carriers of the rare gain-of-function mutation of the gene encoding AMPKgamma(3) subunit, PRKAG3 R225W. METHODS: Aspects of fuel metabolism were studied in vitro in myocytes isolated from vastus lateralis of PRKAG3 R225W carriers and matched control participants. In vivo, muscular strength and fatigue were evaluated by isokinetic dynamometer and surface electromyography, respectively. Glucose uptake in exercising quadriceps was determined using [(18)F]fluorodeoxyglucose and positron emission tomography. RESULTS: Myotubes from PRKAG3 R225W carriers had threefold higher mitochondrial content (p < 0.01) and oxidative capacity, higher leak-dependent respiration (1.6-fold, p < 0.05), higher basal glucose uptake (twofold, p < 0.01) and higher glycogen synthesis rates (twofold, p < 0.05) than control myotubes. They also had higher levels of intracellular glycogen (p < 0.01) and a trend for lower intramuscular triacylglycerol stores. R225W carriers showed remarkable resistance to muscular fatigue and a trend for increased glucose uptake in exercising muscle in vivo. CONCLUSIONS/INTERPRETATION: Through the enhancement of skeletal muscle glucose uptake and increased mitochondrial content, the R225W mutation may significantly enhance exercise performance. These findings are also consistent with the hypothesis that the gamma(3) subunit of AMPK is a promising tissue-specific target for the treatment of type 2 diabetes mellitus, a condition in which glucose uptake and mitochondrial function are impaired.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Adulto , Transporte Biológico/genética , Transporte Biológico/fisiologia , Western Blotting , Células Cultivadas , Ácidos Graxos/metabolismo , Glicogênio/metabolismo , Humanos , Técnicas In Vitro , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Mutação , Consumo de Oxigênio/genética , Consumo de Oxigênio/fisiologia , Tomografia por Emissão de Pósitrons , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIMS/HYPOTHESIS: Obesity is an important risk factor for the development of type 2 diabetes, but not all obese individuals develop this complication. The clinical signs of type 2 diabetes can often be reversed with weight loss; however, it is unknown whether the skeletal muscle oxidative stress associated with type 2 diabetes remains after weight loss. We hypothesised that chronic exposure to high glucose and insulin would re-elicit impaired metabolism in primary myotubes from patients with a history of type 2 diabetes. METHODS: Obese participants with or without type 2 diabetes completed a standardised weight loss protocol, following which all participants were euglycaemic and had similar indices of insulin sensitivity. Satellite cells were isolated from muscle biopsies and differentiated under low or high glucose and insulin conditions (HGI). RESULTS: Cells from participants with no history of type 2 diabetes showed robust increases in mitochondrial content, citrate synthase and cytochrome c oxidase activities when exposed to HGI. This increase in oxidative capacity was absent in cells from patients with a history of type 2 diabetes. High glucose and insulin caused increased oxidative damage in cells from the latter, despite higher superoxide dismutase expression. Cells from patients with a history of type 2 diabetes were unable to decrease mitochondrial membrane potential in response to HGI, potentially due to lower levels of uncoupling protein-3. CONCLUSIONS/INTERPRETATION: This is the first report to note that primary myotubes from patients with a history of type 2 diabetes are unable to adapt to a hyperglycaemic-hyperinsulinaemic challenge. We have demonstrated that impaired mitochondrial biogenesis and an inability to manage oxidative stress define a muscle phenotype at risk of obesity-associated type 2 diabetes.
