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1.
Am J Physiol Cell Physiol ; 326(6): C1669-C1682, 2024 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-38646781

RESUMO

We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase.NEW & NOTEWORTHY Impairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase.


Assuntos
Respiração Celular , Potencial da Membrana Mitocondrial , Mitocôndrias Musculares , Animais , Camundongos , Aspartato Aminotransferase Mitocondrial/metabolismo , Aspartato Aminotransferase Mitocondrial/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Complexo II de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , Succinato Desidrogenase/metabolismo , Succinato Desidrogenase/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo
2.
Am J Physiol Cell Physiol ; 324(6): C1236-C1248, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37125774

RESUMO

We previously found that skeletal muscle mitochondria incubated at low membrane potential (ΔΨ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low, accumulate oxaloacetate (OAA) in amounts sufficient to inhibit complex II respiration. We proposed a mechanism wherein low ΔΨ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance, we carried out studies of mice with inherently different levels of IBAT mitochondrial inner membrane potential. Isolated complex II (succinate)-energized IBAT mitochondria from obesity-resistant 129SVE mice compared with obesity-prone C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower ΔΨ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower ΔΨ, exhibited much lower respiration, twofold greater OAA concentrations, much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared with the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater ΔΨ, ROS, and NADH, but equal or greater O2 flux compared with WT mitochondria. GDP addition, compared with no GDP, increased ΔΨ and complex II respiration in wild-type (WT) mice associated with much less OAA. Respiration on complex I substrates followed the more classical dynamics of greater respiration at lower ΔΨ. These findings support the abovementioned mechanism for OAA- and ΔΨ-dependent complex II respiration and support its physiological relevance.NEW & NOTEWORTHY We examined mitochondrial respiration initiated at mitochondrial complex II in mice with varying degrees of brown adipose tissue UCP1 expression. We show that, by affecting inner membrane potential, UCP1 expression determines reverse electron transport from complex II to complex I and, consequently, the NADH/NAD+ ratio. Accordingly, this regulates the level of oxaloacetate accumulation and the extent of oxaloacetate inhibition of complex II.


Assuntos
Tecido Adiposo Marrom , NAD , Camundongos , Animais , Tecido Adiposo Marrom/metabolismo , NAD/metabolismo , Ácido Oxaloacético/metabolismo , Ácido Oxaloacético/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , Respiração , Obesidade/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Potencial da Membrana Mitocondrial , Succinatos/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
3.
FASEB J ; 33(11): 11696-11705, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31361970

RESUMO

We recently reported that membrane potential (ΔΨ) primarily determines the relationship of complex II-supported respiration by isolated skeletal muscle mitochondria to ADP concentrations. We observed that O2 flux peaked at low ADP concentration ([ADP]) (high ΔΨ) before declining at higher [ADP] (low ΔΨ). The decline resulted from oxaloacetate (OAA) accumulation and inhibition of succinate dehydrogenase. This prompted us to question the effect of incremental [ADP] on respiration in interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low because of uncoupling protein 1 (UCP1). We found that succinate-energized IBAT mitochondria, even in the absence of ADP, accumulate OAA and manifest limited respiration, similar to muscle mitochondria at high [ADP]. This could be prevented by guanosine 5'-diphosphate inhibition of UCP1. NAD+ cycling with NADH requires complex I electron flow and is needed to form OAA. Therefore, to assess the role of electron transit, we perturbed flow using a small molecule, N1-(3-acetamidophenyl)-N2-(2-(4-methyl-2-(p-tolyl)thiazol-5-yl)ethyl)oxalamide. We observed decreased OAA, increased NADH/NAD+, and increased succinate-supported mitochondrial respiration under conditions of low ΔΨ (IBAT) but not high ΔΨ (heart). In summary, complex II-energized respiration in IBAT mitochondria is tempered by complex I-derived OAA in a manner dependent on UCP1. These dynamics depend on electron transit in complex I.-Fink, B. D., Yu, L., Sivitz, W. I. Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.


