Markers Of Mitochondrial Function Research Articles

Pinacidil protects osteoblastic cells against antimycin A-induced oxidative damage.

The present study aimed to investigate the protective effect of a non-selective mitochondrial adenosine triphosphate (ATP)-sensitive potassium channel (mito-KATP) opener, pinacidil, on antimycin A-induced oxidative damage in osteoblastic MC3T3-E1 cells. Antimycin A inhibits mitochondrial electron transport by binding to complex III. Osteoblastic MC3T3-E1 cells were treated with antimycin A in the presence or absence of pinacidil and markers of mitochondrial function and oxidative stress were subsequently examined. The effects of pinacidil on the activation of phosphoinositide 3-kinase (PI3K), Akt and cyclic adenosine monophosphate‑responsive element-binding protein (CREB) were also examined. In osteoblastic MC3T3-E1 cells exposed to antimycin A, pinacidil inhibited antimycin A-induced cell death. The protective effects of pinacidil on cell survival were prevented by the addition of LY294002 (a PI3K inhibitor), an Akt inhibitor or auranofin [a thioredoxin reductase (TrxR) inhibitor], but not by KATP channel inhibitor glibenclamide. Pinacidil inhibited antimycin A-induced inactivation of PI3K and Akt as well as phosphorylation of CREB and TrxR. Furthermore, pinacidil prevented antimycin A-induced mitochondrial superoxide release, mitochondrial membrane potential dissipation, reduced ATP synthesis and intracellular [Ca2+] elevation. In conclusion, these results suggested that pinacidil may rescue osteoblastic cells from antimycin A-induced cellular damage, potentially via antioxidant activity and restoration of mitochondrial function, which are mediated in part by the PI3K/Akt/CREB signaling pathway.

The mitochondrial-targeted antioxidant MitoQ ameliorates metabolic syndrome features in obesogenic diet-fed rats better than Apocynin or Allopurinol

The prevalence of metabolic syndrome (MetS) components including obesity, dyslipidemia, insulin resistance (IR), and hepatic steatosis is rapidly increasing in wealthy societies. It is accepted that inflammation/oxidative stress are involved in the initiation/evolution of the MetS features. The present work was designed to evaluate the effects of three major cellular ROS production systems on obesity, glucose tolerance, and hepatic steatosis development and on oxidative stress onset. To do so, 40 young male Sprague–Dawley rats were divided into 5 groups: 1-control group, 2-high fat (HF) group (60% energy from fat), 3-HF+ MitoQ (mitochondrial ROS scavenger), 4-HF+ Apocynin (NADPH oxidase inhibitor), 5-HF+ Allopurinol (xanthine oxidase inhibitor). After 8 weeks of these treatments, surrogate MetS, mitochondrial function, and oxidative stress markers were measured in blood and liver. As expected, rats that were fed the HF diet exhibited increased body weight, glucose intolerance, overt hepatic steatosis, and increased hepatic oxidative stress. The impacts of the studied ROS inhibitors on these aspects of the MetS were markedly different. MitoQ showed the most clinically relevant effects, attenuating body weight gain and glucose intolerance provoked by the HF diet. Both Apocynin and Allopurinol showed limited effects suggesting secondary roles of xanthine oxidase (XO) or NADPH oxidase-dependent ROS production in the onset of oxidative stress-dependent obesity, glucose intolerance, and hepatic steatosis process. Thus, MitoQ revealed the central role of mitochondrial oxidative stress in the development of MetS and suggested that mitochondria-targeted antioxidants may be worth considering as potentially helpful therapies for MetS features.

Ob/ob mouse livers show decreased oxidative phosphorylation efficiencies and anaerobic capacities after cold ischemia.