Assuntos
Complicações do Diabetes/metabolismo , Diabetes Mellitus Tipo 2/epidemiologia , Fibras Musculares Esqueléticas/patologia , Obesidade/complicações , Obesidade/dietoterapia , Adulto , Composição Corporal , Índice de Massa Corporal , Complicações do Diabetes/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevenção & controle , Feminino , Técnica Clamp de Glucose , Hemoglobinas Glicadas/metabolismo , Glicogênio/metabolismo , Humanos , Hiperinsulinismo , Hipoglicemiantes/uso terapêutico , Insulina/farmacologia , Canais Iônicos/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Estresse Oxidativo , Triglicerídeos/metabolismo , Proteína Desacopladora 3 , Redução de PesoRESUMO
The aim of this study was to determine the biochemical mechanism(s) responsible for enhanced FA utilization (oxidation and esterification) by perfused hearts from type 2 diabetic db/db mice. The plasma membrane content of fatty acid transporters FAT/CD36 and FABPpm was elevated in db/db hearts. Mitochondrial mechanisms that could contribute to elevated rates of FA oxidation were also examined. Carnitine palmitoyl transferase-1 activity was unchanged in mitochondria from db/db hearts, and sensitivity to inhibition by malonyl-CoA was unchanged. Malonyl-CoA content was elevated and AMP kinase activity was decreased in db/db hearts, opposite to what would be expected in hearts exhibiting elevated rates of FA oxidation. Uncoupling protein-3 expression was unchanged in mitochondria from db/db hearts. Therefore, enhanced FA utilization in db/db hearts is most likely due to increased FA uptake caused by increased plasma membrane content of FA transporters; the mitochondrial mechanisms examined do not contribute to elevated FA oxidation observed in db/db hearts.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos/metabolismo , Miocárdio/metabolismo , Animais , Membrana Celular/fisiologia , Diabetes Mellitus Tipo 2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/fisiologia , PerfusãoRESUMO
The lifespan of the nematode, Caenorhabditis elegans, can be extended by mutations affecting components of the insulin-like growth factor (IGF) signaling cascade or by overexpression of SIR2, an NAD+-dependent protein deacetylase. The mammalian homologue of SIR2, Sirt1, has been shown to modulate the activity of FoxO, a transcription factor that is downstream of the IGF signaling system. These results suggest that Sirt1 ought to affect the IGF pathway. We report here evidence that this is the case in mice. The loss of Sirt1 protein in mice results in increased expression of the IGF binding protein IGFBP1, a secreted modulator of IGF function. A number of the anatomical characteristics of Sirt1-null mice closely resemble those of transgenic mice overexpressing IGFBP1. Our data suggest that Sirt1 is part of a regulatory loop that limits the production of IGFBP1 thereby modulating IGF signaling.
Assuntos
Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Transdução de Sinais/fisiologia , Sirtuínas/metabolismo , Somatomedinas/metabolismo , Animais , Caenorhabditis elegans/genética , Longevidade/genética , Camundongos , Camundongos Mutantes , Sirtuína 1 , Sirtuínas/genética , Fatores de Transcrição/metabolismoRESUMO
Uncoupling protein 3 (UCP3) expression is directly correlated to fatty acid oxidation in skeletal muscle. UCP3 has been hypothesized to facilitate high rates of fatty acid oxidation, but evidence thus far is lacking. Our aim was to investigate the effects of UCP3 overexpression and ablation on fatty acid uptake and metabolism in muscle of mice having congenic backgrounds. In mice constitutively expressing the UCP3 protein (human form) at levels just over twofold higher than normal (230% of wild-type levels), indirect calorimetry demonstrated no differences in total energy expenditure (VO2), but a shift toward increased fat oxidation compared with wild-type (WT) mice. Metabolic efficiency (gram weight gain/kcal ingested) was similar between Ucp3 overexpressors, WT and Ucp3 (-/-) mice. In muscle of Ucp3-tg mice, plasma membrane fatty acid binding protein (FABPpm) content was increased compared with WT mice. Although hormone-sensitive lipase activity was unchanged across the genotypes, there were increases in carnitine palmitoyltransferase I, beta-hydroxyacylCoA dehydrogenase, and citrate synthase activities and decreases in intramuscular triacylglycerol in muscle of Ucp3-tg mice. There were no differences in muscle mitochondrial content. High-energy phosphates and total muscle carnitine and CoA were also greater in Ucp3-tg compared with WT mice. Taken together, the findings demonstrate an increased capacity for fat oxidation in the absence of significant increases in thermogenesis in Ucp3-tg mice. Findings from Ucp3 (-/-) mice revealed few differences compared with WT mice, consistent with the possibility of compensatory mechanisms. In conjunction with our observed increases in CoA and carnitine in muscle of Ucp3 overexpressors, the findings support the hypothesized role for Ucp3 in facilitating fatty acid oxidation in muscle.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Ácidos Graxos/metabolismo , Músculo Esquelético/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Difosfato de Adenosina/análise , Trifosfato de Adenosina/análise , Animais , Transporte Biológico , Calorimetria Indireta , Carnitina/análise , Carnitina O-Palmitoiltransferase/metabolismo , Proteínas de Transporte/análise , Citrato (si)-Sintase/metabolismo , Coenzima A/análise , Creatina/análise , Metabolismo Energético , Feminino , Expressão Gênica , Canais Iônicos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/química , Proteínas Mitocondriais , Músculo Esquelético/ultraestrutura , Oxirredução , Consumo de Oxigênio , Fosfatos/análise , Triglicerídeos/análise , Proteína Desacopladora 3RESUMO