Assuntos
Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias/metabolismo , Respiração/efeitos dos fármacos , Succinato Desidrogenase/farmacologia , Difosfato de Adenosina/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Adiposidade/efeitos dos fármacos , Adiposidade/fisiologia , Animais , Complexo I de Transporte de Elétrons/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Miocárdio/metabolismo , Obesidade/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Succinato Desidrogenase/metabolismo , Proteína Desacopladora 1/efeitos dos fármacos , Proteína Desacopladora 1/metabolismo
4.
J Biol Chem ; 293(51): 19932-19941, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30385511

RESUMO

We recently reported a previously unrecognized mitochondrial respiratory phenomenon. When [ADP] was held constant ("clamped") at sequentially increasing concentrations in succinate-energized muscle mitochondria in the absence of rotenone (commonly used to block complex I), we observed a biphasic, increasing then decreasing, respiratory response. Here we investigated the mechanism. We confirmed decades-old reports that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH). We then used an NMR method to assess OAA concentrations (known as difficult to measure by MS) as well as those of malate, fumarate, and citrate in isolated succinate-respiring mitochondria. When these mitochondria were incubated at varying clamped ADP concentrations, respiration increased at low [ADP] as expected given the concurrent reduction in membrane potential. With further increments in [ADP], respiration decreased associated with accumulation of OAA. Moreover, a low pyruvate concentration, that alone was not enough to drive respiration, was sufficient to metabolize OAA to citrate and completely reverse the loss of succinate-supported respiration at high [ADP]. Further, chemical or genetic inhibition of pyruvate uptake prevented OAA clearance and preserved respiration. In addition, we measured the effects of incremental [ADP] on NADH, superoxide, and H2O2 (a marker of reverse electron transport from complex II to I). In summary, our findings, taken together, support a mechanism (detailed within) wherein succinate-energized respiration as a function of increasing [ADP] is initially increased by [ADP]-dependent effects on membrane potential but subsequently decreased at higher [ADP] by inhibition of succinate dehydrogenase by OAA. The physiologic relevance is discussed.


Assuntos
Difosfato de Adenosina/metabolismo , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ácido Oxaloacético/farmacologia , Animais , Respiração Celular/efeitos dos fármacos , Complexo II de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/enzimologia , Células Musculares/citologia , Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
5.
Nature ; 491(7423): 269-73, 2012 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-23051746

RESUMO

Myocardial cell death is initiated by excessive mitochondrial Ca(2+) entry causing Ca(2+) overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm). However, the signalling pathways that control mitochondrial Ca(2+) entry through the inner membrane mitochondrial Ca(2+) uniporter (MCU) are not known. The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (I(MCU)). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced I(MCU) and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing I(MCU). Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca(2+) entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miocárdio/enzimologia , Miocárdio/patologia , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Cálcio/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Ciclosporina/farmacologia , Feminino , Coração/efeitos dos fármacos , Coração/fisiopatologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/prevenção & controle , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Cardíacas/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/prevenção & controle , Miocárdio/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Serina/metabolismo , Estresse Fisiológico/efeitos dos fármacos
6.
Am J Physiol Cell Physiol ; 313(2): C146-C153, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28515085

