线粒体氧化磷酸化抑制剂FCCP体外对人精子动力学和线粒体功能的影响

目的通过线粒体氧化磷酸化(OXPHOS)特异性抑制剂FCCP对人精子活动力及其线粒体功能影响的研究,探讨氧化磷酸化在精子能量代谢中的作用。方法选择来自捐精志愿者的正常精液8份,优选后制备精子悬液,将每份精子悬液分为4组,分别与终浓度为0μmol/L(对照组)、2.5μmol/L、5μmol/L和10μmol/L的FCCP共孵育1h、3h、5h,以精子动力学参数、线粒体膜电位、精子细胞内ATP含量、精子质膜完整性作为评价指标,分析比较各组间的差异。结果 (1)各组精子活动率和其他各项运动参数随着FCCP浓度增高呈下降趋势:孵育1h后,与对照组相比,仅10μmol/L组的精子活动率、前向运动百分率和精子头侧摆幅度(ALH)显著下降(P0.05),其余指标无显著性变化,而其余浓度处理组的各指标变化均无统计学意义(P0.05);孵育3h后,10μmol/L组的精子活动率、前向运动百分率、平均路径速率(VAP)、直线速率(VSL)、曲线速率(VCL)、ALH和鞭打频率(BCF)均显著降低(P0.05),5μmol/L组的ALH和BCF指标显著下降(P0.05);孵育5h后,与对照组相比,10μmol/L组的前述指标继续显著性下降(P0.05),5μmol/L组的精子活动率和前向运动百分率也出现显著降低(P0.05),而2.5μmol/L组各指标差异均无统计学意义(P0.05)。(2)精子线粒体膜电位(MMP)和ATP含量随FCCP浓度增加逐渐降低,10μmol/L组的MMP和ATP含量显著低于对照组(P0.05)。(3)质膜完整性比较中,10μmol/L组比对照组显著降低(73.94%vs.84.53%)(P0.05)。(4)随着FCCP孵育时间延长,各组精子活动率和前向运动百分率呈下降趋势。结论不同浓度的FCCP体外处理精子后,精子活动率和其他各项运动参数,以及反映线粒体活性的线粒体膜电位和ATP含量呈浓度依赖性下降,FCCP所抑制的线粒体氧化磷酸化是精子能量代谢的重要途径。     

Oxidative capacity, lipotoxicity, and mitochondrial damage in type 2 diabetes

Recent evidence points toward decreased oxidative capacity and mitochondrial aberrations as a major contributor to the development of insulin resistance and type 2 diabetes. In this article we will provide an integrative view on the interrelation between decreased oxidative capacity, lipotoxicity, and mitochondrial aberrations in type 2 diabetes. Type 2 diabetes is characterized by disturbances in fatty acid metabolism and is accompanied by accumulation of fatty acids in nonadipose tissues. In metabolically active tissues, such as skeletal muscle, fatty acids are prone to so-called oxidative damage. In addition to producing energy, mitochondria are also a major source of reactive oxygen species, which can lead to lipid peroxidation. In particular, the mitochondrial matrix, which contains DNA, RNA, and numerous enzymes necessary for substrate oxidation, is sensitive to peroxide-induced oxidative damage and needs to be protected against the formation and accumulation of lipids and lipid peroxides. Recent evidence reports that mitochondrial uncoupling is involved in the protection of the mitochondrial matrix against lipid-induced mitochondrial damage. Disturbances in this protection mechanism can contribute to the development of type 2 diabetes.     

The mitochondrial uncoupling agent 2,4-dinitrophenol improves mitochondrial function, attenuates oxidative damage, and increases white matter sparing in the contused spinal cord

The purpose of this study was to investigate the potential neuroprotective efficacy of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) in rats following a mild to moderate spinal cord contusion injury. Animals received intraperitoneal injections of vehicle (DMSO) or 5 mg/mL of DNP prior to injury. Twenty-four hours following surgery, mitochondrial function was assessed in mitochondria isolated from spinal cord synaptosomes. In addition, synaptosomes were used to measure indicators of reactive oxygen species formation, lipid peroxidation, and protein oxidation. Relative to vehicle-treated animals, pretreatment with DNP maintained mitochondrial bioenergetics and significantly decreased reactive oxygen species levels, lipid peroxidation, and protein carbonyl content following spinal cord injury. Furthermore, pretreatment with DNP significantly increased the amount of remaining white matter at the injury epicenter 6 weeks after injury. These results indicate that treatment with mitochondrial uncoupling agents may provide a novel approach for the treatment of secondary injury following spinal cord contusion.     