BackgroundHepatic steatosis is a major risk factor for graft failure in liver transplantation. Hepatic steatosis shows a greater negative influence on graft function following prolonged cold ischaemia. As the impact of steatosis on hepatocyte metabolism during extended cold ischaemia is not well-described, we compared markers of metabolic capacity and mitochondrial function in steatotic and lean livers following clinically relevant durations of cold preservation.MethodsLivers from 10-week old leptin-deficient obese (ob/ob, n = 9) and lean C57 mice (n = 9) were preserved in ice-cold University of Wisconsin solution. Liver mitochondrial function was then assessed using high resolution respirometry after 1.5, 3, 5, 8, 12, 16 and 24 hours of storage. Metabolic marker enzymes for anaerobiosis and mitochondrial mass were also measured in conjunction with non-bicarbonate tissue pH buffering capacity.Results Ob/ob and lean mice livers showed severe (>60%) macrovesicular and mild (<30%) microvesicular steatosis on Oil Red O staining, respectively. Ob/ob livers had lower baseline enzymatic complex I activity but similar adenosine triphosphate (ATP) levels compared to lean livers. During cold storage, the respiratory control ratio and complex I-fueled phosphorylation deteriorated approximately twice as fast in ob/ob livers compared to lean livers. Ob/ob livers also demonstrated decreased ATP production capacities at all time-points analyzed compared to lean livers. Ob/ob liver baseline lactate dehydrogenase activities and intrinsic non-bicarbonate buffering capacities were depressed by 60% and 40%, respectively compared to lean livers.ConclusionsSteatotic livers have impaired baseline aerobic and anaerobic capacities compared to lean livers, and mitochondrial function indices decrease particularly from after 5 hours of cold preservation. These data provide a mechanistic basis for the clinical recommendation of shorter cold storage durations in steatotic donor livers.

Inhibitory effect of paeoniflorin on methylglyoxal-mediated oxidative stress in osteoblastic MC3T3-E1 cells

PurposeMethylglyoxal (MG) has been suggested to be one major source of intracellular reactive carbonyl compounds. In the present study, the effect of paeoniflorin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells. MethodsOsteoblastic MC3T3-E1 cells were pre-incubated with paeoniflorin before treatment with MG, and markers of oxidative damage and mitochondrial function were examined. ResultsPretreatment of MC3T3-E1 cells with paeoniflorin prevented the MG-induced cell death and formation of intracellular reactive oxygen species, cardiolipin peroxidation, and protein adduct in osteoblastic MC3T3-E1 cells. In addition, paeoniflorin increased glutathione level and restored the activity of glyoxalase I to almost the control level. These findings suggest that paeoniflorin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing MG detoxification system. Pretreatment with paeoniflorin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss. Additionally, the nitric oxide and nuclear respiratory factor 1 levels were significantly increased by paeoniflorin, suggesting that paeoniflorin may induce mitochondrial biogenesis. Paeoniflorin treatment decreased the levels of proinflammatory cytokines such as TNF-α and IL-6. ConclusionsThese findings indicate that paeoniflorin might exert its therapeutic effects via upregulation of glyoxalase system and mitochondrial function. Taken together, paeoniflorin may prove to be an effective treatment for diabeteic osteopathy.

Both linoleic and α-linolenic acid prevent insulin resistance but have divergent impacts on skeletal muscle mitochondrial bioenergetics in obese Zucker rats.

The therapeutic use of polyunsaturated fatty acids (PUFA) in preserving insulin sensitivity has gained interest in recent decades; however, the roles of linoleic acid (LA) and α-linolenic acid (ALA) remain poorly understood. We investigated the efficacy of diets enriched with either LA or ALA on attenuating the development of insulin resistance (IR) in obesity. Following a 12-wk intervention, LA and ALA both prevented the shift toward an IR phenotype and maintained muscle-specific insulin sensitivity otherwise lost in obese control animals. The beneficial effects of ALA were independent of changes in skeletal muscle mitochondrial content and oxidative capacity, as obese control and ALA-treated rats showed similar increases in these parameters. However, ALA increased the propensity for mitochondrial H2O2 emission and catalase content within whole muscle and reduced markers of oxidative stress (4-HNE and protein carbonylation). In contrast, LA prevented changes in markers of mitochondrial content, respiratory function, H2O2 emission, and oxidative stress in obese animals, thereby resembling levels seen in lean animals. Together, our data suggest that LA and ALA are efficacious in preventing IR but have divergent impacts on skeletal muscle mitochondrial content and function. Moreover, we propose that LA has value in preserving insulin sensitivity in the development of obesity, thereby challenging the classical view that n-6 PUFAs are detrimental.

