RESUMO
Compensatory growth (CG) is a naturally accelerated growth which occurs upon realimentation, following a prior period of dietary restriction. The process is harnessed worldwide as a management practice to reduce feed costs in beef cattle production. The objective of this study was to assess the potential contribution of hepatic cellular mitochondrial capacity to CG through global hepatic oxidative phosphorylation gene expression analyses as well as functional mitochondrial enzyme activity assays. Holstein-Friesian bulls were separated into two groups: (i) restricted feed allowance for 125â¯days (Period 1) (RES; nâ¯=â¯30) followed by ad-libitum feeding for 55â¯days (Period 2) or (ii) ad-libitum access to feed throughout (Periods 1 and 2) (ADLIB; nâ¯=â¯30). At the end of each period, 15 animals from each treatment group were slaughtered and hepatic tissue was collected. Tissue samples were subjected to RNAseq and spectrophotometric analysis for the functional assessment of mitochondria. RES and ADLIB groups grew at 0.6â¯kg/day and 1.9â¯kg/day, respectively, during Period 1. During Period 2, the RES group underwent CG growing at 2.5â¯kg/day, with ADLIB animals gaining 1.4â¯kg/day. Oxidative phosphorylation genes were differentially expressed in response to both dietary restriction and CG. Spectrophotometric assays indicated that mitochondrial abundance was greater in animals undergoing dietary restriction at the end of Period 1 and subsequently reduced during realimentation (Pâ¯<â¯0.02). Results indicate that mitochondrial capacity may be enhanced during dietary restriction to more effectively utilize diet-derived nutrients. However, enhanced mitochondrial capacity does not appear to be directly contributing to CG in cattle.
Assuntos
Privação de Alimentos , Fosforilação Oxidativa , Ração Animal/análise , Animais , Bovinos , Dieta/veterinária , Fígado/metabolismo , MasculinoRESUMO
Cellular mitochondrial function has been suggested to contribute to variation in feed efficiency (FE) among animals. The objective of this study was to determine mitochondrial abundance and activities of various mitochondrial respiratory chain complexes (complex I (CI) to complex IV (CIV)) in liver and muscle tissue from beef cattle phenotypically divergent for residual feed intake (RFI), a measure of FE. Individual DM intake (DMI) and growth were measured in purebred Simmental heifers (n = 24) and bulls (n = 28) with an initial mean BW (SD) of 372 kg (39.6) and 387 kg (50.6), respectively. All animals were offered concentrates ad libitum and 3 kg of grass silage daily, and feed intake was recorded for 70 days. Residuals of the regression of DMI on average daily gain (ADG), mid-test BW0.75 and backfat (BF), using all animals, were used to compute individual RFI coefficients. Animals were ranked within sex, by RFI into high (inefficient; top third of the population), medium (middle third of population) and low (efficient; bottom third of the population) terciles. Statistical analysis was carried out using the MIXED procedure of SAS v 9.3. Overall mean ADG (SD) and daily DMI (SD) for heifers were 1.2 (0.4) and 9.1 (0.5) kg, respectively, and for bulls were 1.8 (0.3) and 9.5 (1.02) kg, respectively. Heifers and bulls ranked as high RFI consumed 10% and 15% more (P < 0.05), respectively, than their low RFI counterparts. There was no effect of RFI on mitochondrial abundance in either liver or muscle (P > 0.05). An RFI × sex interaction was apparent for CI activity in muscle. High RFI animals had an increased activity (P < 0.05) of CIV in liver tissue compared to their low RFI counterparts; however, the relevance of that observation is not clear. Our data provide no clear evidence that cellular mitochondrial function within either skeletal muscle or hepatic tissue has an appreciable contributory role to overall variation in FE among beef cattle.
