Mitochondrial energetics is impaired in vivo in aged skeletal muscle

With aging, most skeletal muscles undergo a progressive loss of mass and strength, a process termed sarcopenia. Aging‐related defects in mitochondrial energetics have been proposed to be causally involved in sarcopenia. However, changes in muscle mitochondrial oxidative phosphorylation with aging remain a highly controversial issue, creating a pressing need for integrative approaches to determine whether mitochondrial bioenergetics are impaired in aged skeletal muscle. To address this issue, mitochondrial bioenergetics was first investigated in vivo in the gastrocnemius muscle of adult (6 months) and aged (21 months) male Wistar rats by combining a modular control analysis approach with ³¹P magnetic resonance spectroscopy measurements of energetic metabolites. Using this innovative approach, we revealed that the in vivo responsiveness (‘elasticity’) of mitochondrial oxidative phosphorylation to contraction‐induced increase in ATP demand is significantly reduced in aged skeletal muscle, a reduction especially pronounced under low contractile activities. In line with this in vivo aging‐related defect in mitochondrial energetics, we found that the mitochondrial affinity for ADP is significantly decreased in mitochondria isolated from aged skeletal muscle. Collectively, the results of this study demonstrate that mitochondrial bioenergetics are effectively altered in vivo in aged skeletal muscle and provide a novel cellular basis for this phenomenon.     

Impaired resting muscle energetics studied by (31)P-NMR in diet-induced obese rats

Objective: Mitochondrial activity is altered in skeletal muscle of obese, insulin‐resistant or type 2 diabetic patients. We hypothesized that this situation was associated with profound adaptations in resting muscle energetics. For that purpose, we used in vivo ³¹P‐nuclear magnetic resonance (³¹P‐NMR) in male sedentary Wistar rats fed with obesogenic diets known to induce alterations in muscle mitochondrial activity. Methods and Procedures: Two experimental diets (high sucrose and high fat) were provided for 6 weeks at two levels of energy (standard, N and high, H) and compared to control diet. The rates of the adenosine triphosphate (ATP) exchange between phosphocreatine (PCr) and γ ‐ATP (k_a) and β ‐adenosine diphosphate ( β ‐ADP) to β ‐ATP (k_b) were evaluated using ³¹P‐NMR in resting gastrocnemius muscle. Muscle contents in phosphorylated compounds as well as creatine, were assessed using ³¹P‐NMR and biochemical assays, respectively. Results: ATP content increased by 6.7–8.5% in standard‐energy high‐sucrose (NSU), high‐energy high‐fat (HF) and high‐energy high‐sucrose (HSU) groups compared to control (P < 0.05), whereas PCr content decreased by 4.2–6.4% (P < 0.01). Consequently, PCr to ATP ratio decreased in NSU, HF, and HSU groups, compared to control (P < 0.01). Furthermore in high‐energy groups (HF and HSU) compared to control, creatine contents were decreased by 14–19% (P < 0.001), whereas k_a and k_b fluxes were increased by 89–133% (P < 0.001) and 243–277% (P < 0.01), respectively. Discussion: Our in vivo data showed adaptations of resting skeletal muscle energetics in response to high‐energy diets. Increased activity of enzymes catalyzing ATP production may reflect a compensatory mechanism to face impaired mitochondrial ATP synthesis in order to preserve intracellular energy homeostasis.     

Treatment with the mitochondrial‐targeted antioxidant peptide SS‐31 rescues neurovascular coupling responses and cerebrovascular endothelial function and improves cognition in aged mice