RESUMO

Nanomolar free calcium enhances oxidative phosphorylation. However, the effects over a broad concentration range, at different respiratory states, or on specific energy substrates are less clear. We examined the action of varying [Ca2+] over respiratory states ranging 4 to 3 on skeletal muscle mitochondrial respiration, potential, ATP production, and H2O2 production using ADP recycling to clamp external [ADP]. Calcium at 450 nM enhanced respiration in mitochondria energized by the complex I substrates, glutamate/malate (but not succinate), at [ADP] of 4-256 µM, but more substantially at intermediate respiratory states and not at all at state 4. Using varied [Ca2+], we found that the stimulatory effects on respiration and ATP production were most prominent at nanomolar concentrations, but inhibitory at 10 µM or higher. ATP production decreased more than respiration at 10 µM calcium. However, potential continued to increase up to 10 µM; suggesting a calcium-induced inability to utilize potential for phosphorylation independent of opening of the mitochondrial permeability transition pore (MTP). This effect of 10 µM calcium was confirmed by direct determination of ATP production over a range of potential created by differing substrate concentrations. Consistent with past reports, nanomolar [Ca2+] had a stimulatory effect on utilization of potential for phosphorylation. Increasing [Ca2+] was positively and continuously associated with H2O2 production. In summary, the stimulatory effect of calcium on mitochondrial function is substrate dependent and most prominent over intermediate respiratory states. Calcium stimulates or inhibits utilization of potential for phosphorylation dependent on concentration with inhibition at higher concentration independent of MTP opening.


Assuntos
Trifosfato de Adenosina/biossíntese , Cálcio/metabolismo , Mitocôndrias/metabolismo , Respiração , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , Ácido Glutâmico/metabolismo , Peróxido de Hidrogênio/metabolismo , Malatos/metabolismo , Camundongos , Mitocôndrias/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Fosforilação Oxidativa , Consumo de Oxigênio
7.
Am J Physiol Regul Integr Comp Physiol ; 311(4): R756-R763, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27558314

RESUMO

Recently, we used an ADP recycling approach to examine mouse skeletal muscle (SkM) mitochondrial function over respiratory states intermittent between state 3 and 4. We showed that respiration energized at complex II by succinate, in the presence of rotenone to block complex I, progressively increased with incremental additions of ADP. However, in the absence of rotenone, respiration peaked at low [ADP] but then dropped markedly as [ADP] was further increased. Here, we tested the hypothesis that these respiratory dynamics would differ between mitochondria of mice fed high fat (HF) and treated with a low dose of streptozotocin to mimic Type 2 diabetes and mitochondria from controls. We found that respiration and ATP production on succinate alone for both control and diabetic mice increased to a maximum at low [ADP] but dropped markedly as [ADP] was incrementally increased. However, peak respiration by the diabetic mitochondria required a higher [ADP] (right shift in the curve of O2 flux vs. [ADP]). ATP production by diabetic mitochondria respiring on succinate alone was significantly less than controls, whereas membrane potential trended higher, indicating that utilization of potential for oxidative phosphorylation was impaired. The rightward shift in the curve of O2 flux versus [ADP] is likely a consequence of these changes in ATP production and potential. In summary, using an ADP recycling approach, we demonstrated that ATP production by SkM mitochondria of HF/streptozotocin diabetic mice energized by succinate is impaired due to decreased utilization of ΔΨ and that more ADP is required for peak O2 flux.


Assuntos
Difosfato de Adenosina/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Complexo II de Transporte de Elétrons/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Musculares/metabolismo , Obesidade/fisiopatologia , Consumo de Oxigênio , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/induzido quimicamente , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estreptozocina
9.
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
10.
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
11.
Diabetes Care ; 47(4): 580-588, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38211595