RESUMO
In mitochondria ATP synthesis is not perfectly coupled to oxygen consumption due to proton leak across the mitochondrial inner membrane. Quantitative studies have shown that proton leak contributes to approximately 25% of the resting oxygen consumption of mammals. Proton leak plays a role in accounting for differences in basal metabolic rate. Thyroid studies, body mass studies, phylogenic studies and obesity studies have all shown that increased mass-specific metabolic rate is linked to increased mitochondrial proton leak. The mechanism of the proton leak is unclear. Evidence suggests that proton leak occurs by a non-specific diffusion process across the mitochondrial inner membrane. However, the high degree of sequence homology of the recently cloned uncoupling proteins UCP 2 and UCP 3 to brown adipose tissue UCP 1, and their extensive tissue distribution, suggest that these novel uncoupling proteins play a role in proton leak. Early indications from reconstitution experiments and several in vitro expression studies suggest that the novel uncoupling proteins uncouple mitochondria. Furthermore, mice overexpressing UCP 3 certainly show a phenotype consistent with increased metabolism. The evidence for a role for these novel UCPs in mitochondrial proton leak is reviewed.
Assuntos
Proteínas de Transporte/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Proteínas/metabolismo , Prótons , Tecido Adiposo Marrom/metabolismo , Sequência de Aminoácidos , Animais , Metabolismo Basal , Difusão , Humanos , Canais Iônicos , Cinética , Mitocôndrias Hepáticas/metabolismo , Dados de Sequência Molecular , Fosforilação Oxidativa , Consumo de Oxigênio , Alinhamento de Sequência , Desacopladores/metabolismo , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMO
To date, UCP 3 has only been associated with skeletal muscle and brown adipose tissue (BAT). Using RT-PCR/PCR methodology, we show that human spleen and human thymus contain UCP 3. In addition, using peptide antibodies, previously demonstrated to be selective for UCP 3, we show that UCP 3 protein is present in mitochondria isolated from rat thymus and mitochondria isolated from reticulocytes, monocytes and lymphocytes of rat spleen. UCP 3 protein expression is also starvation-sensitive. UCP 3 abundance is augmented in mitochondria isolated from thymus and mitochondria isolated from lymphocytes of the spleen from fasted rats when compared to fed controls. The results are consistent with a role for UCP 3 in developing lymphocytes, thymus atrophy and fatty acid utilisation in spleen and thymus.
Assuntos
Proteínas de Transporte/biossíntese , Mitocôndrias/metabolismo , Baço/metabolismo , Inanição/metabolismo , Timo/metabolismo , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Canais Iônicos , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Monócitos/metabolismo , Monócitos/ultraestrutura , Ratos , Ratos Wistar , Reticulócitos/metabolismo , Reticulócitos/ultraestrutura , Baço/ultraestrutura , Timo/ultraestrutura , Proteína Desacopladora 3RESUMO
In order to investigate the control of endogenous betaine supply to the cytoplasmic enzyme betaine-homocysteine methyltransferase, it was necessary to understand how betaine synthesized within the mitochondrial matrix is transported across the mitochondrial inner membrane. Mitochondria were loaded with radiolabelled betaine and efflux was measured in a medium at physiological ionic strength. Efflux of radiolabelled betaine occurred continuously with time. The efflux rate was unaffected by the presence or absence of a source of energy except at high membrane potentials, where betaine efflux rate increased 2-3-fold. Titration of the membrane potential demonstrated a non-ohmic relationship between betaine efflux rate and membrane potential. The rate of betaine efflux was proportional to the matrix betaine concentration up to 9 mM. Efflux was unaffected by addition of analogues of betaine and known mitochondrial transport inhibitors. N-Ethylmaleimide did inhibit efflux by 50%, but evidence suggested that the effect was non-specific. The lack of saturability or other evidence for a transport system suggests that betaine escapes from mitochondria by simple diffusion. The relative diffusion rates of glycine, sarcosine, dimethylglycine and betaine suggest that increasing the degree of N-methylation lowers diffusion rate.