Relationship between sperm mitochondrial membrane potential,sperm motility,and fertility potential

AIM:To analyze the relationship between sperm mitochondrial membrane potential and sperm motility parameters by means of a computer-assisted sperm analyzer (CASA) and in-vitro fertilization rate(%FR). METHODS: Semen samples were obtained from 26 men undergoing in vitro fertilization-embryo transfer (IVF-ET). Informed consent was obtained from all men prior to the study. Samples were prepared using wash and swim-up method in HEPES-HTF medium. The sperm motility (%MOT), progressive motility (%PMOT), average path velocity (VAP) microm/s), straight line velocity (VSL) (micro m/s), curvilinear velocity (VCL) (microm/s) and %hyperactivated sperm (%HA), and the %FR were assessed. The samples were incubated in the presence of 2.0 mciromol/L of 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) for 30 min at 37 degrees C in air and washed in PBS before flow cytometry (FACSCalibur: Becton Dickinson) analysis. The mitochondrial probe JC-1 was used to identify the mitochondrial membrane potential. The sperm was divided into three populations according to the fluorescence pattern as follows: the high mitochondrial membrane potential group (n=8), the moderate group (n=5), and the low group (n=13). Statistical analysis was performed using unpaired t-test. RESULTS:Significant differences were found between the high and the low groups in %MOT (91.1+/-8.5 vs 63.0+/-32.7, mean+/-SD), VAP (73.0+/-14.2 vs 52.1+/-12.5), VCL (127.0+/-28.1 vs 87.0+/-22.6), %HA (27.3+/-23.6 vs 7.2+/-9.0) and %FR [73.2 (48/56) vs 59.0 (69/117)]. No significant differences were found in other CASA parameters. CONCLUSION: When the sperm mitochondrial membrane potential increases, sperm motility parameters and fertility potential will also increase. The JC-1 dye method is useful to predict sperm fertility potential.     

Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis

The aim of this study was to evaluate conventional semen parameters (density, morphology, and progressive motility) and the flow-cytometric parameters of DNA fragmentation, mitochondrial membrane potential, phosphatidylserine externalization, and chromatin compactness in patients with varicocele before and after varicocelectomy. Thirty men (26.5 ± 3.2 years old, range 20-32 years) with oligoasthenoteratozoospermia and grade 3 left varicocele were selected (without other causes of male infertility). Each of them underwent sperm analysis and flow cytometric evaluation before and 4 months after subinguinal microsurgical varicocelectomy (SMV). After varicocelectomy, men had significantly higher sperm density, progressive motility, and normal forms compared with baseline. They also had a significantly lower percentage of spermatozoa with low mitochondrial membrane potential. After SMV, they showed a significantly lower percentage of spermatozoa with phosphatidylserine externalization, an early sign of apoptosis. Significantly decreased percentages of spermatozoa with abnormal chromatin compactness and spermatozoa with DNA fragmentation were found after SMV compared with baseline. Subinguinal microsurgical varicocelectomy improves sperm function in oligoasthenoteratozoospermia secondary to grade 3 left varicocele. Improvements are seen in conventional parameters and biofunctional parameters not routinely evaluated.     

Proximal tubular cytochrome c efflux: determinant, and potential marker, of mitochondrial injury

BACKGROUND: Cytochrome c (cyt c) is released from mitochondria after tissue injury, but little is known of its subsequent fate. This study was undertaken to ascertain: (1) does cyt c readily gain access to the extracellular space; (2) if so, what are some determinants of this process; and (3) might cyt c release be a potentially useful marker of in vivo tissue damage. METHODS: Isolated mouse proximal tubules (PT) were subjected to site 1 (rotenone; Rot), site 2 (antimycin A, AA), or site 3 (hypoxic) respiratory chain blockade (+/- 2 mmol/L glycine, to prevent plasma membrane disruption/cell death). Alternatively, oxidant injury was imposed (Fe(2+) or cholesterol oxidase). Extra- and intracellular cyt c levels were quantified by Western blot. Plasma or urine cyt c levels were also determined after rhabdomyolysis or ischemic acute renal failure (ARF) (in mice), or clinical ARF. RESULTS: AA, Rot, and hypoxia caused variable degrees of PT cyt c release (AA > rot approximately hypoxia), but at most, <20% of total cell content was involved. In contrast, Fe(2+) evoked approximately 65% cyt c efflux, and cholesterol oxidation caused approximately 100% cyt c release. Glycine did not block cyt c efflux, dissociating this process from plasma membrane disruption/necrotic cell death. After rhabdomyolysis, plasma cyt c levels rose and correlated with the severity of ARF (r, 0.93 vs. BUNs). Cyt c was detected in urine after both experimental and clinical ARF. CONCLUSION: Cell cyt c release is dependent on the site and the type of mitochondrial injury sustained. Oxidative injury, in general, and cholesterol oxidation, in particular, seem particularly relevant in this regard. After mitochondrial release, cyt c traverses plasma membranes, eventuating in the extracellular space. The data suggest that plasma and/or urine cyt c appearance might function as a clinically useful in vivo marker of mitochondrial stress and the tissue injury sustained.     

