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1.
J Pharmacol Exp Ther ; 351(3): 699-708, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25301169

RESUMO

We hypothesized that the mitochondrial-targeted antioxidant, mitoquinone (mitoQ), known to have mitochondrial uncoupling properties, might prevent the development of obesity and mitigate liver dysfunction by increasing energy expenditure, as opposed to reducing energy intake. We administered mitoQ or vehicle (ethanol) to obesity-prone C57BL/6 mice fed high-fat (HF) or normal-fat (NF) diets. MitoQ (500 µM) or vehicle (ethanol) was added to the drinking water for 28 weeks. MitoQ significantly reduced total body mass and fat mass in the HF-fed mice but had no effect on these parameters in NF mice. Food intake was reduced by mitoQ in the HF-fed but not in the NF-fed mice. Average daily water intake was reduced by mitoQ in both the NF- and HF-fed mice. Hypothalamic expression of neuropeptide Y, agouti-related peptide, and the long form of the leptin receptor were reduced in the HF but not in the NF mice. Hepatic total fat and triglyceride content did not differ between the mitoQ-treated and control HF-fed mice. However, mitoQ markedly reduced hepatic lipid hydroperoxides and reduced circulating alanine aminotransferase, a marker of liver function. MitoQ did not alter whole-body oxygen consumption or liver mitochondrial oxygen utilization, membrane potential, ATP production, or production of reactive oxygen species. In summary, mitoQ added to drinking water mitigated the development of obesity. Contrary to our hypothesis, the mechanism involved decreased energy intake likely mediated at the hypothalamic level. MitoQ also ameliorated HF-induced liver dysfunction by virtue of its antioxidant properties without altering liver fat or mitochondrial bioenergetics.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Hepatopatias/prevenção & controle , Mitocôndrias Hepáticas/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Ubiquinona/análogos & derivados , Aumento de Peso/efeitos dos fármacos , Animais , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Hepatopatias/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/enzimologia , Compostos Organofosforados/uso terapêutico , Ubiquinona/farmacologia , Ubiquinona/uso terapêutico , Aumento de Peso/fisiologia
2.
J Bioenerg Biomembr ; 46(1): 33-44, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24121995

RESUMO

Fat intake alters mitochondrial lipid composition which can affect function. We used novel methodology to assess bioenergetics, including simultaneous ATP and reactive oxygen species (ROS) production, in liver and heart mitochondria of C57BL/6 mice fed diets of variant fatty acid content and saturation. Our methodology allowed us to clamp ADP concentration and membrane potential (ΔΨ) at fixed levels. Mice received a control diet for 17­19 weeks, a high-fat (HF) diet (60% lard) for 17­19 weeks, or HF for 12 weeks followed by 6­7 weeks of HF with 50% of fat as menhaden oil (MO) which is rich in n-3 fatty acids. ATP production was determined as conversion of 2-deoxyglucose to 2-deoxyglucose phosphate by NMR spectroscopy. Respiration and ATP production were significantly reduced at all levels of ADP and resultant clamped ΔΨ in liver mitochondria from mice fed HF compared to controls. At given ΔΨ, ROS production per mg mitochondrial protein, per unit respiration, or per ATP generated were greater for liver mitochondria of HF-fed mice compared to control or MO-fed mice. Moreover, these ROS metrics began to increase at a lower ΔΨ threshold. Similar, but less marked, changes were observed in heart mitochondria of HF-fed mice compared to controls. No changes in mitochondrial bioenergetics were observed in studies of separate mice fed HF versus control for only 12 weeks. In summary, HF feeding of sufficient duration impairs mitochondrial bioenergetics and is associated with a greater ROS "cost" of ATP production compared to controls. These effects are, in part, mitigated by MO.


Assuntos
Gorduras na Dieta/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Animais , Respiração Celular/fisiologia , Metabolismo Energético , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo
3.
J Pharmacol Exp Ther ; 342(3): 709-19, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22661629