Mitochondrial changes in platelets are not related to those in skeletal muscle during human septic shock.

Platelets can serve as general markers of mitochondrial (dys)function during several human diseases. Whether this holds true even during sepsis is unknown. Using spectrophotometry, we measured mitochondrial respiratory chain biochemistry in platelets and triceps brachii muscle of thirty patients with septic shock (within 24 hours from admission to Intensive Care) and ten surgical controls (during surgery). Results were expressed relative to citrate synthase (CS) activity, a marker of mitochondrial density. Patients with septic shock had lower nicotinamide adenine dinucleotide dehydrogenase (NADH)/CS (p = 0.015), complex I/CS (p = 0.018), complex I and III/CS (p<0.001) and complex IV/CS (p = 0.012) activities in platelets but higher complex I/CS activity (p = 0.021) in triceps brachii muscle than controls. Overall, NADH/CS (r2 = 0.00; p = 0.683) complex I/CS (r2 = 0.05; p = 0.173), complex I and III/CS (r2 = 0.01; p = 0.485), succinate dehydrogenase (SDH)/CS (r2 = 0.00; p = 0.884), complex II and III/CS (r2 = 0.00; p = 0.927) and complex IV/CS (r2 = 0.00; p = 0.906) activities in platelets were not associated with those in triceps brachii muscle. In conclusion, several respiratory chain enzymes were variably inhibited in platelets, but not in triceps brachii muscle, of patients with septic shock. Sepsis-induced mitochondrial changes in platelets do not reflect those in other organs.

Synaptic Proteins in the Postmortem Anterior Cingulate Cortex in Schizophrenia: Relationship to Treatment and Treatment Response

The anterior cingulate cortex (ACC) is one of several brain regions that are abnormal in schizophrenia (SZ). Here we compared markers of synapse and mitochondrial function using western blots of postmortem ACC in: 1) normal controls (NCs, n=13) vs subjects with SZ (n=25); NC, treatment-resistant SZ, and treatment-responsive SZ; and 3) NC and SZ treated with typical or atypical antipsychotic drugs (APDs). Protein levels of synaptophysin, mitofusin-2, vGLUT1, and calcineurin did not differ between the NC and SZ group as a whole, or the NCs vs the SZ group divided by treatment response or type of APDs. In several cases, the levels of vGLUT1 were minuscule or absent. The proportion of NCs lacking vGLUT1 was significantly less than that of the SZ groups. There were several positive correlations across all subjects between: 1) synaptophysin and vGLUT1; 2) synaptophysin and calcineurin; 3) synaptophysin and mitofusin; and 4) calcineurin and mitofusin. Synaptophysin and calcineurin were positively correlated in responders, and this correlation was significantly stronger than that in treatment-resistant SZ subjects or in NCs. Synaptophysin and calcineurin were positively correlated in SZ patients on atypical APDs; this correlation was significantly stronger than that in SZ patients on typical APDs or in NCs. Mitofusin-2 and calcineurin were positively correlated in SZ patients on atypical APDs and in NCs; this correlation was stronger in SZ patients on atypical rather than typical APDs or in NCs. The correlation between these proteins, which have roles in synaptic vesicle cycling, glutamate transmission, mitochondrial fusion, and calcium buffering, is complex and was differentially regulated among the groups.