Moment‐to‐moment adjustment of cerebral blood flow (CBF) via neurovascular coupling has an essential role in maintenance of healthy cognitive function. In advanced age, increased oxidative stress and cerebromicrovascular endothelial dysfunction impair neurovascular coupling, likely contributing to age‐related decline of higher cortical functions. There is increasing evidence showing that mitochondrial oxidative stress plays a critical role in a range of age‐related cellular impairments, but its role in neurovascular uncoupling remains unexplored. This study was designed to test the hypothesis that attenuation of mitochondrial oxidative stress may exert beneficial effects on neurovascular coupling responses in aging. To test this hypothesis, 24‐month‐old C57BL/6 mice were treated with a cell‐permeable, mitochondria‐targeted antioxidant peptide (SS‐31; 10 mg kg^?1 day^?1, i.p.) or vehicle for 2 weeks. Neurovascular coupling was assessed by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that neurovascular coupling responses were significantly impaired in aged mice. Treatment with SS–31 significantly improved neurovascular coupling responses by increasing NO‐mediated cerebromicrovascular dilation, which was associated with significantly improved spatial working memory, motor skill learning, and gait coordination. These findings are paralleled by the protective effects of SS–31 on mitochondrial production of reactive oxygen species and mitochondrial respiration in cultured cerebromicrovascular endothelial cells derived from aged animals. Thus, mitochondrial oxidative stress contributes to age‐related cerebromicrovascular dysfunction, exacerbating cognitive decline. We propose that mitochondria‐targeted antioxidants may be considered for pharmacological microvascular protection for the prevention/treatment of age‐related vascular cognitive impairment (VCI).     

In vivo and in vitro assessment of brain bioenergetics in aging rats

Brain energy disorders can be present in aged men and animals. To this respect, the mitochondrial and free radical theory of aging postulates that age‐associated brain energy disorders are caused by an imbalance between pro‐ and anti‐oxidants that can result in oxidative stress. Our study was designed to investigate brain energy metabolism and the activity of endogenous antioxidants during their lifespan in male Wistar rats. In vivo brain bioenergetics were measured using ³¹P nuclear magnetic resonance (NMR) spectroscopy and in vitro by polarographic analysis of mitochondrial oxidative phosphorylation. When compared to the young controls, a significant decrease of age‐dependent mitochondrial respiration and adenosine‐3‐phosphate (ATP) production measured in vitro correlated with significant reduction of forward creatine kinase reaction (kfor) and with an increase in phosphocreatine (PCr)/ATP, PCr/Pi and PME/ATP ratio measured in vivo. The levels of enzymatic antioxidants catalase, GPx and GST significantly decreased in the brain tissue as well as in the peripheral blood of aged rats. We suppose that mitochondrial dysfunction and oxidative inactivation of endogenous enzymes may participate in age‐related disorders of brain energy metabolism.     

Mitochondrial function in vivo: spectroscopy provides window on cellular energetics

Mitochondria integrate the key metabolic fluxes in the cell. This role places this organelle at the center of cellular energetics and, hence, mitochondrial dysfunction underlies a growing number of human disorders and age-related degenerative diseases. Here we present novel analytical and technical methods for evaluating mitochondrial metabolism and (dys)function in human muscle in vivo. Three innovations involving advances in optical spectroscopy (OS) and magnetic resonance spectroscopy (MRS) permit quantifying key compounds in energy metabolism to yield mitochondrial oxidation and phosphorylation fluxes. The first of these uses analytical methods applied to optical spectra to measure hemoglobin (Hb) and myoglobin (Mb) oxygenation states and relative contents ([Hb]/[Mb]) to determine mitochondrial respiration (O₂ uptake) in vivo. The second uses MRS methods to quantify key high-energy compounds (creatine phosphate, PCr, and adenosine triphosphate, ATP) to determine mitochondrial phosphorylation (ATP flux) in vivo. The third involves a functional test that combines these spectroscopic approaches to determine mitochondrial energy coupling (ATP/O₂), phosphorylation capacity (ATP_max) and oxidative capacity (O_2max) of muscle. These new developments in optical and MR tools allow us to determine the function and capacity of mitochondria noninvasively in order to identify specific defects in vivo that are associated with disease in human and animal muscle. The clinical implication of this unique diagnostic probe is the insight into the nature and extent of dysfunction in metabolic and degenerative disorders, as well as the ability to follow the impact of interventions designed to reverse these disorders.     