RESUMO

Mitochondrial-targeted analogs of coenzyme Q (CoQ) are under development to reduce oxidative damage induced by a variety of disease states. However, there is a need to understand the bioenergetic effects of these agents and whether or not these effects are related to redox properties, including their known pro-oxidant effects. We examined the bioenergetic effects of two mitochondrial-targeted CoQ analogs in their quinol forms, mitoquinol (MitoQ) and plastoquinonyl-decyl-triphenylphosphonium (SkQ1), in bovine aortic endothelial cells. We used an extracellular oxygen and proton flux analyzer to assess mitochondrial action at the intact-cell level. Both agents, in dose-dependent fashion, reduced the oxygen consumption rate (OCR) directed at ATP turnover (OCR(ATP)) (IC50 values of 189 ± 13 nM for MitoQ and 181 ± 7 for SKQ1; difference not significant) while not affecting or mildly increasing basal oxygen consumption. Both compounds increased extracellular acidification in the basal state consistent with enhanced glycolysis. Both compounds enhanced mitochondrial superoxide production assessed by using mitochondrial-targeted dihydroethidium, and both increased H2O2 production from mitochondria of cells treated before isolation of the organelles. The manganese superoxide dismutase mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin did not alter or actually enhanced the actions of the targeted CoQ analogs to reduce OCR(ATP). In contrast, N-acetylcysteine mitigated this effect of MitoQ and SkQ1. In summary, our data demonstrate the important bioenergetic effects of targeted CoQ analogs. Moreover, these effects are mediated, at least in part, through superoxide production but depend on conversion to H2O2. These bioenergetic and redox actions need to be considered as these compounds are developed for therapeutic purposes.


Assuntos
Células Endoteliais/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Acetilcisteína/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/fisiologia , Bovinos , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Metabolismo Energético/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Peróxido de Hidrogênio/metabolismo , Metaloporfirinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Plastoquinona/análogos & derivados , Plastoquinona/farmacologia , Prótons , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Compostos de Tritil/farmacologia , Ubiquinona/farmacologia
4.
Am J Physiol Regul Integr Comp Physiol ; 301(6): R1616-24, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21940403

RESUMO

Mitochondrial superoxide is important in the pathogeneses of diabetes and its complications. However, there is uncertainty regarding the intrinsic propensity of mitochondria to generate this radical. Studies to date suggest that superoxide production by mitochondria of insulin-sensitive target tissues of insulin-deficient rodents is reduced or unchanged. Moreover, little is known of the role of the Coenzyme Q (CoQ), whose semiquinone form reacts with molecular oxygen to generate superoxide. We measured reactive oxygen species (ROS) production, respiratory parameters, and CoQ content in mitochondria from gastrocnemius muscle of control and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between mitochondria isolated from vehicle- or STZ-treated animals. CoQ also was unaffected by weight loss in the absence of diabetes (induced by caloric restriction). Under state 4 or state 3 conditions, both respiration and ROS release were reduced in diabetic mitochondria fueled with succinate, glutamate plus malate, or with all three substrates (continuous TCA cycle). However, H(2)O(2) and directly measured superoxide production were substantially increased in gastrocnemius mitochondria of diabetic rats when expressed per unit oxygen consumed. On the basis of substrate and inhibitor effects, the mechanism involved multiple electron transport sites. More limited results using heart mitochondria were similar. ROS per unit respiration was greater in muscle mitochondria from diabetic compared with control rats during state 3, as well as state 4, while the reduction in ROS per unit respiration on transition to state 3 was less for diabetic mitochondria. In summary, ROS production is, in fact, increased in mitochondria from insulin-deficient muscle when considered relative to electron transport. This is evident on multiple energy substrates and in different respiratory states. CoQ is not reduced in diabetic mitochondria or with weight loss due to food restriction. The implications of these findings are discussed.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Superóxidos/metabolismo , Ubiquinona/metabolismo , Animais , Transporte de Elétrons , Regulação da Expressão Gênica , Peróxido de Hidrogênio , Masculino , Potencial da Membrana Mitocondrial , Consumo de Oxigênio , Prótons , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/genética
5.
Endocrinology ; 150(1): 46-55, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18772240