Near infrared radiation rescues mitochondrial dysfunction in cortical neurons after oxygen-glucose deprivation

Near infrared radiation (NIR) is known to penetrate and affect biological systems in multiple ways. Recently, a series of experimental studies suggested that low intensity NIR may protect neuronal cells against a wide range of insults that mimic diseases such as stroke, brain trauma and neurodegeneration. However, the potential molecular mechanisms of neuroprotection with NIR remain poorly defined. In this study, we tested the hypothesis that low intensity NIR may attenuate hypoxia/ischemia-induced mitochondrial dysfunction in neurons. Primary cortical mouse neuronal cultures were subjected to 4 h oxygen-glucose deprivation followed by reoxygenation for 2 h, neurons were then treated with a 2 min exposure to 810-nm NIR. Mitochondrial function markers including MTT reduction and mitochondria membrane potential were measured at 2 h after treatment. Neurotoxicity was quantified 20 h later. Our results showed that 4 h oxygen-glucose deprivation plus 20 h reoxygenation caused 33.8 ± 3.4 % of neuron death, while NIR exposure significantly reduced neuronal death to 23.6 ± 2.9 %. MTT reduction rate was reduced to 75.9 ± 2.7 % by oxygen-glucose deprivation compared to normoxic controls, but NIR exposure significantly rescued MTT reduction to 87.6 ± 4.5 %. Furthermore, after oxygen-glucose deprivation, mitochondria membrane potential was reduced to 48.9 ± 4.39 % of normoxic control, while NIR exposure significantly ameliorated this reduction to 89.6 ± 13.9 % of normoxic control. Finally, NIR significantly rescued OGD-induced ATP production decline at 20 min after NIR. These findings suggest that low intensity NIR can protect neurons against oxygen-glucose deprivation by rescuing mitochondrial function and restoring neuronal energetics.

Effects of Switching from Stavudine to Raltegravir on Subcutaneous Adipose Tissue in HIV-Infected Patients with HIV/HAART-Associated Lipodystrophy Syndrome (HALS). A Clinical and Molecular Study

HIV-1/HAART-associated lipodystrophy syndrome (HALS) has been associated with exposure to stavudine (d4T) through mitochondrial dysfunction. We performed a 48-week study to assess the effects of switching from d4T to raltegravir (RAL) on metabolic and fat molecular parameters of patients with HALS. Forty-two patients with HALS and a median exposure to d4T > 7 years were switched to RAL and followed for 48 weeks. Fasting metabolic tests, HIV RNA, CD4 cell count, and fat measured by DEXA were obtained at baseline and week 48. mtDNA and gene transcripts for PPAR gamma, adiponectin, cytochrome b, Cox IV, TNF alpha, MCP-1 and CD68 were assessed in paired subcutaneous fat tissue biopsies. Lipid parameters, fasting glucose, insulin, and HOMA-IR did not change significantly. Whole body fat (P = 0.0027) and limb fat mass (P<0.0001) increased from baseline. Trunk/limb fat ratio (P = 0.0022), fat mass ratio (P = 0.0020), fat mass index (P = 0.0011) and percent leg fat normalized to BMI (P<0.0001) improved after 48 weeks. Relative abundance of mtDNA, expression of PPAR gamma, adiponectin, Cyt b, and MCP-1 genes increased, whereas Cox IV, TNF alpha, and CD68 did not change significantly from baseline. Switching from d4T to RAL in patients with HALS is associated with an increase in limb fat mass and an improvement in markers of adipocyte differentiation and mitochondrial function in SAT.