MITOCHONDRIA: Investigation of in vivo muscle mitochondrial function by 31P magnetic resonance spectroscopy

The most important function of mitochondria is the production of energy in the form of ATP. The socio-economic impact of human diseases that affect skeletal muscle mitochondrial function is growing, and improving their clinical management critically depends on the development of non-invasive assays to assess mitochondrial function and monitor the effects of interventions. ³¹P magnetic resonance spectroscopy provides two approaches that have been used to assess in vivo ATP synthesis in skeletal muscle: measuring P_i ⟶ ATP exchange flux using saturation transfer in resting muscle, and measuring phosphocreatine recovery kinetics after exercise. However, P_i ⟶ ATP exchange does not represent net mitochondrial ATP synthesis flux and has no simple relationship with mitochondrial function. Post-exercise phosphocreatine recovery kinetics, on the other hand, yield reliable measures of muscle mitochondrial capacity in vivo, whose ability to define the site of functional defects is enhanced by combination with other non-invasive techniques.     

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo

Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (³¹P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.     

A tissue‐specific screen of ceramide expression in aged mice identifies ceramide synthase‐1 and ceramide synthase‐5 as potential regulators of fiber size and strength in skeletal muscle

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi‐organ investigation of age‐associated changes of ceramide species, a unique and distinctive change pattern of C_16:0 and C_18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C_16:0 and C_18:0 acyl chains, respectively, was down‐regulated in skeletal muscle of aged mice. Similarly, an age‐dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1^Δ/Δ and CerS5^Δ/Δ knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1‐ and CerS5‐deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age‐related muscle wasting disorders in humans.     

Deletion of junctional adhesion molecule A from platelets increases early‐stage neointima formation after wire injury in hyperlipidemic mice

Platelets play an important role in the pathogenesis of vascular remodelling after injury. Junctional adhesion molecule A (JAM‐A) was recently described to regulate platelet activation. Specific deletion of JAM‐A from platelets resulted in increased reactivity and in accelerated progression of atherosclerosis. The aim of this study was to investigate the specific contribution of platelet‐derived JAM‐A to neointima formation after vascular injury. Mice with or without platelet‐specific (tr)JAM‐A‐deficiency in an apolipoprotein e (apoe^?/?) background underwent wire‐induced injury of the common carotid artery. Ex vivo imaging by two‐photon microscopy revealed increased platelet coverage at the site of injury in trJAM‐A‐deficient mice. Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM‐A‐deficient platelets than to control platelets. Inhibition of α_Mβ₂ or GPIbα, but not of CD62P, suppressed those differences. Up to 4 weeks after wire injury, intimal neoplasia and neointimal cellular content were analysed. Neointimal lesion area was increased in trJAM‐A^?/? apoe^?/? mice and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM‐A^+/+ apoe^?/? littermates 2 weeks, but not 4 weeks after injury. Re‐endothelialization was decreased in trJAM‐A^?/? apoe^?/? mice compared with controls 2 weeks after injury, yet it was complete in both groups after 4 weeks. A platelet gain of function by deletion of JAM‐A accelerates neointima formation only during earlier phases after vascular injury, through an increased recruitment of mononuclear cells. Thus, the contribution of platelets might become less important when neointima formation progresses to later stages.     

Glutaredoxin-2 Is Required to Control Oxidative Phosphorylation in Cardiac Muscle by Mediating Deglutathionylation Reactions

Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation of mitochondrial proteins. Deletion of Grx2 (Grx2^−/−) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring the deglutathionylated states of proteins. Grx2^−/− hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from Grx2 heterozygotes (Grx2^+/−) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2^+/− mice were far less hypertensive than Grx2^−/− mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel redox-related therapies in cardiac disease.     

