ESTABLISHMENT OF THE PYRUVATE DEHYDROGENASE COMPLEX AS A CENTRAL REGULATOR OF MITOCHONDRIAL REDOX WITHIN SKELETAL MUSCLE

Once regarded as "byproducts" of aerobic metabolism, the production of superoxide/Hâ‚‚Oâ‚‚ is now understood to be a highly specialized and extensively regulated process responsible for exerting control over a vast number of thiol-containing proteins, collectively referred to as the redox-sensitive proteome. Although disruptions within this process, secondary to elevated peroxide exposure, have been linked to disease, delineation of the sources and mechanisms regulating this increased peroxide burden remain poorly defined and as such difficult to target using pharmacotherapy. Herein we demonstrate a role for pyruvate dehydrogenase (PDH) as a key source of Hâ‚‚Oâ‚‚ under physiological constraints in which respiratory chain-dependent electron leak is negligible. PDH is shown to generate Hâ‚‚Oâ‚‚ as a function of glutathione content, matrix metabolic balance, as well as antioxidant reductase activity. With respect to the latter, manipulation of matrix redox buffering reveals a novel mechanism whereby Hâ‚‚Oâ‚‚ producing NADH-linked dehydrogenases, such as PDH, are functionally linked to the redox buffering network within skeletal muscle through the activity of nicotinamide nucleotide transhydrogenase (NNT). These findings highlight the importance of NNT and the entire redox buffering system in regulating cytosolic peroxide emission and suggest a novel and pivotal role for PDH as a redox-sensitive reporter of matrix redox buffering integrity and nutrient status.Ph.D

Effect of eicosapentaenoic and docosahexaenoic acid on resting and exercise-induced inflammatory and oxidative stress biomarkers: a randomized, placebo controlled, cross-over study

<p>Abstract</p> <p>Background</p> <p>The purpose of the present investigation was to determine the effects of EPA/DHA supplementation on resting and exercise-induced inflammation and oxidative stress in exercise-trained men. Fourteen men supplemented with 2224 mg EPA+2208 mg DHA and a placebo for 6 weeks in a random order, double blind cross-over design (with an 8 week washout) prior to performing a 60 minute treadmill climb using a weighted pack. Blood was collected pre and post exercise and analyzed for a variety of oxidative stress and inflammatory biomarkers. Blood lactate, muscle soreness, and creatine kinase activity were also measured.</p> <p>Results</p> <p>Treatment with EPA/DHA resulted in a significant increase in blood levels of both EPA (18 ± 2 μmol·L^-1vs. 143 ± 23 μmol·L^-1; p < 0.0001) and DHA (67 ± 4 μmol·L^-1vs. 157 ± 13 μmol·L^-1; p < 0.0001), while no differences were noted for placebo. Resting levels of CRP and TNF-α were lower with EPA/DHA compared to placebo (p < 0.05). Resting oxidative stress markers were not different (p > 0.05). There was a mild increase in oxidative stress in response to exercise (XO and H₂O₂) (p < 0.05). No interaction effects were noted. However, a condition effect was noted for CRP and TNF-α, with lower values with the EPA/DHA condition.</p> <p>Conclusion</p> <p>EPA/DHA supplementation increases blood levels of these fatty acids and results in decreased resting levels of inflammatory biomarkers in exercise-trained men, but does not appear necessary for exercise-induced attenuation in either inflammation or oxidative stress. This may be due to the finding that trained men exhibit a minimal increase in both inflammation and oxidative stress in response to moderate duration (60 minute) aerobic exercise.</p

Effect of the dietary supplement Meltdown on catecholamine secretion, markers of lipolysis, and metabolic rate in men and women: a randomized, placebo controlled, cross-over study