Partial Reversal of Skeletal Muscle Aging by Restoration of Normal NAD+ Levels

That some aging-associated phenotypes may be reversible is an emerging theme in contemporary aging research. Gomes et al. report that age-associated oxidative phosphorylation (OXPHOS) defects in murine skeletal muscle are biphasic. In the first phase, OXPHOS is decreased because of reduced expression of mitochondrially encoded genes. Treatment of moderately old mice (first-phase OXPHOS defects) with nicotinamide adenine dinucleotide (NAD⁺) precursor nicotinamide mononucleotide (NMN) for 1 week restores oxidative phosphorylation activity and other markers of mitochondrial function in skeletal muscle. However, muscle strength is not restored. In very old animals (second-phase OXPHOS defects), expression of OXPHOS genes from both the nucleus and mitochondria is reduced and mitochondrial DNA integrity is diminished. Gomes et al. propose a model linking decreased NAD⁺ to loss of nuclear SIRT1 activity to stabilization of the hypoxia-associated transcription factor hypoxia-inducible factor 1-alpha (HIF-1a). HIF-1a promotes an hypoxic-like (Warburg effect) state in the cell. The HIF-1a protein interacts with c-Myc, decreasing c-Myc-regulated transcription of the key mitochondrial regulator mitochondrial transcription factor A (TFAM). Low levels of TFAM lead to first-phase OXPHOS dysfunction. The transition to irreversible phase 2 dysfunction remains to be characterized, but may be related to increased reactive oxygen species (ROS) production. This model suggests that intervention in mitochondrial aging may be possible using appropriate NAD⁺ precursors such as nicotinamide riboside. Restoring NAD⁺ levels may be beneficial throughout the organism. For example, aging-associated disturbances in circadian rhythm are linked to diminished SIRT1 activity, and loss of hematopoietic stem cell function to reduced SIRT3. Work to elucidate other biphasic aging mechanisms is strongly encouraged.

Freshly isolated mitochondria from failing human hearts exhibit preserved respiratory function

In heart failure mitochondrial dysfunction is thought to be responsible for energy depletion and contractile dysfunction. The difficulties in procuring fresh left ventricular (LV) myocardium from humans for assessment of mitochondrial function have resulted in the reliance on surrogate markers of mitochondrial function and limited our understanding of cardiac energetics. We isolated mitochondria from fresh LV wall tissue of patients with heart failure and reduced systolic function undergoing heart transplant or left ventricular assist device placement, and compared their function to mitochondria isolated from the non-failing LV (NFLV) wall tissue with normal systolic function from patients with pulmonary hypertension undergoing heart–lung transplant. We performed detailed mitochondrial functional analyses using 4 substrates: glutamate–malate (GM), pyruvate–malate (PM) palmitoyl carnitine–malate (PC) and succinate. NFLV mitochondria showed preserved respiratory control ratios and electron chain integrity with only few differences for the 4 substrates. In contrast, HF mitochondria had greater respiration with GM, PM and PC substrates and higher electron chain capacity for PM than for PC. Surprisingly, HF mitochondria had greater respiratory control ratios and lower ADP-independent state 4 rates than NFLV mitochondria for GM, PM and PC substrates demonstrating that HF mitochondria are capable of coupled respiration ex vivo. Gene expression studies revealed decreased expression of key genes in pathways for oxidation of both fatty acids and glucose. Our results suggest that mitochondria from the failing LV myocardium are capable of tightly coupled respiration when isolated and supplied with ample substrates. Thus energy starvation in the failing heart may be the result of dysregulation of metabolic pathways, impaired substrate supply or reduced mitochondrial number but not the result of reduced mitochondrial electron transport capacity.

Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E

In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE-19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3-methyladenine (3MA), with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real-time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.