RESUMO

OBJECTIVE: To compare the long-term effects of glucose-lowering medications (insulin glargine U-100, glimepiride, liraglutide, and sitagliptin) when added to metformin on insulin sensitivity and ß-cell function. RESEARCH DESIGN AND METHODS: In the Glycemia Reduction Approaches in Diabetes: A Comparative Effectiveness Study (GRADE) cohort with type 2 diabetes (n = 4,801), HOMA2 was used to estimate insulin sensitivity (HOMA2-%S) and fasting ß-cell function (HOMA2-%B) at baseline and 1, 3, and 5 years on treatment. Oral glucose tolerance test ß-cell responses (C-peptide index [CPI] and total C-peptide response [incremental C-peptide/incremental glucose over 120 min]) were evaluated at the same time points. These responses adjusted for HOMA2-%S in regression analysis provided estimates of ß-cell function. RESULTS: HOMA2-%S increased from baseline to year 1 with glargine and remained stable thereafter, while it did not change from baseline in the other treatment groups. HOMA2-%B and C-peptide responses were increased to variable degrees at year 1 in all groups but then declined progressively over time. At year 5, CPI was similar between liraglutide and sitagliptin, and higher for both than for glargine and glimepiride [0.80, 0.87, 0.74, and 0.64 (nmol/L)/(mg/dL) * 100, respectively; P < 0.001], while the total C-peptide response was greatest with liraglutide, followed in descending order by sitagliptin, glargine, and glimepiride [1.54, 1.25, 1.02, and 0.87 (nmol/L)/(mg/dL) * 100, respectively, P < 0.001]. After adjustment for HOMA2-%S to obtain an estimate of ß-cell function, the nature of the change in ß-cell responses reflected those in ß-cell function. CONCLUSIONS: The differential long-term effects on insulin sensitivity and ß-cell function of four different glucose-lowering medications when added to metformin highlight the importance of the loss of ß-cell function in the progression of type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Metformina , Compostos de Sulfonilureia , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Insulina Glargina/uso terapêutico , Hipoglicemiantes/uso terapêutico , Glucose/uso terapêutico , Liraglutida/farmacologia , Liraglutida/uso terapêutico , Resistência à Insulina/fisiologia , Peptídeo C , Glicemia , Metformina/uso terapêutico , Fosfato de Sitagliptina/uso terapêutico
12.
Diabetes Care ; 47(4): 571-579, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38190619

RESUMO

OBJECTIVE: To compare the effects of insulin sensitivity and ß-cell function over time on HbA1c and durability of glycemic control in response to dual therapy. RESEARCH DESIGN AND METHODS: GRADE participants were randomized to glimepiride (n = 1,254), liraglutide (n = 1,262), or sitagliptin (n = 1,268) added to baseline metformin and followed for mean ± SD 5.0 ± 1.3 years, with HbA1c assessed quarterly and oral glucose tolerance tests at baseline, 1, 3, and 5 years. We related time-varying insulin sensitivity (HOMA 2 of insulin sensitivity [HOMA2-%S]) and early (0-30 min) and total (0-120 min) C-peptide (CP) responses to changes in HbA1c and glycemic failure (primary outcome HbA1c ≥7% [53 mmol/mol] and secondary outcome HbA1c >7.5% [58 mmol/mol]) and examined differential treatment responses. RESULTS: Higher HOMA2-%S was associated with greater initial HbA1c lowering (3 months) but not subsequent HbA1c rise. Greater CP responses were associated with a greater initial treatment response and slower subsequent HbA1c rise. Higher HOMA2-%S and CP responses were each associated with lower risk of primary and secondary outcomes. These associations differed by treatment. In the sitagliptin group, HOMA2-%S and CP responses had greater impact on initial HbA1c reduction (test of heterogeneity, P = 0.009 HOMA2-%S, P = 0.018 early CP, P = 0.001 total CP) and risk of primary outcome (P = 0.005 HOMA2-%S, P = 0.11 early CP, P = 0.025 total CP) but lesser impact on HbA1c rise (P = 0.175 HOMA2-%S, P = 0.006 early CP, P < 0.001 total CP) in comparisons with the glimepiride and liraglutide groups. There were no differential treatment effects on secondary outcome. CONCLUSIONS: Insulin sensitivity and ß-cell function affected treatment outcomes irrespective of drug assignment, with greater impact in the sitagliptin group on initial (short-term) HbA1c response in comparison with the glimepiride and liraglutide groups.


Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Metformina , Compostos de Sulfonilureia , Humanos , Hipoglicemiantes/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Liraglutida/uso terapêutico , Hemoglobinas Glicadas , Metformina/uso terapêutico , Fosfato de Sitagliptina/uso terapêutico , Resultado do Tratamento , Glicemia , Quimioterapia Combinada
13.
Biochim Biophys Acta Bioenerg ; 1864(1): 148930, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36272463

RESUMO

At low inner mitochondrial membrane potential (ΔΨ) oxaloacetate (OAA) accumulates in the organelles concurrently with decreased complex II-energized respiration. This is consistent with ΔΨ-dependent OAA inhibition of succinate dehydrogenase. To assess the metabolic importance of this process, we tested the hypothesis that perturbing metabolic clearance of OAA in complex II-energized mitochondria would alter O2 flux and, further, that this would occur in both ΔΨ and tissue-dependent fashion. We carried out respiratory and metabolite studies in skeletal muscle and interscapular brown adipose tissue (IBAT) directed at the effect of OAA transamination to aspartate (catalyzed by the mitochondrial form of glutamic-oxaloacetic transaminase, Got2) on complex II-energized respiration. Addition of low amounts of glutamate to succinate-energized mitochondria at low ΔΨ increased complex II (succinate)-energized respiration in muscle but had little effect in IBAT mitochondria. The transaminase inhibitor, aminooxyacetic acid, increased OAA concentrations and impaired succinate-energized respiration in muscle but not IBAT mitochondria at low but not high ΔΨ. Immunoblotting revealed that Got2 expression was far greater in muscle than IBAT mitochondria. Because we incidentally observed metabolism of OAA to pyruvate in IBAT mitochondria, more so than in muscle mitochondria, we also examined the expression of mitochondrial oxaloacetate decarboxylase (ODX). ODX was detected only in IBAT mitochondria. In summary, at low but not high ΔΨ, mitochondrial transamination clears OAA preventing loss of complex II respiration: a process far more active in muscle than IBAT mitochondria. We also provide evidence that OAA decarboxylation clears OAA to pyruvate in IBAT mitochondria.


Assuntos
Ácido Oxaloacético , Succinato Desidrogenase , Ácido Oxaloacético/metabolismo , Succinato Desidrogenase/metabolismo , Tecido Adiposo Marrom , Músculo Esquelético/metabolismo , Respiração , Ácido Pirúvico/metabolismo , Ácido Succínico/metabolismo
14.
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
15.
FASEB Bioadv ; 4(3): 197-210, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35392250

RESUMO

Classically, mitochondrial respiration responds to decreased membrane potential (ΔΨ) by increasing respiration. However, we found that for succinate-energized complex II respiration in skeletal muscle mitochondria (unencumbered by rotenone), low ΔΨ impairs respiration by a mechanism culminating in oxaloacetate (OAA) inhibition of succinate dehydrogenase (SDH). Here, we investigated whether this phenomenon extends to far different mitochondria of a tissue wherein ΔΨ is intrinsically low, i.e., interscapular brown adipose tissue (IBAT). Also, to advance our knowledge of the mechanism, we performed isotopomer studies of metabolite flux not done in our previous muscle studies. In additional novel work, we addressed possible ways ADP might affect the mechanism in IBAT mitochondria. UCP1 activity, and consequently ΔΨ, were perturbed both by GDP, a well-recognized potent inhibitor of UCP1 and by the chemical uncoupler carbonyl cyanide m-chlorophenyl hydrazone (FCCP). In succinate-energized mitochondria, GDP increased ΔΨ but also increased rather than decreased (as classically predicted under low ΔΨ) O2 flux. In GDP-treated mitochondria, FCCP reduced potential but also decreased respiration. Metabolite studies by NMR and flux analyses by LC-MS support a mechanism, wherein ΔΨ effects on the production of reactive oxygen alters the NADH/NAD+ ratio affecting OAA accumulation and, hence, OAA inhibition of SDH. We also found that ADP-altered complex II respiration in complex fashion probably involving decreased ΔΨ due to ATP synthesis, a GDP-like nucleotide inhibition of UCP1, and allosteric enzyme action. In summary, complex II respiration in IBAT mitochondria is regulated by UCP1-dependent ΔΨ altering substrate flow through OAA and OAA inhibition of SDH.