Mitoprotective therapy prevents rapid,strain-dependent mitochondrial dysfunction after articular cartilage injury

Posttraumatic osteoarthritis (PTOA) involves the mechanical and biological deterioration of articular cartilage that occurs following joint injury. PTOA is a growing problem in health care due to the lack of effective therapies combined with an aging population with high activity levels. Recently, acute mitochondrial dysfunction and altered cellular respiration have been associated with cartilage degeneration after injury. This finding is particularly important because recently developed mitoprotective drugs, including SS peptides, can preserve mitochondrial structure and function after acute injury in other tissues. It is not known, however, if cartilage injury induces rapid structural changes in mitochondria, to what degree mitochondrial dysfunction in cartilage depends on the mechanics of injury or the time frame over which such dysfunction develops. Similarly, it is unknown if SS-peptide treatment can preserve mitochondrial structure and function after cartilage injury. Here, we combined fast camera elastography, longitudinal fluorescence assays, and computer vision techniques to track the fates of thousands of individual cells. Our results show that impact induces mechanically dependent mitochondrial depolarization within a few minutes after injury. Electron microscopy revealed that impact causes rapid structural changes in mitochondria that are related to reduced mitochondrial function, namely, fission and loss of cristae structure. We found that SS-peptide treatment prior to impact protects the mitochondrial structure and preserves mitochondrial function at levels comparable with that of unimpacted control samples. Overall, this study reveals the vital role of mitochondria in mediating cartilage's peracute (within minutes) response to traumatic injury and demonstrates mitoprotection as a promising therapeutic strategy for injury-induced cartilage damage.     

Anaesthesia and mitochondrial disease

Mitochondrial diseases, or encephalomyopathies, are an uncommon, heterogeneous group of disorders with variable clinical course and presentation. Many of these patients present for surgery, or undergo anaesthesia in the course of investigation of their illness. Unfortunately, little information exists on their management in anaesthetic texts and the literature. We report on the anaesthetic management of a paediatric patient with mitochondrial disease, and briefly discuss the pathophysiology and anaesthetic implications of these disorders.     

乙醇诱导肝细胞线粒体质量和跨膜电位变化的研究

目的：探讨酒精性肝病过程中乙醇诱导肝细胞线粒体质量和跨膜电位变化.方法：将20只健康雄性大鼠随机分为2组：酒精性肝病模型组10只,用高度白酒灌胃建立动物模型;对照组10只,给予相同体积的生理盐水代替白酒灌胃.分离大鼠肝细胞用Rhodamine123和NAO标记,以流式细胞仪测定线粒体质量和跨膜电位.结果：与对照组相比,模型组的线粒体跨膜电位（△ψm）显著降低（P＜0.01）;模型组的线粒体膜中心磷脂含量显著减少（P＜0.01）.结论：长期摄入酒精可导致肝脏线粒体跨膜电位降低以及线粒体膜损伤,从而引发肝细胞的破坏.     

Anesthesia in mitochondrial encephalomyopathies

A 6-year-old boy with a rare mitochondrial disease (MELAS: mitochondrial encephalopathy, lactic acidosis, stroke-like episodes) was presented to undergo adenoid resection and bilateral paracentesis. ENT surgery was performed without complications under general anaesthesia using propofol, fentanyl, and ventilation with nitrous oxide and oxygen. Routine intraoperative monitoring (ECG, noninvasive blood pressure, oxymetry and capnometry) was supplemented by frequent body temperature measurements and repeated laboratory analysis of venous blood gases, lactate, and glucose. Clinically, the postoperative course was uneventful and the boy was discharged from hospital on the first postoperative day. Signs or symptoms of malignant hyperthermia never occurred. Laboratory analysis only showed a remarkable serum lactate elevation postoperatively (6 mmol/l) which decreased on the first postoperative day (3.7 mmol/l). The present anaesthesiologic experiences with MELAS-syndrome are limited, and recommendations are mainly based on case reports. Careful preoperative physical examination with special regard to all available medical records, and anaesthetic management comparable with that in malignant hyperthermia susceptible resulted in an uneventful course in our patient. Pathogenetic aspects of mitochondrial diseases focussing on anaesthetic considerations are briefly discussed.     