Assuntos
Betaína/metabolismo , Mitocôndrias Hepáticas/metabolismo , Animais , Difusão , Etilmaleimida/farmacologia , Feminino , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Dilatação Mitocondrial , Ratos , Ratos WistarRESUMO
The non-linear relationship between respiration rate and protonmotive force in isolated mitochondria is explained entirely by delta p-dependent changes in the proton conductance of the mitochondrial inner membrane and is not caused by redox slip in the proton pumps. Mitochondrial proton leak occurs in intact cells and tissues: the futile cycle of proton pumping and proton leak accounts for 26% +/- 7% of the total oxygen consumption rate or 33% +/- 7% of the mitochondrial respiration rate of isolated hepatocytes (mean +/- S.D. for 43 rats); 52% of the oxygen consumption rate of resting perfused muscle and up to 38% of the basal metabolic rate of a rat, suggesting that heat production may be an important function in the proton leak in homeotherms. Together with non-mitochondrial oxygen consumption, it lowers the effective P/O ratio in cells from maximum possible values of 2.33 (palmitate oxidation) or 2.58 (glucose oxidation) to as low as 1.1 in liver or 0.8 in muscle. The effective P/O ratio increases in response to ATP demand; the ability to allow rapid switching of flux from leak to ATP turnover may be an even more important function of the leak reaction than heat production. The mitochondrial proton conductance in isolated mitochondria and in hepatocytes is greatly modulated by thyroid hormones, by phylogeny and by body mass. Usually the reactions of ATP turnover change in parallel so that the coupling ratio is not greatly affected. Changes in proton leak in tissues are brought about in the short term by changes in mitochondrial protonmotive force and in the longer term by changes in the surface area and proton permeability of the mitochondrial inner membrane. Permeability changes are probably caused by changes in the fatty acid composition of the membrane phospholipids.
Assuntos
Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Músculos/metabolismo , Prótons , Trifosfato de Adenosina/metabolismo , Animais , Metabolismo Basal , Mitocôndrias Hepáticas/metabolismo , Oxirredução , Consumo de Oxigênio , Bombas de Próton/metabolismoRESUMO
A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.
Assuntos
Aclimatação/fisiologia , Proteínas de Transporte/análise , Músculo Esquelético/química , Hormônios Tireóideos/farmacologia , Tecido Adiposo Marrom/química , Animais , Células COS , Canais Iônicos , Proteínas Mitocondriais , Peroxissomos/química , Ratos , Proteína Desacopladora 3RESUMO
The degree of control exerted by the mitochondrial choline transporter over the oxidation pathway was measured in isolated rat liver mitochondria. Choline transporter activity was titrated with hemicholinium-3, a known competitive inhibitor of the transporter. It was shown that the rate of betaine efflux from mitochondria was an accurate measure of choline oxidation. The relative rate of choline oxidation was measured as a function of the relative degree of inhibition of the transporter. The resulting data gave a flux control coefficient over choline oxidation of 0.9 for the choline transporter. It is concluded that the choline transporter is the major site for control of choline oxidation in isolated rat liver mitochondria.
Assuntos
Proteínas de Transporte/metabolismo , Colina/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias Hepáticas/metabolismo , Animais , Oxirredução , Ratos , Ratos WistarRESUMO
In this investigation, the effects on proton leak of leptin administration to ob/ob mice was measured for liver mitochondria. We and others have shown that proton leak is approximately 3 times greater in liver mitochondria from ob/ob mice compared to lean controls at any given membrane potential. The results are consistent with obese mammals having higher lean mass-specific metabolic rates compared to lean controls. The increase in proton leak rate at any given membrane potential cannot be explained by the presence of free fatty acids associated with mitochondria isolated from the obese animals. The difference in proton leak must therefore represent a real difference in inner membrane permeability. Acute leptin (OB protein) administration restores the liver mitochondrial proton leak rate of ob/ob mice to that of lean controls. There was no effect on proton leak rate of liver mitochondria from sham-treated ob/ob mice. These novel results indicate a role for leptin, either directly or indirectly, in regulating the efficiency of oxidative phosphorylation.
Assuntos
Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas/administração & dosagem , Prótons , Animais , Esquema de Medicação , Metabolismo Energético/efeitos dos fármacos , Injeções Intraperitoneais , Cinética , Leptina , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Obesos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Obesidade/metabolismo , Fosforilação Oxidativa/efeitos dos fármacosAssuntos
Proteínas de Transporte/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Sistemas de Transporte de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos , Animais , Arginina/metabolismo , Transporte Biológico , Carnitina Aciltransferases/metabolismo , Citrulina/metabolismo , Glutamina/metabolismo , Glicina/metabolismo , HumanosRESUMO
In the 1930s, Max Kleiber and Samuel Brody established that the interspecies correlation between mammalian body mass and metabolic rate (alphaM(0.75)) cannot be explained (solely) by whole body surface area (alphaM(0.66)) to volume ratios. Metabolic considerations must also be taken into account. Decreases in the proportion of visceral organ mass to whole body mass can account for some of the whole body metabolic differences. However, superimposed upon these anatomical differences, the metabolism of tissues and cells has been demonstrated to decrease with increasing body mass. These decreases in oxygen consumption rates (with increasing body mass) in cells and tissues can be explained by a decrease in ATP turnover and mitochondrial density and an increase in mitochondrial functional efficiency (decrease in proton leak). The majority of the proton leak differences reflect differences in mitochondrial inner membrane surface area. Indeed, liver metabolism correlates directly with liver mitochondrial inner membrane surface area. Apart from being a significant contributor (approximately 25%) to basal metabolism, mitochondrial proton leak is a major factor determining the differences in basal metabolism between mammals of different body mass.