Mesenchymal stem cells conditioned with glucose depletion augments their ability to repair-infarcted myocardium

Mesenchymal stem cells (MSCs) are an attractive candidate for autologous cell therapy, but their ability to repair damaged myocardium is severely compromised with advanced age. Development of viable autologous cell therapy for treatment of heart failure in the elderly requires the need to address MSC ageing. In this study, MSCs from young (2 months) and aged (24 months) C57BL/6 mice were characterized for gene expression of IGF‐1, FGF‐2, VEGF, SIRT‐1, AKT, p16^INK4a, p21 and p53 along with measurements of population doubling (PD), superoxide dismutase (SOD) activity and apoptosis. Aged MSCs displayed senescent features compared with cells isolated from young animals and therefore were pre‐conditioned with glucose depletion to enhance age affected function. Pre‐conditioning of aged MSCs led to an increase in expression of IGF‐1, AKT and SIRT‐1 concomitant with enhanced viability, proliferation and delayed senescence. To determine the myocardial repair capability of pre‐conditioned aged MSCs, myocardial infarction (MI) was induced in 24 months old C57BL/6 wild type mice and GFP expressing untreated and pre‐conditioned aged MSCs were transplanted. Hearts transplanted with pre‐conditioned aged MSCs showed increased expression of paracrine factors, such as IGF‐1, FGF‐2, VEGF and SDF‐1α. This was associated with significantly improved cardiac performance as measured by dp/dt_max, dp/dt_min, LVEDP and LVDP, declined left ventricle (LV) fibrosis and apoptosis as measured by Masson's Trichrome and TUNEL assays, respectively, after 30 days of transplantation. In conclusion, pre‐conditioning of aged MSCs with glucose depletion can enhance proliferation, delay senescence and restore the ability of aged cells to repair senescent infarcted myocardium.     

High-level resistance to gentamicin: genetic transfer between Enterococcus faecalis isolated from food of animal origin and human microbiota

Aims: To investigate the in vivo gene transfer of high‐level gentamicin resistance (HLRG) from Enterococcus faecalis isolated from the food of animal origin to a human isolate, using a mouse model of intestinally colonized human microbiota. Methods and Results: In vitro study: The presence of plasmids involved in HLRG coding was investigated. After the conjugation experiment, the recipient strain, Ent. faecalis JH2‐SS, acquired a plasmid responsible for HLRG [minimal inhibitory concentration (MIC) >800 μg ml^?1], in a similar position to the donor cells. In vivo study: Seven BALB/c mice were dosed with ceftriaxone (400 mg kg^?1) and then inoculated with a dilution of 1/100 of human faeces (HFc). After 72 h, Ent. faecalis JH2‐SS (recipient) was inoculated and then, after a further 72 h, the animals were given Ent. faecalis CS19, isolated from the food of animal origin, involved in HLRG (donor). The presence of transconjugant strains in HFc was subsequently recorded on a daily basis until the end of the experiment. The clonal relationship between Ent. faecalis and Escherichia coli in faeces was assessed by RAPD‐PCR. Both the in vitro and in vivo studies showed that the receptor strain acquired a plasmid responsible for HLRG (MICs >800 μg ml^?1), which migrated with a similar relative mobility value. Transconjugant strains were detected from 24 h after the donor strain inoculation and persisted until the end of the experiment. Conclusions: The in vivo gene transfer of HLRG from Ent. faecalis strains, isolated from the food of animal origin, to human microbiota has been demonstrated in a mouse model. Significance and Impact of the Study: The complexity found on the therapeutic responses of invasive infectious diseases caused by Ent. faecalis facilitates the assessment of food of animal origin as a resistant pathogen reservoir. In addition, this study may contribute to the understanding of antimicrobials’ resistance gene transfer between Ent. faecalis strains from food and human GI tract.     

Endogenous reactive oxygen species cause astrocyte defects and neuronal dysfunctions in the hippocampus: a new model for aging brain