Recent evidence demonstrates that growth factor networks are highly interactive with the estrogen receptor (ER) in the control of breast cancer growth and development. As such, tumor responses to anti-hormones are likely to be a composite of the ER and growth factor inhibitory activity of these agents, with alterations/aberrations in growth factor signalling providing a mechanism for the development of anti-hormone resistance. In this light, the current article focuses on illustrating the relationship between growth factor signalling and anti-hormone failure in our in-house tumor models of breast cancer and describes how we are now beginning to successfully target their actions to improve the effects of anti-hormonal drugs and to block aggressive disease progression.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Substâncias de Crescimento/metabolismo , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Receptores ErbB/metabolismo , Feminino , Antagonistas de Hormônios/uso terapêutico , Humanos , Invasividade Neoplásica , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Tamoxifeno/uso terapêutico , Falha de TratamentoRESUMO
Mitochondria are a cell's single greatest source of reactive oxygen species. Reactive oxygen species are important for many life sustaining processes of cells and tissues, but they can also induce cell damage and death. If their production and levels within cells is not effectively controlled, then the detrimental effects of oxidative stress can accumulate. Oxidative stress is widely thought to underpin many ageing processes, and the oxidative stress theory of ageing is one of the most widely acknowledged theories of ageing. As well as being the major source of reactive oxygen species, mitochondria are also a major site of oxidative damage. The purpose of this review is a concise and current review of the effects of oxidative stress and ageing on mitochondrial function. Emphasis is placed upon the roles of mitochondrial proton leak, the uncoupling proteins, and the anti-ageing effects of caloric restriction.
Assuntos
Envelhecimento/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Mitocôndrias/fisiologia , Estresse Oxidativo/fisiologia , Animais , Restrição Calórica , Regulação da Expressão Gênica , Humanos , Canais Iônicos , Proteínas Mitocondriais , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 1RESUMO
SETTING: Twenty-three Gambian villages. OBJECTIVE: To evaluate the feasibility of involving traditional healers in tuberculosis diagnosis and treatment in The Gambia. DESIGN: Twenty-eight traditional healers were educated in the recognition of signs and symptoms of tuberculosis and indications for referral. They administered medications to confirmed cases, and were revisited after 1 year to assess knowledge retention. RESULTS: Over 6 months, the traditional healers referred 66 suspects, from whom eight cases were diagnosed. All were successfully treated. Twenty-three of 24 traditional healers re-interviewed retained appropriate knowledge; 16 continued to refer suspects. CONCLUSIONS: Traditional healers can play a positive role in tuberculosis control.
Assuntos
Medicinas Tradicionais Africanas , Tuberculose/diagnóstico , Tuberculose/terapia , Adulto , Idoso , Estudos de Viabilidade , Feminino , Gâmbia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
AIMS/HYPOTHESIS: It has been suggested that uncoupling protein 3 (UCP3) can increase energy expenditure, thereby regulating body weight. Although studies on UCP3 knock-out mice suggest that lack of UCP3 function does not cause obesity or Type 2 diabetes, it is possible that up-regulation of UCP3 function improves these disorders or their clinical sequelae. A 10- to 20-fold increase of UCP3 gene expression is achievable through physiological or pharmacological stimuli. We examined the phenotype of transgenic mice with approximately 18-fold overexpression of mouse UCP3 mRNA in skeletal muscle. METHODS: We generated transgenic mice with approximately 18-fold overexpression of mouse UCP3 mRNA in skeletal muscle under control of the skeletal muscle-specific muscle creatine kinase gene promoter. The phenotype of these mice was analysed either on a standard diet or on a 4-week high-fat diet. RESULTS: In mice on standard chow, there was no difference in body weight, oxygen consumption and mitochondrial protonmotive force between transgenic mice and non-transgenic littermates. However, transgenic mice tended to have lower body weight, increased oxygen consumption and decreased mitochondrial protonmotive force than the control mice. Transgenic mice on a 4-week high-fat diet consumed much more oxygen and had noticeably less weight gain and less epididymal fat, as well as better glucose tolerance than non-transgenic littermates. CONCLUSIONS/INTERPRETATION: Our study shows that 18-fold overexpression of UCP3 mRNA in the skeletal muscle reduced diet-induced obesity. An 18-fold increase of UCP3 mRNA can be attained by physiological or pharmacological stimuli, suggesting that UCP3 has therapeutic potential in the treatment of obesity.