Assuntos
Peso Corporal , Consumo de Oxigênio , Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Metabolismo Basal , Fígado/citologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Mitocôndrias/metabolismo , Tamanho do Órgão , Fosforilação Oxidativa , PrótonsRESUMO
The standard metabolic rate of an animal is the rate of heat production under conditions that minimize known extra requirements for energy. In tissues and cells from aerobic organisms, energy expenditure can conveniently be measured as oxygen consumption. Measurements made using isolated rat hepatocytes have shown that a significant contribution to resting oxygen consumption (and hence heat production) is made by a futile cycle of proton pumping and proton leak across the mitochondrial inner membrane. Two important factors affecting standard metabolic rate, thyroid status and phylogeny, also affect the proton permeability. A third major factor affecting standard metabolic rate is body mass. Here we show that proton leak decreases with increasing body mass in mammals. We suggest that differences in proton leak may partly explain the differences in standard metabolic rate between mammals of different mass.
Assuntos
Peso Corporal , Mitocôndrias Hepáticas/metabolismo , Prótons , Animais , Cricetinae , Furões , Cavalos , Camundongos , Consumo de Oxigênio , Coelhos , Ovinos , SuínosRESUMO
Resting O2 consumption of hepatocytes isolated from mammals ranging in mass from 20-g mice to 200-kg horses decreases with increasing body mass. The substrate oxidation system increases in activity with increasing body mass and mitochondrial proton leak and phosphorylation system decrease in activity, resulting in a higher mitochondrial membrane potential in hepatocytes from larger mammals. The absolute rates of O2 consumption due to nonmitochondrial processes, substrate oxidation, mitochondrial proton leak, and the phosphorylation system decrease with increasing body mass. These decreases are due partly to a decrease in mitochondrial number per cell and partly to decrease in mitochondrial inner membrane proton leakiness and in ATP turnover by cells from larger mammals. Quantitatively, the proportion of total cell O2 consumption by nonmitochondrial processes (13%) and oxidation of substrates (87%) and the proportions used to drive mitochondrial proton leak (19%) and the phosphorylation system (68%) are the same for hepatocytes from all mammals investigated. The effect of matched decreases in the rates of proton leak and of ATP turnover is to keep the effective amount of ATP synthesized per unit of O2 consumed relatively constant with body mass, suggesting that the observed value is optimal.
Assuntos
Peso Corporal , Fígado/metabolismo , Mamíferos/anatomia & histologia , Mamíferos/metabolismo , Consumo de Oxigênio , Trifosfato de Adenosina/metabolismo , Animais , Furões , Cavalos , Cinética , Fígado/citologia , Fígado/ultraestrutura , Camundongos , Microscopia Eletrônica , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Oxirredução , Fosforilação , Prótons , Ratos , Ovinos , SuínosRESUMO
Hepatocytes were isolated from nine species of mammal of different body mass (and standard metabolic rate). The cells were incubated under identical conditions and oxygen consumption measured. The rate of oxygen consumption (per unit mass of cells) scaled with body mass with exponent -0.18. In general, there was a 5.5-fold decrease in oxygen consumption rate with a 12,500-fold increase in body mass. The decrease in oxygen consumption rate was not due to an increase in cell volume with increasing body mass but to a decrease in intrinsic metabolic activity of the cells. This novel finding confirms and explains the decrease in oxygen consumption rate measured in tissue slices from larger mammals by H. A. Krebs (Biochim. Biophys. Acta 4: 249-269, 1950) and recently by P. Couture and A. J. Hulbert [Am. J. Physiol. 268 (Regulatory Integrative Comp. Physiol. 37): R641-R650, 1995].