<p>Abstract</p> <p>Background</p> <p>We have recently reported that the dietary supplement Meltdown^®increases plasma norepinephrine (NE), epinephrine (EPI), glycerol, free fatty acids (FFA), and metabolic rate in men. However, in that investigation measurements ceased at 90 minutes post ingestion, with values for blood borne variables peaking at this time. It was the purpose of the present investigation to extend the time course of measurement to 6 hours, and to include women within the design to determine if sex differences to treatment exist.</p> <p>Methods</p> <p>Ten men (24 ± 4 yrs) and 10 women (22 ± 2 yrs) ingested Meltdown^®or a placebo, using a randomized, cross-over design with one week separating conditions. Blood samples were collected immediately before supplementation and at one hour intervals through 6 hours post ingestion. A standard meal was provided after the hour 3 collection. Samples were assayed for EPI, NE, glycerol, and FFA. Five minute breath samples were collected at each time for measurement of metabolic rate and substrate utilization. Area under the curve (AUC) was calculated. Heart rate and blood pressure were recorded at all times. Data were also analyzed using a 2 (sex) × 2 (condition) × 7 (time) repeated measures analysis of variance, with Tukey <it>post hoc </it>testing.</p> <p>Results</p> <p>No sex × condition interactions were noted for AUC for any variable (p > 0.05). Hence, AUC data are collapsed across men and women. AUC was greater for Meltdown^®compared to placebo for EPI (367 ± 58 pg·mL^-1·6 hr^-1vs. 183 ± 27 pg·mL^-1·6 hr^-1; p = 0.01), NE (2345 ± 205 pg·mL^-1·6 hr^-1vs. 1659 ± 184 pg·mL^-1·6 hr^-1; p = 0.02), glycerol (79 ± 8 μg·mL^-1·6 hr^-1vs. 59 ± 6 μg·mL^-1·6 hr^-1; p = 0.03), FFA (2.46 ± 0.64 mmol·L^-1·6 hr^-1vs. 1.57 ± 0.42 mmol·L^-1·6 hr^-1; p = 0.05), and kilocalorie expenditure (439 ± 26 kcal·6 hrs^-1vs. 380 ± 14 kcal·6 hrs^-1; p = 0.02). No effect was noted for substrate utilization (p = 0.39). Both systolic and diastolic blood pressure (p < 0.0001; 1–16 mmHg), as well as heart rate (p = 0.01; 1–9 bpm) were higher for Meltdown^®. No sex × condition × time interactions were noted for any variable (p > 0.05).</p> <p>Conclusion</p> <p>Ingestion of Meltdown^®results in an increase in catecholamine secretion, lipolysis, and metabolic rate in young men and women, with a similar response for both sexes. Meltdown^®may prove to be an effective intervention strategy for fat loss, assuming individuals are normotensive and their treatment is monitored by a qualified health care professional.</p

Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

Chronic metabolic diseases have been linked to molecular signatures of mitochondrial dysfunction. Nonetheless, molecular remodeling of the transcriptome, proteome, and/or metabolome does not necessarily translate to functional consequences that confer physiologic phenotypes. The work here aims to bridge the gap between molecular and functional phenomics by developing and validating a multiplexed assay platform for comprehensive assessment of mitochondrial energy transduction. The diagnostic power of the platform stems from a modified version of the creatine kinase energetic clamp technique, performed in parallel with multiplexed analyses of dehydrogenase activities and ATP synthesis rates. Together, these assays provide diagnostic coverage of the mitochondrial network at a level approaching that gained by molecular “-omics� technologies. Application of the platform to a comparison of skeletal muscle versus heart mitochondria reveals mechanistic insights into tissue-specific distinctions in energy transfer efficiency. This platform opens exciting opportunities to unravel the connection between mitochondrial bioenergetics and human disease

The transcriptional response to oxidative stress is part of, but not sufficient for, insulin resistance in adipocytes.

Insulin resistance is a major risk factor for metabolic diseases such as Type 2 diabetes. Although the underlying mechanisms of insulin resistance remain elusive, oxidative stress is a unifying driver by which numerous extrinsic signals and cellular stresses trigger insulin resistance. Consequently, we sought to understand the cellular response to oxidative stress and its role in insulin resistance. Using cultured 3T3-L1 adipocytes, we established a model of physiologically-derived oxidative stress by inhibiting the cycling of glutathione and thioredoxin, which induced insulin resistance as measured by impaired insulin-stimulated 2-deoxyglucose uptake. Using time-resolved transcriptomics, we found > 2000 genes differentially-expressed over 24 hours, with specific metabolic and signalling pathways enriched at different times. We explored this coordination using a knowledge-based hierarchical-clustering approach to generate a temporal transcriptional cascade and identify key transcription factors responding to oxidative stress. This response shared many similarities with changes observed in distinct insulin resistance models. However, an anti-oxidant reversed insulin resistance phenotypically but not transcriptionally, implying that the transcriptional response to oxidative stress is insufficient for insulin resistance. This suggests that the primary site by which oxidative stress impairs insulin action occurs post-transcriptionally, warranting a multi-level 'trans-omic' approach when studying time-resolved responses to cellular perturbations