RESUMO

Mitochondrial reactive oxygen species have been implicated in both diabetic complications and the progression of the underlying diabetic state. However, it is not clear whether mitochondria of diabetic origin are intrinsically altered to generate excess reactive oxygen species independent of the surrounding diabetic milieu. Mitochondria were isolated from gastrocnemius, heart, and liver of 2-wk and 2-month streptozotocin diabetic rats and controls. We rigidly quantified mitochondrial superoxide, respiration and ATP production, respiratory coupling, the expression of several proteins with antioxidant properties, and the redox state of glutathione. Both fluorescent assessment and electron paramagnetic spectroscopy revealed that superoxide production was unchanged or reduced in the 2-month diabetic mitochondria compared with controls. Kinetic analysis of the proton leak showed that diabetic heart and muscle mitochondria were actually more coupled compared with control despite an approximate 2- to 4-fold increase in uncoupling protein-3 content. Adenine nucleotide translocator type 1 expression was reduced by approximately 50% in diabetic muscle mitochondria. Catalase was significantly up-regulated in muscle and heart tissue and in heart mitochondria, whereas glutathione peroxidase expression was increased in liver mitochondria of diabetic rats. We conclude that gastrocnemius, heart, and liver mitochondria of streptozotocin diabetic rats are not irrevocably altered toward excess superoxide production either by complex I or complex III. Moreover, gastrocnemius and heart mitochondria demonstrate increased, not decreased, respiratory coupling. Mitochondria of insulin-deficient diabetic rats do show signs of adaptation to antecedent oxidative stress manifested as tissue-specific enzyme and uncoupling protein expression but remain remarkably robust with respect to superoxide production.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Insulina/deficiência , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Musculares/metabolismo , Consumo de Oxigênio/fisiologia , Superóxidos/metabolismo , Animais , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Coração/fisiopatologia , Fígado/fisiopatologia , Malatos/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Rotenona/farmacologia , Succinatos/metabolismo
6.
Endocrinology ; 147(6): 2781-8, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16527844

RESUMO

Antecedent hypoglycemia is well known to impair sympathetic responses to subsequent hypoglycemia. However, it is less clear whether this occurs through altered sympathetic neural traffic or through decreased adrenal catecholamine release per se. It is also not clear whether antecedent hypoglycemia impairs sympathetic responsiveness to subsequent nonhypoglycemic sympathetic stimuli. We exposed rats to two episodes of insulin-induced hypoglycemia or sham hypoglycemia (n = 15 per group) on d -2 and -1 before exposure to transient (10 min) hypotension on d 0. Adrenal sympathetic nerve activity (SNA) was directly recorded in the conscious state and plasma catecholamine concentrations were assessed. We also examined the effect of antecedent hypoglycemia on phosphorylated and nonphosphorylated tyrosine hydroxylase (TH) protein expression as well as the expression of phenylethanolamine N-methyltransferase. Adrenal SNA was not significantly altered by antecedent hypoglycemia either at baseline of d 0 (before hypotension) or in response to hypotension. In contrast, plasma epinephrine (EPI) responsiveness was impaired by more than 50% (P = 0.025) in rats exposed to antecedent vs. sham hypoglycemia. Antecedent hypoglycemia had no effect on norepinephrine responsiveness to hypotension. In studies of adrenal tissue from separate rats, antecedent hypoglycemia decreased adrenal EPI content but did not significantly alter the expression of TH, phosphorylated TH, or phenylethanolamine N-methyltransferase. In summary, antecedent hypoglycemia impaired EPI responsiveness to subsequent hypotension despite no reduction in adrenal SNA and in association with reduced adrenal EPI content. Thus, antecedent hypoglycemia impaired responsiveness to a subsequent nonhypoglycemic sympathetic stimulus, an effect mediated at the level of the adrenal medullae.


Assuntos
Glândulas Suprarrenais/química , Catecolaminas/análise , Hipoglicemia/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Animais , Catecolaminas/metabolismo , Hipotensão/etiologia , Feniletanolamina N-Metiltransferase/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/metabolismo
7.
Metabolism ; 52(11): 1484-90, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14624411

RESUMO

We previously showed, through direct neural recording in conscious rats, that hypoglycemia increases adrenal sympathetic nerve activity (SNA) both acutely and 24 hours following the second of 2 daily antecedent hypoglycemic episodes. Nonetheless, antecedent hypoglycemia impaired catecholamine responsiveness to subsequent acute hypoglycemia. Here we hypothesized that antecedent, nonhypoglycemic adrenal sympathetic stimulation by leptin would impair acute adrenal catecholamine responsiveness to subsequent hypoglycemia. We also hypothesized that acute leptin administration (after 2 days of antecedent hypoglycemia) would enhance adrenal SNA and thereby enhance catecholamine responsiveness to concurrent hypoglycemia. Leptin or saline was administered to normal rats in repeated subcutaneous injections for 2 days prior to acute insulin-induced hypoglycemia. In contrast to our hypothesis, antecedent leptin did not change catecholamine responsiveness or glycemic change in response to subsequent acute insulin administration. In additional studies, intravenous leptin or saline was acutely administered beginning 1 hour before insulin-induced hypoglycemia. All rats had been exposed to antecedent hypoglycemia. In these experiments, acute leptin did not alter catecholamine responses to insulin or glycemic change during or after termination of insulin. We conclude that antecedent nonhypoglycemic sympathetic stimulation by leptin does not alter subsequent catecholamine or glycemic responses to insulin. Moreover, concurrent leptin does not enhance catecholamine responses to insulin in rats exposed to antecedent hypoglycemia.