The etiology of astrocyte dysfunction is not well understood even though neuronal defects have been extensively studied in a variety of neuronal degenerative diseases. Astrocyte defects could be triggered by the oxidative stress that occurs during physiological aging. Here, we provide evidence that intracellular or mitochondrial reactive oxygen species (ROS) at physiological levels can cause hippocampal (neuronal) dysfunctions. Specifically, we demonstrate that astrocyte defects occur in the hippocampal area of middle‐aged Tet‐mev‐1 mice with the SDHC^V69E mutation. These mice are characterized by chronic oxidative stress. Even though both young adult and middle‐aged Tet‐mev‐1 mice overproduced MitoSOX Red‐detectable mitochondrial ROS compared to age‐matched wild‐type C57BL/6J mice, only young adult Tet‐mev‐1 mice upregulated manganese and copper/zinc superoxide dismutase (Mn‐ and Cu/Zn‐SODs) activities to eliminate the MitoSOX Red‐detectable mitochondrial ROS. In contrast, middle‐aged Tet‐mev‐1 mice accumulated both MitoSOX Red‐detectable mitochondrial ROS and CM‐H₂DCFDA‐detectable intracellular ROS. These ROS levels appeared to be in the physiological range as shown by normal thiol and glutathione disulfide/glutathione concentrations in both young adult and middle‐aged Tet‐mev‐1 mice relative to age‐matched wild‐type C57BL/6J mice. Furthermore, only middle‐aged Tet‐mev‐1 mice showed JNK/SAPK activation and Ca²⁺ overload, particularly in astrocytes. This led to decreasing levels of glial fibrillary acidic protein and S100β in the hippocampal area. Significantly, there were no pathological features such as apoptosis, amyloidosis, and lactic acidosis in neurons and astrocytes. Our findings suggest that the age‐dependent physiologically relevant chronic oxidative stress caused astrocyte defects in mice with impaired mitochondrial electron transport chain functionality.     

AMP‐activated protein kinase deficiency exacerbates aging‐induced myocardial contractile dysfunction

Aging is associated with myocardial dysfunction although the underlying mechanism is unclear. AMPK, a key cellular fuel sensor for energy metabolism, is compromised with aging. This study examined the role of AMPK deficiency in aging‐associated myocardial dysfunction. Young or old wild‐type (WT) and transgenic mice with overexpression of a mutant AMPK α₂ subunit (kinase dead, KD) were used. AMPK α isoform activity, myocardial function and morphology were examined. DCF and JC‐1 fluorescence probes were employed to quantify reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm), respectively. KD mice displayed significantly reduced α₂ but not α₁ AMPK isoform activity at both ages with a greater effect at old age. Aging itself decreased α₁ isoform activity. Cardiomyocyte contractile function, intracellular Ca²⁺ handling, and SERCA2a levels were compromised with aging, the effects of which were exacerbated by AMPK deficiency. H&E staining revealed cardiomyocyte hypertrophy with aging, which was more pronounced in KD mice. TEM micrographs displayed severe disruption of mitochondrial ultrastructure characterized by swollen, irregular shape and disrupted cristae in aged KD compared with WT mice. Aging enhanced ROS production and reduced ΔΨm, the effects of which were accentuated by AMPK deficiency. Immunoblotting data depicted unchanged Akt phosphorylation and a significant decrease in mitochondrial biogenesis cofactor PGC‐1α in aged groups. AMPK deficiency but not aging decreased the phosphorylation of ACC and eNOS. Expression of membrane Glut4 and HSP90 was decreased in aged KD mice. Moreover, treatment of the AMPK activator metformin attenuated aging‐induced cardiomyocyte contractile defects. Collectively, our data suggest a role for AMPK deficiency in aging‐induced cardiac dysfunction possibly through disrupted mitochondrial function and ROS production.     

Low plasma lysophosphatidylcholines are associated with impaired mitochondrial oxidative capacity in adults in the Baltimore Longitudinal Study of Aging