16.
JAMA Netw Open ; 5(9): e2230710, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36074461

RESUMO

Importance: The lower risk of cardiovascular disease (CVD) among women compared with men in the general population may be diminished among those with diabetes. Objective: To evaluate cardiometabolic risk factors and their management in association with CVD events in women vs men with type 1 diabetes enrolled in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Design, Setting, and Participants: This cohort study used data obtained during the combined DCCT (randomized clinical trial, conducted 1983-1993) and EDIC (observational study, conducted 1994 to present) studies through April 30, 2018 (mean [SD] follow-up, 28.8 [5.8] years), at 27 clinical centers in the US and Canada. Data analyses were performed between July 2021 and April 2022. Exposure: During the DCCT phase, patients were randomized to intensive vs conventional diabetes therapy. Main Outcomes and Measures: Cardiometabolic risk factors and CVD events were assessed via detailed medical history and focused physical examinations. Blood and urine samples were assayed centrally. CVD events were adjudicated by a review committee. Linear mixed models and Cox proportional hazards models evaluated sex differences in cardiometabolic risk factors and CVD risk over follow-up. Results: A total of 1441 participants with type 1 diabetes (mean [SD] age at DCCT baseline, 26.8 [7.1] years; 761 [52.8%] men; 1390 [96.5%] non-Hispanic White) were included. Over the duration of the study, compared with men, women had significantly lower body mass index (BMI, calculated as weight in kilograms divided by height in meters squared; ß = -0.43 [SE, 0.16]; P = .006), waist circumference (ß = -10.56 cm [SE, 0.52 cm]; P < .001), blood pressure (systolic: ß = -5.77 mm Hg [SE, 0.35 mm Hg]; P < .001; diastolic: ß = -3.23 mm Hg [SE, 0.26 mm Hg]; P < .001), and triglyceride levels (ß = -10.10 mg/dL [SE, 1.98 mg/dL]; P < .001); higher HDL cholesterol levels (ß = 9.36 mg/dL [SE, 0.57 mg/dL]; P < .001); and similar LDL cholesterol levels (ß = -0.76 mg/dL [SE, 1.22 mg/dL]; P = .53). Women, compared with men, achieved recommended targets more frequently for blood pressure (ie, <130/80 mm Hg: 90.0% vs 77.4%; P < .001) and triglycerides (ie, <150 mg/dL: 97.3% vs 90.5%; P < .001). However, sex-specific HDL cholesterol targets (ie, ≥50 mg/dL for women, ≥40 mg/dL for men) were achieved less often (74.3% vs 86.6%; P < .001) and cardioprotective medications were used less frequently in women than men (ie, angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker: 29.6% [95% CI, 25.7%-33.9%] vs 40.0% [95% CI, 36.1%-44.0%]; P = .001; lipid-lowering medication: 25.3% [95% CI, 22.1%-28.7%] vs 39.6% [95% CI, 36.1%-43.2%]; P < .001). Women also had significantly higher pulse rates (mean [SD], 75.2 [6.8] beats per minute vs 71.8 [6.9] beats per minute; P < .001) and hemoglobin A1c levels (mean [SD], 8.3% [1.0%] vs 8.1% [1.0%]; P = .01) and achieved targets for tighter glycemic control less often than men (ie, hemoglobin A1c <7%: 11.2% [95% CI, 9.3%-13.3%] vs 14.0% [95% CI, 12.0%-16.3%]; P = .03). Conclusions and Relevance: These findings suggest that despite a more favorable cardiometabolic risk factor profile, women with type 1 diabetes did not have a significantly lower CVD event burden than men, suggesting a greater clinical impact of cardiometabolic risk factors in women vs men with diabetes. These findings call for conscientious optimization of the control of CVD risk factors in women with type 1 diabetes.


Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 1 , Adulto , Fatores de Risco Cardiometabólico , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , HDL-Colesterol , Estudos de Coortes , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/epidemiologia , Feminino , Hemoglobinas Glicadas/análise , Humanos , Masculino , Fatores de Risco , Adulto Jovem
17.
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
18.
Methods Mol Biol ; 2276: 271-283, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060049

RESUMO

Several methods are available to measure ATP production by isolated mitochondria or permeabilized cells but have several limitations, depending upon the particular assay employed. These limitations may include poor sensitivity or specificity, complexity of the method, poor throughput, changes in mitochondrial inner membrane potential as ATP is consumed, and/or inability to simultaneously assess other mitochondrial functional parameters. Here we describe a novel nuclear magnetic resonance (NMR)-based assay that can be carried out with high efficiency in a manner that alleviates the above problems.


Assuntos
Trifosfato de Adenosina/metabolismo , Peróxido de Hidrogênio/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Desoxiglucose/metabolismo , Metabolismo Energético , Hexoquinase/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo
19.
J Med Chem ; 64(1): 662-676, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33395531

RESUMO

Mitochondrial dysfunction is an underlying pathology in numerous diseases. Delivery of diagnostic and therapeutic cargo directly into mitochondria is a powerful approach to study and treat these diseases. The triphenylphosphonium (TPP+) moiety is the most widely used mitochondriotropic carrier. However, studies have shown that TPP+ is not inert; TPP+ conjugates uncouple mitochondrial oxidative phosphorylation. To date, all efforts toward addressing this problem have focused on modifying lipophilicity of TPP+-linker-cargo conjugates to alter mitochondrial uptake, albeit with limited success. We show that structural modifications to the TPP+ phenyl rings that decrease electron density on the phosphorus atom can abrogate uncoupling activity as compared to the parent TPP+ moiety and prevent dissipation of mitochondrial membrane potential. These alterations of the TPP+ structure do not negatively affect the delivery of cargo to mitochondria. Results here identify the 4-CF3-phenyl TPP+ moiety as an inert mitochondria-targeting carrier to safely target pharmacophores and probes to mitochondria.


Assuntos
Portadores de Fármacos , Mitocôndrias/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Humanos , Mitocôndrias/metabolismo , Compostos Organofosforados/metabolismo , Fosforilação Oxidativa
20.
Pharmacol Res Perspect ; 9(1): e00701, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33547885

RESUMO

Previous work by ourselves and others showed that mitoquinone (mitoQ) reduced oxidative damage and prevented hepatic fat accumulation in mice made obese with high-fat (HF) feeding. Here we extended these studies to examine the effect of mitoQ on parameters affecting liver function in rats treated with HF to induce obesity and in rats treated with HF plus streptozotocin (STZ) to model a severe form of type 2 diabetes. In prior reported work, we found that mitoQ significantly improved glycemia based on glucose tolerance data in HF rats but not in the diabetic rats. Here we found only non-significant reductions in insulin and glucose measured in the fed state at sacrifice in the HF mice treated with mitoQ. Metabolomic data showed that mitoQ altered several hepatic metabolic pathways in HF-fed obese rats toward those observed in control normal chow-fed non-obese rats. However, mitoQ had little effect on pathways observed in the diabetic rats, wherein diabetes itself induced marked pathway aberrations. MitoQ did not alter respiration or membrane potential in isolated liver mitochondria. MitoQ reduced liver fat and liver hydroperoxide levels but did not improve liver function as marked by circulating levels of aspartate and alanine aminotransferase (ALT). In summary, our results for HF-fed rats are consistent with past findings in HF-fed mice indicating decreased liver lipid hydroperoxides (LPO) and improved glycemia. However, in contrast to the HF obese mice, mitoQ did not improve glycemia or reset perturbed metabolic pathways in the diabetic rats.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Fígado Gorduroso/metabolismo , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Compostos Organofosforados/farmacologia , Ubiquinona/análogos & derivados , Animais , Glicemia/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Dieta Hiperlipídica , Fígado Gorduroso/sangue , Insulina/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metabolômica , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Mitocôndrias Hepáticas/fisiologia , Obesidade/sangue , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Ubiquinona/farmacologia
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