Assuntos
Catecolaminas/sangue , Hipoglicemia/sangue , Hipoglicemia/induzido quimicamente , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Leptina/farmacologia , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/inervação , Animais , Glicemia/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Injeções Intravenosas , Camundongos , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/efeitos dos fármacos
8.
Invest Ophthalmol Vis Sci ; 55(12): 8330-41, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25468886

RESUMO

PURPOSE: Traumatic brain injury (TBI) frequently leads to chronic visual dysfunction. The purpose of this study was to investigate the effect of TBI on retinal ganglion cells (RGCs), and to test whether treatment with the novel neuroprotective compound P7C3-S243 could prevent in vivo functional deficits in the visual system. METHODS: Blast-mediated TBI was modeled using an enclosed over-pressure blast chamber. The RGC physiology was evaluated using a multielectrode array and pattern electroretinogram (PERG). Histological analysis of RGC dendritic field and cell number were evaluated at the end of the study. Visual outcome measures also were evaluated based on treatment of mice with P7C3-S243 or vehicle control. RESULTS: We show that deficits in neutral position PERG after blast-mediated TBI occur in a temporally bimodal fashion, with temporary recovery 4 weeks after injury followed by chronically persistent dysfunction 12 weeks later. This later time point is associated with development of dendritic abnormalities and irreversible death of RGCs. We also demonstrate that ongoing pathologic processes during the temporary recovery latent period (including abnormalities of RGC physiology) lead to future dysfunction of the visual system. We report that modification of PERG to provocative postural tilt testing elicits changes in PERG measurements that correlate with a key in vitro measures of damage: the spontaneous and light-evoked activity of RGCs. Treatment with P7C3-S243 immediately after injury and throughout the temporary recovery latent period protects mice from developing chronic visual system dysfunction. CONCLUSIONS: Provocative PERG testing serves as a noninvasive test in the living organism to identify early damage to the visual system, which may reflect corresponding damage in the brain that is not otherwise detectable by noninvasive means. This provides the basis for developing an earlier diagnostic test to identify patients at risk for developing chronic CNS and visual system damage after TBI at an earlier stage when treatments may be more effective in preventing these sequelae. In addition, treatment with the neuroprotective agent P7C3-S243 after TBI protects from visual system dysfunction after TBI.


Assuntos
Traumatismos por Explosões/tratamento farmacológico , Lesões Encefálicas/tratamento farmacológico , Carbazóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Transtornos da Visão/prevenção & controle , Análise de Variância , Animais , Traumatismos por Explosões/complicações , Traumatismos por Explosões/fisiopatologia , Lesões Encefálicas/complicações , Lesões Encefálicas/fisiopatologia , Contagem de Células , Dendritos/patologia , Modelos Animais de Doenças , Eletrorretinografia/efeitos dos fármacos , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/fisiologia , Transtornos da Visão/etiologia , Transtornos da Visão/fisiopatologia
9.
Diabetes ; 62(6): 1833-42, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23328129

RESUMO

Interpreting mitochondrial function as affected by comparative physiologic conditions is confounding because individual functional parameters are interdependent. Here, we studied muscle mitochondrial function in insulin-deficient diabetes using a novel, highly sensitive, and specific method to quantify ATP production simultaneously with reactive oxygen species (ROS) at clamped levels of inner mitochondrial membrane potential (ΔΨ), enabling more detailed study. We used a 2-deoxyglucose (2DOG) energy clamp to set ΔΨ at fixed levels and to quantify ATP production as 2DOG conversion to 2DOG-phosphate measured by one-dimensional (1)H and two-dimensional (1)H/(13)C heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. These techniques proved far more sensitive than conventional (31)P nuclear magnetic resonance and allowed high-throughput study of small mitochondrial isolates. Over conditions ranging from state 4 to state 3 respiration, ATP production was lower and ROS per unit of ATP generated was greater in mitochondria isolated from diabetic muscle. Moreover, ROS began to increase at a lower threshold for inner membrane potential in diabetic mitochondria. Further, ATP production in diabetic mitochondria is limited not only by respiration but also by limited capacity to use ΔΨ for ATP synthesis. In summary, we describe novel methodology for measuring ATP and provide new mechanistic insight into the dysregulation of ATP production and ROS in mitochondria of insulin-deficient rodents.


Assuntos
Diabetes Mellitus/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Peróxido de Hidrogênio/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
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