Assuntos
Proteínas de Transporte/genética , Dieta , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Obesidade/genética , Envelhecimento , Ração Animal , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , Primers do DNA , Dieta Redutora , Teste de Tolerância a Glucose , Canais Iônicos , Cinética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais , Consumo de Oxigênio , Proteína Desacopladora 3 , Redução de PesoRESUMO
Although many estrogen receptor-positive breast cancers initially respond to antihormones, responses are commonly incomplete with resistance ultimately emerging. Delineation of signaling mechanisms underlying these phenomena would allow development of therapies to improve antihormone response and compromise resistance. This in vitro investigation in MCF-7 breast cancer cells examines whether epidermal growth factor receptor (EGFR) signaling limits antiproliferative and proapoptotic activity of antihormones and ultimately supports development of resistance. It addresses whether the anti-EGFR agent gefitinib (ZD1839/Iressa; TKI: 1 mum) combined with the antihormones 4-hydroxytamoxifen (TAM: 0.1 mum) or fulvestrant (Faslodex; 0.1 mum) enhances growth inhibition and prevents resistance. TAM significantly suppressed MCF-7 growth over wk 2-5, reducing proliferation detected by immunocytochemistry and fluorescence-activated cell sorter cell cycle analysis. A modest apoptotic increase was observed by fluorescence-activated cell sorter and fluorescence microscopy, with incomplete bcl-2 suppression. EGFR induction occurred during TAM response, as measured by immunocytochemistry and Western blotting, with EGFR-positive, highly proliferative resistant growth subsequently emerging. Although TKI alone was ineffective on growth, TAM plus TKI cotreatment exhibited superior antigrowth activity vs. TAM, with no viable cells by wk 12. Cotreatment was more effective in inhibiting proliferation, promoting apoptosis, and eliminating bcl-2. Cotreatment blocked EGFR induction, markedly depleted ERK1/2 MAPK and protein kinase B phosphorylation, and prevented emergence of EGFR-positive resistance. Faslodex plus TKI cotreatment was also a superior antitumor strategy. Thus, increased EGFR evolves during treatment with antihormones, limiting their efficacy and promoting resistance. Gefitinib addition to antihormonal therapy could prove more effective in treating estrogen receptor-positive breast cancer and may combat development of resistance.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Fator de Crescimento Epidérmico/antagonistas & inibidores , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Proteínas Serina-Treonina Quinases , Quinazolinas/farmacologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Combinação de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Receptores ErbB/fisiologia , Feminino , Fulvestranto , Gefitinibe , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/efeitos dos fármacosRESUMO
An increasing body of evidence demonstrates that growth factor networks are highly interactive with estrogen receptor signaling in the control of breast cancer growth. As such, tumor responses to antihormones are likely to be a composite of the estrogen receptor and growth factor inhibitory activity of these agents. The modulation of growth factor networks during endocrine response is examined, and in vitro and clinical evidence is presented that epidermal growth factor receptor signaling, maintained in either an estrogen receptor-dependent or a receptor-independent manner, is critical to antihormone-resistant breast cancer cell growth. The considerable potential of the epidermal growth factor receptor-selective tyrosine kinase inhibitor Iressa (ZD 1839) to efficiently treat, and perhaps even prevent, endocrine-resistant breast cancer is highlighted.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores ErbB/metabolismo , Estradiol/análogos & derivados , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Divisão Celular/efeitos dos fármacos , Glândulas Endócrinas , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Feminino , Fulvestranto , Gefitinibe , Humanos , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Receptor ErbB-2/fisiologia , Transdução de Sinais/fisiologia , Tamoxifeno/farmacologia , Trastuzumab , Células Tumorais CultivadasRESUMO