The decrease in skeletal muscle mitochondrial oxidative capacity with age adversely affects muscle strength and physical performance. Factors that are associated with this decrease have not been well characterized. Low plasma lysophosphatidylcholines (LPC), a major class of systemic bioactive lipids, are predictive of aging phenotypes such as cognitive impairment and decline of gait speed in older adults. Therefore, we tested the hypothesis that low plasma LPC are associated with impaired skeletal muscle mitochondrial oxidative capacity. Skeletal muscle mitochondrial oxidative capacity was measured using in vivo phosphorus magnetic resonance spectroscopy (³¹P‐MRS) in 385 participants (256 women, 129 men), aged 24–97 years (mean 72.5) in the Baltimore Longitudinal Study of Aging. Postexercise recovery rate of phosphocreatine (PCr), k_PCr, was used as a biomarker of mitochondrial oxidative capacity. Plasma LPC were measured using liquid chromatography–tandem mass spectrometry. Adults in the highest quartile of k_PCr had higher plasma LPC 16:0 (p = 0.04), 16:1 (p = 0.004), 17:0 (p = 0.01), 18:1 (p = 0.0002), 18:2 (p = 0.002), and 20:3 (p = 0.0007), but not 18:0 (p = 0.07), 20:4 (p = 0.09) compared with those in the lower three quartiles in multivariable linear regression models adjusting for age, sex, and height. Multiple machine‐learning algorithms showed an area under the receiver operating characteristic curve of 0.638 (95% confidence interval, 0.554, 0.723) comparing six LPC in adults in the lower three quartiles of k_PCr with the highest quartile. Low plasma LPC are associated with impaired mitochondrial oxidative capacity in adults.     

Microwave irradiation decreases ATP,increases free [Mg2+], and alters in vivo intracellular reactions in rat brain

Rapid inactivation of metabolism is essential for accurately determining the concentrations of metabolic intermediates in the in vivo state. We compared a broad spectrum of energetic intermediate metabolites and neurotransmitters in brains obtained by microwave irradiation to those obtained by freeze blowing, the most rapid method of extracting and freezing rat brain. The concentrations of many intermediates, cytosolic free NAD(P)⁺/NAD(P)H ratios, as well as neurotransmitters were not affected by the microwave procedure. However, the brain concentrations of ATP were about 30% lower, whereas those of ADP, AMP, and GDP were higher in the microwave‐irradiated compared with the freeze‐blown brains. In addition, the hydrolysis of approximately 1 μmol/g of ATP, a major in vivo Mg²⁺‐binding site, was related to approximately five‐fold increase in free [Mg²⁺] (0.53 ± 0.07 mM in freeze blown vs. 2.91 mM ± 0.48 mM in microwaved brains), as determined from the ratio [citrate]/[isocitrate]. Consequently, many intracellular properties, such as the phosphorylation potential and the ?G' of ATP hydrolysis were significantly altered in microwaved tissue. The determinations of some glycolytic and TCA cycle metabolites, the phosphorylation potential, and the ?G' of ATP hydrolysis do not represent the in vivo state when using microwave‐fixed brain tissue.     

Control of mitochondrial respiration in muscle

Summary Control of mitochondrial respiration depends on ADP availability to the F₁ATPase. An electrochemical gradient of ADP and ATP across the mitochondrial inner membrane is maintained by the adenine nucleotide translocase which provides ADP to the matrix for ATP synthesis and ATP for energy-dependent processes in the cytosol. Mitochondrial respiration is responsive to the cytosolic phosphorylation potential, ATP/ADP · P_i which is in apparent equilibrium with the first two sites in the electron transport chain. Conventional measures of free adenine nucleotides is a confounding issue in determining cytosolic and mitochondrial phosphorylation potentials. The advent of phosphorus-31 nuclear magnetic resonance (P-31 NMR) allows the determination of intracellular free concentrations of ATP, creatine-P and Pi in perfused muscle in situ. In the glucose-perfused heart, there is an absence of correlation between the cytosolic phosphorylation potential as determined by P-31 NMR and cardiac oxygen consumption over a range of work loads. These data suggest that contractile work leads to increased generation of mitochondrial NADH so that ATP production keeps pace with myosin ATPase activity. The mechanism of increased ATP synthesis is referred to as stimulusre-sponse-metabolism coupling. In muscle, increased contractility is a result of interventions which increase cytosolic free Ca²⁺ concentrations. The Ca^2- signal thus generated increases glycogen breakdown and myosin ATPase in the cytosol. This signal is concomitantly transmitted to the mitochondria which respond to small increases in matrix Ca²⁺ by activation of Ca²⁺-sensitive dehydrogenases. The Ca²⁺-activated dehydrogenase activities are key rate-controlling enzymes in tricarboxylic acid cycle flux, and their activation by Ca^2- leads to increased pyridine nucleotide reduction and oxidative phosphorylation. These observations which have been consistent in preparations both in vitro and in situ do not obviate a role for ADP control of muscle respiration, but do explain, in part, the lack of dramatic fluctuations in the cytosolic phosphorylation potential over a large range of contractile activities.     