Serum Levels of Acylcarnitines Are Altered in Prediabetic Conditions

ObjectiveThe role of mitochondrial function in the complex pathogenesis of type 2 diabetes is not yet completely understood. Therefore, the aim of this study was to investigate serum concentrations of short-, medium- and long-chain acylcarnitines as markers of mitochondrial function in volunteers with normal, impaired or diabetic glucose control.MethodsBased on a 75 g oral glucose tolerance test, 1019 studied subjects were divided into a group with normal glucose tolerance (NGT; n = 636), isolated impaired fasting glycaemia (IFG; n = 184), impaired glucose tolerance (IGT; n = 87) or type 2 diabetes (T2D; n = 112). Serum concentrations of free carnitine and 24 acylcarnitines were measured by mass spectrometry.ResultsSerum levels of acetylcarnitine (C2), propionylcarnitine (C3), octanoylcarnitine (C8), malonylcarnitine/hydroxybutyrylcarnitine (C3DC+C4OH), hexanoylcarnitine (C6), octenoylcarnitine (C8:1), decanoylcarnitine (C10), decenoylcarnitine (C10:1), dodecanoylcarnitine (C12), tetradecenoylcarnitine (C14:1), tetradecadienylcarnitine (C14:2), hydroxytetradecanoylcarnitine (C14OH), hydroxyhexadecanoylcarnitine (C16OH) and octadecenoylcarnitine (C18:1) were significantly different among the groups (all p<0.05 adjusted for age, gender and BMI). Between the prediabetic states C14:1, C14:2 and C18:1 showed significantly higher serum concentrations in persons with IGT (p<0.05). Compared to T2D the IFG and the IGT subjects showed lower serum concentrations of malonylcarnitine/hydroxybutyrylcarnitine (C3DC+C4OH) (p<0.05).ConclusionAlterations in serum concentrations of several acylcarnitines, in particular tetradecenoylcarnitine (C14:1), tetradecadienylcarnitine (C14:2), octadecenoylcarnitine (C18:1) and malonylcarnitine/hydroxybutyrylcarnitine (C3DC+C4OH) are associated not only with T2D but also with prediabetic states.

Therapeutic targeting of the mitochondrial dysfunction in septic acute kidney injury

Acute kidney injury (AKI) is a common and serious complication of severe sepsis. No targeted therapies exist for sepsis-associated AKI, suggesting a pressing need for elucidation of the underlying pathogenic mechanisms. Emerging studies of human and experimental septic AKI kidneys have affirmed the longstanding observation that cell death in the tubule is uncommon despite often severe impairment of filtration. Rather than cell death, there appears to be widespread sublethal injury to tubular epithelial mitochondria. These organelles efficiently harness energy through controlled oxidation of metabolic fuels, they house pro-apoptotic proteins, and they produce reactive oxygen species. Derangement in one or more of these functions may contribute to the large reduction in renal function in septic AKI despite only scant cell death. In experimental septic AKI, molecular markers of mitochondrial biogenesis and function - whose renal expression dips during injury - rebound to normal levels as kidney function improves. Results from knockout mice suggest that restoration of mitochondrial function within the nephron may be critical to functional recovery. Recent findings from human and experimental septic AKI studies strongly implicate the mitochondrion as an important target for sublethal kidney injury. Stimulating the natural pathways through which mitochondrial function is normally recovered following sepsis represents a promising strategy for the development of novel therapies.

Liquiritigenin Restores Osteoblast Damage through Regulating Oxidative Stress and Mitochondrial Dysfunction

We investigated the protective effect of liquiritigenin, one of the flavonoids present in Glycyrrhizae radix, against antimycin A-induced mitochondrial dysfunction in MC3T3-E1 osteoblast cells. Osteoblastic MC3T3-E1 cells were pre-incubated with liquiritigenin before treatment with antimycin A, and markers of mitochondrial function and oxidative damage were examined. In addition, the effects of liquiritigenin on the activation of phosphoinositide 3-kinase (PI3K) were examined in MC3T3-E1 cells. Liquiritigenin protected MC3T3-E1 cells from antimycin A-induced cell death. However, the PI3K inhibitor, LY294002, significantly attenuated liquiritigenin-mediated cell survival, indicating the involvement of PI3K in the cytoprotective effect of liquiritigenin. Pretreatment with liquiritigenin prior to antimycin A exposure significantly reduced antimycin A-induced PI3K inactivation, mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Liquiritigenin also reduced mitochondrial superoxide generation, nitrotyrosine production, and cardiolipin peroxidation during mitochondrial complex inhibition with antimycin A. Taken together, the results of this study show that modulation of PI3K, antioxidant effects, and the attenuation of mitochondrial dysfunction by liquiritigenin represent an important mechanism for its protection of osteoblasts against cytotoxicity resulting from mitochondrial oxidative stress.