Assuntos
Peso Corporal , Fígado/metabolismo , Consumo de Oxigênio , Animais , Fígado/citologia , MamíferosRESUMO
In this paper we examine the non-linearity of the relationship between the proton electrochemical gradient across the mitochondrial inner membrane (delta p) and oxygen consumption of non-phosphorylating mitochondria in situ in hepatocytes. Models proposing to explain the non-linear relationship were tested experimentally. It was shown that the mitochondrial proton conductance and the number of protons pumped to the cytosolic side of the mitochondrial inner membrane by the electron transport complexes per oxygen atom consumed (H+/O ratio) are independent of electron transport rate in mitochondria in isolated hepatocytes. The non-linearity of the plot of delta p against the non-phosphorylating oxygen consumption is due to either a potential-dependent slippage of the proton pumps of the mitochondrial inner membrane and/or a potential-dependent leakage of protons back across the mitochondrial inner membrane.
Assuntos
Transporte de Elétrons , Membranas Intracelulares/fisiologia , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Consumo de Oxigênio , Animais , Células Cultivadas , Transporte de Elétrons/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Potenciais da Membrana/efeitos dos fármacos , Metacrilatos , Mitocôndrias Hepáticas/efeitos dos fármacos , Oligomicinas/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Tiazóis/farmacologia , Valinomicina/farmacologiaRESUMO
Rat liver mitochondria possess a specific choline transporter in the inner membrane. The transporter shows saturable kinetics at high membrane potential with a Km of 220 microM and a Vmax of 0.4 nmol/mg of protein/min at pH 7.0 and 25 degrees C. At physiological concentrations of choline, the rate of choline uptake by the transporter shows a linear dependence on membrane potential; uptake is distinct from the nonspecific cation diffusion process. Hemicholinium-3, hemicholinium-15, quinine, and quinidine, all analogues of choline, are high affinity competitive inhibitors of choline transport with Ki values of 17, 55, 15, and 127 microM, respectively. The choline transporter is distinct from other known mitochondrial transporters. Rat heart mitochondria do not appear to possess a choline transporter. Evidence suggests that the transporter is an electrophoretic uniporter. Analogue studies have shown that the hydroxyl and the quaternary ammonium groups of choline are necessary for binding to the transporter. A comparison of molecular models of choline and the high affinity inhibitors has provided evidence for the preferred conformation of choline for binding to the transporter. The presence of a choline transporter in the mitochondrial inner membrane provides a potential site for control of choline oxidation and hence supply of endogenous betaine.
Assuntos
Colina/metabolismo , Mitocôndrias Hepáticas/metabolismo , Animais , Transporte Biológico , Colina/análogos & derivados , Cromatografia em Camada Fina , Membranas Intracelulares/fisiologia , Cinética , Potenciais da Membrana , Mitocôndrias Cardíacas/metabolismo , Dilatação Mitocondrial , Modelos Moleculares , RatosRESUMO
We investigated why liver mitochondria from small mammals are leakier to protons than those from larger mammals. Sixty-nine percent (+/-23%) of the proton leak differences appeared to relate to membrane area (less inner membrane surface area in larger animals); any residual differences must reflect differences in membrane properties. There were differences in phospholipid fatty acid composition; unsaturation index, monounsaturates, palmitate (16:0), stearate (18:0), docosahexaenoate [22:6(n-3)], and the 22:6(n-3)/22:5(n-3) ratio all correlated with body mass. Proton flux per square centimeter did not correlate significantly with body mass or, in general, with phospholipid fatty acid composition, suggesting little role for fatty acid composition in determining proton leak in mammals of different body mass. However, unsaturation index and n-3 polyunsaturated fatty acid content correlated significantly with proton leak per milligram phospholipid when literature data from reptiles and rats in different thyroid states were included, giving some support to suggestions of a general role for phospholipid fatty acid composition in determining mitochondrial proton leak.