The relationship between glycolysis, mitochondrial respiration, protein-carboxyl methylation and motility in hamster epididymal spermatozoa

Endogenous protein-carboxyl methylase activity can be measured in intact motile spermatozoa by using [³H]methionine as a precursor of the methyl donor, S-adenosylmethionine (SAM). Since the conversion of methionine to SAM requires ATP, the relationship between the energy metabolism of spermatozoa and methylation was investigated using inhibitors of glycolysis and mitochondrial respiration. When hamster spermatozoa from cauda epididymides were incubated in 12.2 mM glucose, glycolysis was progressively inhibited as concentrations of 2-deoxyglucose (2-DOG) increased. On the other hand, endogenous protein-carboxyl methylation showed a biphasic response being stimulated at low concentrations of 2-DOG and inhibited at higher concentrations. Sperm movement was also altered by 2-DOG. Increasing concentrations of 2-DOG in the incubation medium resulted in an increase in beat amplitude and a corresponding decrease in beat frequency. When the glucose concentration of the medium was reduced to 5 mM, protein methylation was inhibited at all concentrations of 2-DOG. The biphasic effect of 2-DOG on methylation at 12.2 mM and its monophasic effect at 5 mM suggested that this reaction was related to the effective glucose concentration. To investigate this possibility, endogenous protein-carboxyl methylation was measured after incubation of sperm in glucose concentrations ranging from 0–48.8 mM. Low glucose concentrations stimulated protein methylation (up to 6.1 mM for maximal effect), but further increases in the glucose concentration***     

The relationship of glycogen,glucose, and lactate to mitochondrial dysfunction in late hemorrhagic shock

The relationship of glycogen depletion, hypoglycemia, and lactic acidemia to mitochondrial dysfunction in late hemorrhagic shock was examined. Anesthetized rats were hemorrhaged through a femoral artery catheter using a modified Wiggers' technique. Animals were sacrificed after 0, 15, 30, 45, or 60% uptake of the maximum shed volume. Hepatic mitochondrial energy-linked functions, hepatic glycogen and lactate, and serum glucose and lactate were determined at the time of sacrifice.The frequency of uncoupled oxidative phosphorylation increased with percentage of shed blood taken up. Although blood glucose was significantly lower in animals in whom uncoupling occurred, there was a wide range of values and a poor coefficient of correlation. A borderline difference between the coupled and uncoupled groups was noted with glycogen, and no difference was observed with blood or hepatic lactate.Glycogen depletion, hypoglycemia, and lactic acidemia although all manifest in late, experimental, hemorrhagic shock do not cause hepatic mitochondrial dysfunction. Effective treatment of late shock requires a more precise definition of the underlying metabolic pathology.     

Transgenic control of mitochondrial fission induces mitochondrial uncoupling and relieves diabetic oxidative stress

Mitochondria are the essential eukaryotic organelles that produce most cellular energy. The energy production and supply by mitochondria appear closely associated with the continuous shape change of mitochondria mediated by fission and fusion, as evidenced not only by the hereditary diseases caused by mutations in fission/fusion genes but also by aberrant mitochondrial morphologies associated with numerous pathologic insults. However, how morphological change of mitochondria is linked to their energy-producing activity is poorly understood. In this study, we found that perturbation of mitochondrial fission induces a unique mitochondrial uncoupling phenomenon through a large-scale fluctuation of a mitochondrial inner membrane potential. Furthermore, by genetically controlling mitochondrial fission and thereby inducing mild proton leak in mice, we were able to relieve these mice from oxidative stress in a hyperglycemic model. These findings provide mechanistic insight into how mitochondrial fission participates in regulating mitochondrial activity. In addition, these results suggest a potential application of mitochondrial fission to control mitochondrial reactive oxygen species production and oxidative stress in many human diseases.     

Anesthetic considerations in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome: a case series

Purpose

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome (MELAS) is a rare inherited mitochondrial disorder associated with severe multiorgan pathology and stress-induced episodes of metabolic decompensation and lactic acidosis. The purpose of this case series is to review the medical records of patients with MELAS who underwent anesthetic care at the Mayo Clinic to observe their perioperative responses to anesthesia and to assess outcomes.

Principal findings

From September 1997 to October 2010, nine patients with MELAS were identified who underwent 20 general anesthetics, 12 prior to MELAS diagnosis. Debilitating neurologic symptoms involved eight patients, and three patients had substantial cardiac comorbidities. The patients tolerated commonly used anesthetics and muscle relaxants, including succinylcholine. Lactated Ringer’s solution was used frequently. One patient was noted to have elevated postoperative serum lactate, but his serum lactate was chronically elevated. Metabolic acidosis was not observed in any patient. Hyponatremia and hyperkalemia, sometimes profound, were observed in seven patients, but these abnormalities also occurred at times remote from surgery. Two patients developed renal dysfunction following cardiac surgery and abdominal surgery for severe sepsis.

Conclusion

The MELAS patients developed episodes of hyponatremia and hyperkalemia of variable severity unrelated to the timing of surgery, suggesting these patients are prone to major electrolyte disturbances. Given the propensity to develop acid-base disturbances and lactacidemia, it is prudent to review and normalize electrolyte abnormalities and to adjust the anesthetic plan accordingly. Fortunately, the limited data suggest that patients with MELAS tolerate commonly used anesthetic drugs well.