Abstract A36: Protein markers of tumor-initiating cells and mitochondrial function in ovarian cancer

Abstract Background: Epithelial ovarian carcinoma (EOC) is the 5th leading cause of total cancer death in women. Despite initial responsiveness to treatment, &amp;gt;75% of patients relapse into metastatic and essentially incurable chemoresistant disease. In carcinomas, metastasis is associated with the dedifferentiation process termed epithelial-to-mesenchymal transition (EMT). EMT is molecularly related to a cancer stem cell phenotype. Cancer stem cells, or tumor-initiating cells (TICs) as they are now called, are increasingly believed to underlie relapse, and are by definition highly chemoresistant and able to give rise to metastases. By repeated cisplatin treatment of the EOC cell line SKOV-3, originating from malignant ascites, we have created a stable subline (SKOV-3-R) expressing TIC markers, and showing EMT, increased motility, sphere formation in stem cell medium. They also show increased mitochondrial content and expression of mitochondrial VDAC, and hexokinase-II (HK-II) known to be upregulated in glycolytic tumor cells [1]. The chemoresistance of SKOV-3-R was found to depend on HK-II [1]. The role of altered mitochondrial functions in tumor progression and in TICs is attracting increased attention. Aim: To examine and correlate expression of TIC markers and mitochondrial markers in clinical samples, and to relate these to EOC subtype and hormone receptor status. Results: SKOV-3-R expresses TIC markers CD117, CD44 and ALDH1. Cells freshly isolated from malignant EOC ascites were analysed for TIC markers CD117, CD44, EpCAM and Nanog, and loss of E-cadherin as a marker of EMT. In an immunohistochemical pilot study on 60 patient samples we now examine expression of CD117, CD44, ALDH1, and VDAC and HK-II. Discussion: The SKOV-3-R results indicate that these cells constitute an in vitro model of TICs and that they have increased mitochondrial content. The analysis of malignant ascites shows potentially high levels of TICs in ascites. Results of immunhistochemical analysis of tumors are pending (August, 2013). 1. Wintzell M, Lofstedt L, Johansson J, Pedersen AB, Fuxe J, Shoshan M: Repeated cisplatin treatment can lead to a multiresistant tumor cell population with stem cell features and sensitivity to 3-bromopyruvate. Cancer Biol Ther 2012, 13(14):1454-1462. Citation Format: Marike Gabrielson, My Björklund, Joseph Carlson, Maria Shoshan. Protein markers of tumor-initiating cells and mitochondrial function in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A36.

L-Carnitine Exposure and Mitochondrial Function in Human Neuronal Cells

L-Carnitine is a naturally occurring substance required in mammalian energy metabolism that functions by facilitating long-chain fatty acid entry into cellular mitochondria, thereby delivering substrate for oxidation and subsequent energy production. It has been purposed that L-carnitine may improve and preserve cognitive performance, and may lead to better cognitive aging through the life span, and several controlled human clinical trials with L-carnitine support the hypothesis that this substance has the ability to improve cognitive function. We further hypothesized that, since L-carnitine is an important co-factor of mammalian mitochondrial energy metabolism, acute administration of L-carnitine to human tissue culture cells should result in detectable increases in mitochondrial function. Cultures of SH-SY-5Y human neuroblastoma and 1321N1 human astrocytoma cells grown in 96-well cell culture plates were acutely administered L-carnitine hydrochloride, and then, mitochondrial function was assayed using the colorimetric 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt cell assay kit in a VERSAmax tunable microplate reader. Significant increases in mitochondrial function were observed when human neuroblastoma or human astrocytoma cells were exposed to 100 nM (20 μg L-carnitine hydrochloride/L) to 100 μM (20 mg L-carnitine hydrochloride/L) concentrations of L-carnitine hydrochloride in comparison to unexposed cells, whereas no significant positive effects were observed at lower or higher concentrations of L-carnitine hydrochloride. The results of the present study provide insights for how L-carnitine therapy may significantly improve human neuronal function, but we recommend that future studies further explore different derivatives of L-carnitine compounds in different in vitro cell-based systems using different markers of mitochondrial function.