Pharmacokinetics of oxytetracycline in yellow catfish [Pelteobagrus fulvidraco (Richardson, 1846)] with a single and multiple‐dose oral administration

A pharmacokinetic study of oxytetracycline (OTC) following a single (100 mg kg^?1) or a multi‐dose (100 mg kg^?1 for 5 days) oral administration was carried out in yellow catfish, Pelteobagrus fulvidraco. After oral administration at 25°C, a one‐compartment model was developed. The absorption half‐life (t_1/2(ka)) was 3.92, 1.44, 2.75, and 3.34 h in plasma, muscle, liver, and kidney after the single dose, and 0.35, 0.22, 0.42, 0.32 h after the multi‐dose, respectively. The order of peak concentration (C_max) was liver > kidney > plasma > muscle, at 3.48 μg g^?1, 2.90 μg g^?1, 1.46 μg ml^?1, and 1.39 μg g^?1 after the single dose, and 14.02 μg g^?1, 8.51 μg g^?1, 4.17 μg ml^?1, and 3.84 μg g^?1 after the multi‐dose, respectively. The elimination half‐lives (t_1/2(ke)) of OTC in plasma, muscle, liver, and kidney were calculated to be 7.64, 26.29, 19.08, and 10.61 h after the single dose, and 47.54, 70.99, 49.87, and 47.73 h after the multi‐dose, respectively. The results suggest that OTC was absorbed faster after the multi‐dose than after the single dose, suggesting that OTC could be more efficacious after the multi‐dose and more effective in the control bacterial diseases in aquaculture, with the side effects of longer withdrawal periods.     

AnxA5 reduces plaque inflammation of advanced atherosclerotic lesions in apoE−/− mice

Annexin A5 (AnxA5) exerts anti‐inflammatory, anticoagulant and anti‐apoptotic effects through binding cell surface expressed phosphatidylserine. The actions of AnxA5 on atherosclerosis are incompletely understood. We investigated effects of exogenous AnxA5 on plaque morphology and phenotype of advanced atherosclerotic lesions in apoE^?/? mice. Advanced atherosclerotic lesions were induced in 12 weeks old Western type diet fed apoE^?/? mice using a collar placement around the carotid artery. After 5 weeks mice were injected either with AnxA5 (n = 8) or vehicle for another 4 weeks. AnxA5 reduced plaque macrophage content both in the intima (59% reduction, P < 0.05) and media (73% reduction, P < 0.01) of advanced atherosclerotic lesions of the carotid artery. These findings corroborated with advanced lesions of the aortic arch, where a 67% reduction in plaque macrophage content was observed with AnxA5 compared to controls (P < 0.01). AnxA5 did not change lesion extension, plaque apoptosis, collagen content, smooth muscle cell content or acellular plaque composition after 4 weeks of treatment as determined by immunohistochemistry in advanced carotid lesions. In vitro, AnxA5 exhibited anti‐inflammatory effects in macrophages and a flow chamber based assay demonstrated that AnxA5 significantly inhibited capture, rolling, adhesion as well as transmigration of peripheral blood mononuclear cells on a TNF‐α‐activated endothelial cell layer. In conclusion, short‐term treatment with AnxA5 reduces plaque inflammation of advanced lesions in apoE^?/? mice likely through interfering with recruitment and activation of monocytes to the inflamed lesion site. Suppressing chronic inflammation by targeting exposed phosphatidylserine may become a viable strategy to treat patients suffering from advanced atherosclerosis.