The physiological function of uncoupling protein 3 (UCP3) is as yet unknown. Based on its 57% homology to UCP1 whose physiologic function is uncoupling and thermogenesis, UCP3 was attributed with the function of mitochondrial uncoupling through proton-leak reactions. UCP3 is expressed selectively in muscle, a tissue in which it has been estimated that proton leak accounts for approx. 50% of resting energy metabolism. Genetic linkage, association and variant studies suggest a role for UCP3 in obesity and/or diabetes. Studies of the heterologous expression of UCP3 in yeast provide support for the idea that UCP3 can uncouple mitochondrial oxidative phosphorylation, but the physiological relevance of these results is questionable. In vitro studies of mitochondria from Ucp3(-/-) mice provide support, but there are no changes in resting metabolic rate (RMR) of mice. In vivo studies demonstrate increased ATP synthesis, but estimates of substrate oxidation rate indicate no change. Mice that greatly overexpress Ucp3 in muscle have increased RMR. Inconsistent with the function of uncoupling are the observations that fasting results in increased expression of UCP3, but no change in muscle proton leak. Moreover, fasting decreases energy expenditure in muscle. Expression patterns for Ucp3 and lipid-metabolism genes support a physiological role in fatty acid oxidation. Overall, findings support a role for Ucp3 in fatty acid metabolism that may have implications for obesity and/or Type II diabetes.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Mitocôndrias/metabolismo , Animais , Proteínas de Transporte/genética , Clonagem Molecular , Canais Iônicos , Camundongos , Proteínas Mitocondriais , Músculos/metabolismo , Prótons , Proteína Desacopladora 3RESUMO
TENB2 encodes a putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin, which has been identified through the application of a differential display technique to a xenograft model of prostate cancer. Northern analysis and competitive PCR were used to demonstrate significantly increased TENB2 expression (p = 0.0003) on the acquisition of androgen independence in the model system. TENB2 is also overexpressed in clinical prostate carcinoma vs. its benign counterpart (p < 0.0001), with particular prominence in high-grade tumours, and shows a high degree of tissue specificity, being detected on a multitissue Northern array exclusively in brain and prostate material. Studies of recombinant protein expression demonstrate that TENB2 is a chondroitin sulphate proteoglycan. The presence of an EGF and 2 follistatin domains suggests a role in the regulation of growth factor signalling either as a ligand precursor, a membrane-bound receptor or as a binding protein for growth factors. These data are indicative of a significant role for TENB2 in the progression of poorly differentiated tumour types, with implications for prostate cancer detection, prognosis and therapy.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/análise , Neoplasias da Próstata/química , Sequência de Aminoácidos , Animais , Células CHO , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/genética , Sulfatos de Condroitina/análise , Cricetinae , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Neoplasias da Próstata/patologiaRESUMO
Uncoupling protein-3 (UCP3) is a mitochondrial carrier protein of as yet undefined physiological function. To elucidate characteristics of its function, we studied the effects of fasting on resting metabolic rate, respiratory quotient, muscle Ucp3 expression, and mitochondrial proton leak in wild-type and Ucp3(-/-) mice. Also analyzed were the fatty acid compositions of skeletal muscle mitochondria in fed and fasted Ucp3(-/-) and wild-type mice. In wild-type mice, fasting caused significant increases in Ucp3 (4-fold) and Ucp2 (2-fold) mRNA but did not significantly affect mitochondrial proton leak. State 4 oxygen consumption was not affected by fasting in either of the two groups. However, protonmotive force was consistently higher in mitochondria of Ucp3(-/-) animals (P = 0.03), and fasting further augmented protonmotive force in Ucp3(-/-) mice; there was no effect in wild-type mitochondria. Resting metabolic rates decreased with fasting in both groups. Ucp3(-/-) mice had higher respiratory quotients than wild-type mice in fed resting states, indicating impaired fatty acid oxidation. Altogether, results show that the fasting-induced increases in Ucp2 and Ucp3 do not correlate with increased mitochondrial proton leak but support a role for UCP3 in fatty acid metabolism.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Metabolismo Energético , Jejum , Ácidos Graxos/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais , Músculo Esquelético/ultraestrutura , Animais , Ácido Araquidônico/análise , Metabolismo Basal , Peso Corporal , Calorimetria Indireta , Ácidos Docosa-Hexaenoicos/análise , Alimentos , Canais Iônicos , Cinética , Ácido Linoleico/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/química , Tamanho do Órgão , Consumo de Oxigênio , Ácido Palmítico/análise , Proteínas/análise , Prótons , RNA Mensageiro/análise , Ácidos Esteáricos/análise , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMO
All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.