Two weeks of reduced‐volume sprint interval or traditional exercise training does not improve metabolic functioning in sedentary obese men

To investigate the effects of short-term, reduced-volume sprint interval training (SIT) compared to traditional exercise recommendations (TER) in sedentary obese men. Sixteen subjects [37.8 ± 5.8 years; body mass index (BMI) 32.8 ± 4.7 kg/m(2)] were randomly allocated to 2 weeks of either SIT (6 sessions of 8-12 × 10 s sprints) or TER [10 sessions of 30 min at 65% peak oxygen consumption (VO(2peak))] cycle exercise. Fasting plasma glucose, insulin, non-esterified fatty acids (NEFA), homeostasis model assessment of insulin sensitivity (HOMA-IR), body composition and VO(2peak) were assessed at baseline and approximately 72 h after the final training bout. Skeletal muscle biopsy samples were also obtained before and 72 h after training and analysed for AS160 phosphorylation and COX II, COX IV, GLUT-4, Nur77 and SIRT1 protein expression. No changes in BMI, body composition, VO(2peak), glucose, insulin, NEFA and HOMA-IR were observed after training, either within or between groups. Skeletal muscle markers of glucose metabolism and mitochondrial function also remained unaltered after 2 weeks of exercise training. Our findings show that 2 weeks of reduced-volume SIT or TER did not elicit any measurable metabolic adaptations in sedentary obese men. Further work is needed to determine the minimal amount of exercise required for short-term adaptations in this population.

Synaptic Aging is Associated with Mitochondrial Dysfunction, Reduced Antioxidant Contents and Increased Vulnerability to Amyloid-&amp;beta; Toxicity

Synaptic loss is the major neuropathological correlate of memory decline as a result of Alzheimer's Disease (AD). Synaptic failure appears to depend on the toxic actions of small and soluble amyloid-β (Aβ) oligomers. However, few studies have addressed the mechanism by which aging makes synapses more vulnerable to Aβ toxicity. In the present study we analyzed mitochondrial function and morphology and markers of oxidative stress in isolated presynaptic nerve endings from the hippocampus that were exposed to Aβ peptide at different ages. We found an age-related decline in mitochondrial activity, reduced antioxidant contents and increased oxidative stress markers in resting and depolarized synaptic terminals. Ultrastructural changes including an increase in mitochondrial size and a significant reduction of synaptic vesicles contents were also observed. In addition, synaptosomes obtained from 24 month old rats were more sensitive to Aβ toxicity. These data provide evidence of morphological and biochemical synaptic changes associated with aging that may contribute to exacerbate the damaging effects of Aβ.

Interactions between L‐arginine supplementation and physical training in the mitochondrial biomarkers and nitric oxide bioavailability in rat skeletal muscle

Nitric oxide (NO) and physical exercise regulate mitochondrial function with improvement of resistance to fatigue and muscular wasting. We studied the relationship between physical performance and the protein expressions of mitochondrial function markers (mtTFA and ATP synthase subunit c) in gastrocnemius muscle from exercised rats supplemented with L‐arginine during 8 weeks. We also analyzed the nNOS/NO pathway by measuring the content of tissue nitrite/nitrate as well as proteins expression of Cu/Zn superoxide dismutase (Cu/Zn‐SOD), neuronal nitric oxide synthase (nNOS) and mitochondrial manganese superoxide dismutase (Mn‐SOD) in the cytoplasm and mitochondrial fractions from skeletal muscle. Wistar rats were divided into: control sedentary (SD), sedentary supplemented with L‐arginine (SDLA), trained (TR) and trained supplemented with L‐arginine (TRLA). L‐arginine supplementation‐plus physical training promotes an up‐regulation of the protein expressions of ATP synthase subunit c, nNOS, Cu/Zn‐SOD, Mn‐SOD and mtTFA in gastrocnemius muscle that was associated with improvement in fatigue resistance and reduction in body weight. Our data shows that run physical training in combination with L‐arginine supplementation initiate a sequence of biochemical events in isolated gastrocnemius muscle that culminate in improvement of fatigue resistance as well as in lowering body weight gain.