Assuntos
Ácidos Graxos/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias Hepáticas/metabolismo , Prótons , Animais , Peso Corporal , Membranas Intracelulares/ultraestrutura , Mamíferos , Permeabilidade , Fosfolipídeos/metabolismo , Proteínas/metabolismo , Propriedades de SuperfícieRESUMO
Synaptosomes, isolated from the whole brain of young (3 months) and old (24 months) rats were used to study the major bioenergetic systems of neuronal mitochondria in situ, within the synaptosome. Approximately 85% of the resting oxygen consumption of synaptosomes from both young and old rats was a result of proton leak (and possibly other ion cycling) across the mitochondrial inner membrane. There were no significant differences between synaptosomes from the young and old rats in the kinetic responses of the substrate oxidation system, the mitochondrial proton leak and the phosphorylation system to changes in the proton electrochemical gradient. Flux control coefficients of 0.71, 0.27 and 0.02 were calculated for substrate oxidation system, phosphorylation system and the proton leak, respectively, at maximal ATP producing capacity in synaptosomes from young animals. The corresponding values calculated for synaptosomes from old animals were 0.53, 0.43 and 0.05. Thus substrate oxidation had greatest control over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain, with proton leak, having little control under maximal ATP producing capacity. The uncoupled rate of oxygen consumption, in the presence of the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), was significantly lower (p = 0.0124) in synaptosomes from old rats (6.08 +/- 0.42, n = 11) when compared with those from the young rats (7.87 +/- 0.48, n = 8). Thus, there is an impaired flux through the substrate oxidation system is synaptosomes from old rats, as compared to synaptosomes from the young animals. These in situ results may have important implications for the interpretation of theories that age-dependent impairment of mitochondrial energy production may result in increased susceptibility to neurodegeneration.
Assuntos
Encéfalo/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Sinaptossomos/metabolismo , Fatores Etários , Animais , Química Encefálica , Cinética , Masculino , Potenciais da Membrana/fisiologia , Mitocôndrias/química , Oxirredução , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar , Sinaptossomos/químicaRESUMO
Thymi were dissected from rats and connective tissue was removed. Mitochondria were purified from isolated thymocytes and immunoblot analysis was performed using an antibody specific for uncoupling protein 1, which detected a 32.5 kDa protein associated with mitochondria from the thymocytes. This implies that rat thymocytes contain uncoupling protein 1.
Assuntos
Proteínas de Transporte/análise , Proteínas de Membrana/análise , Timo/metabolismo , Animais , Proteínas de Transporte/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Imunoensaio , Canais Iônicos , Proteínas de Membrana/isolamento & purificação , Proteínas Mitocondriais , Ratos , Ratos Wistar , Proteína Desacopladora 1RESUMO
In mitochondria, ATP synthesis is coupled to oxygen consumption by the proton electrochemical gradient established across the mitochondrial inner membrane in a process termed oxidative phosphorylation. It has long been known from stoichiometric studies that ATP synthesis is not perfectly coupled to oxygen consumption. The major inefficiency in the system is leakage of protons across the mitochondrial inner membrane driven by the proton electrochemical gradient. The kinetics of the proton leak can be determined indirectly, by measuring the oxygen consumption of mitochondria under non-phosphorylating conditions (plus oligomycin) as a function of the proton electrochemical gradient. This experimental system provides a convenient means to investigate inner membrane permeability to protons and the effect of factors that may effect that permeability. In this paper we review some results from our laboratory of indirect measurement of mitochondrial proton leak and how it has been applied to investigate the effect of aging, obesity and thyroid status on proton leak. The results show that (i) proton leak in isolated liver mitochondria is not significantly different in a comparison of young and old rats, in contrast (ii) there is an apparent increase in proton leak in in situ mitochondria in hepatocytes from old rats when compared to those from young rats, (iii) proton leak in neuronal mitochondria in situ in synaptosomes is not significantly different in young and old rats, (iv) proton leak is greater in isolated liver mitochondria from ob/ob mice compared to lean controls, (v) acute leptin (OB protein) administration restores the increased leak rate in isolated liver mitochondria from ob/ob mice to that of lean controls, (vi) administration of thyroid hormone (T3) increases proton leak in rat muscle mitochondria, and (vii) proton leak in muscle mitochondria is insensitive to the presence of GDP. It is proposed that the experimental system described here for measuring proton leak, is an ideal functional assay for determining whether the novel uncoupling proteins increase inner membrane permeability to protons.