Lactate shuttling as an allostatic means of thermoregulation in the brain.

Lactate, the redox-balanced end product of glycolysis, travels within and between cells to fulfill an array of physiologic functions. While evidence for the centrality of this lactate shuttling in mammalian metabolism continues to mount, its application to physical bioenergetics remains underexplored. Lactate represents a metabolic "cul-de-sac," as it can only re-enter metabolism by first being converted back to pyruvate by lactate dehydrogenase (LDH). Given the differential distribution of lactate producing/consuming tissues during metabolic stresses (e.g., exercise), we hypothesize that lactate shuttling vis-à-vis the exchange of extracellular lactate between tissues serves a thermoregulatory function, i.e., an allostatic strategy to mitigate the consequences of elevated metabolic heat. To explore this idea, the rates of heat and respiratory oxygen consumption in saponin-permeabilized rat cortical brain samples fed lactate or pyruvate were measured. Heat and respiratory oxygen consumption rates, and calorespirometric ratios were lower during lactate vs. pyruvate-linked respiration. These results support the hypothesis of allostatic thermoregulation in the brain with lactate.

Mitochondrial lactate metabolism: history and implications for exercise and disease.

Mitochondrial structures were probably observed microscopically in the 1840s, but the idea of oxidative phosphorylation (OXPHOS) within mitochondria did not appear until the 1930s. The foundation for research into energetics arose from Meyerhof's experiments on oxidation of lactate in isolated muscles recovering from electrical contractions in an O2 atmosphere. Today, we know that mitochondria are actually reticula and that the energy released from electron pairs being passed along the electron transport chain from NADH to O2 generates a membrane potential and pH gradient of protons that can enter the molecular machine of ATP synthase to resynthesize ATP. Lactate stands at the crossroads of glycolytic and oxidative energy metabolism. Based on reported research and our own modelling in silico, we contend that lactate is not directly oxidized in the mitochondrial matrix. Instead, the interim glycolytic products (pyruvate and NADH) are held in cytosolic equilibrium with the products of the lactate dehydrogenase (LDH) reaction and the intermediates of the malate-aspartate and glycerol 3-phosphate shuttles. This equilibrium supplies the glycolytic products to the mitochondrial matrix for OXPHOS. LDH in the mitochondrial matrix is not compatible with the cytoplasmic/matrix redox gradient; its presence would drain matrix reducing power and substantially dissipate the proton motive force. OXPHOS requires O2 as the final electron acceptor, but O2 supply is sufficient in most situations, including exercise and often acute illness. Recent studies suggest that atmospheric normoxia may constitute a cellular hyperoxia in mitochondrial disease. As research proceeds appropriate oxygenation levels should be carefully considered.

S29434, a Quinone Reductase 2 Inhibitor: Main Biochemical and Cellular Characterization.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.

Autotaxin-LPA signaling contributes to obesity-induced insulin resistance in muscle and impairs mitochondrial metabolism.

Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid (LPA). ATX-LPA signaling has been implicated in diet-induced obesity and systemic insulin resistance. However, it remains unclear whether the ATX-LPA pathway influences insulin function and energy metabolism in target tissues, particularly skeletal muscle, the major site of insulin-stimulated glucose disposal. The objective of this study was to test whether the ATX-LPA pathway impacts tissue insulin signaling and mitochondrial metabolism in skeletal muscle during obesity. Male mice with heterozygous ATX deficiency (ATX+/-) were protected from obesity, systemic insulin resistance, and cardiomyocyte dysfunction following high-fat high-sucrose (HFHS) feeding. HFHS-fed ATX+/- mice also had improved insulin-stimulated AKT phosphorylation in white adipose tissue, liver, heart, and skeletal muscle. Preserved insulin-stimulated glucose transport in muscle from HFHS-fed ATX+/- mice was associated with improved mitochondrial pyruvate oxidation in the absence of changes in fat oxidation and ectopic lipid accumulation. Similarly, incubation with LPA decreased insulin-stimulated AKT phosphorylation and mitochondrial energy metabolism in C2C12 myotubes at baseline and following palmitate-induced insulin resistance. Taken together, our results suggest that the ATX-LPA pathway contributes to obesity-induced insulin resistance in metabolically relevant tissues. Our data also suggest that LPA directly impairs skeletal muscle insulin signaling and mitochondrial function.

A regionally-adapted implementation of conservation agriculture delivers rapid improvements to soil properties associated with crop yield stability.

Climate models predict increasing weather variability, with negative consequences for crop production. Conservation agriculture (CA) may enhance climate resilience by generating certain soil improvements. However, the rate at which these improvements accrue is unclear, and some evidence suggests CA can lower yields relative to conventional systems unless all three CA elements are implemented: reduced tillage, sustained soil cover, and crop rotational diversity. These cost-benefit issues are important considerations for potential adopters of CA. Given that CA can be implemented across a wide variety of regions and cropping systems, more detailed and mechanistic understanding is required on whether and how regionally-adapted CA can improve soil properties while minimizing potential negative crop yield impacts. Across four US states, we assessed short-term impacts of regionally-adapted CA systems on soil properties and explored linkages with maize and soybean yield stability. Structural equation modeling revealed increases in soil organic matter generated by cover cropping increased soil cation exchange capacity, which improved soybean yield stability. Cover cropping also enhanced maize minimum yield potential. Our results demonstrate individual CA elements can deliver rapid improvements in soil properties associated with crop yield stability, suggesting that regionally-adapted CA may play an important role in developing high-yielding, climate-resilient agricultural systems.

Lactate metabolism: historical context, prior misinterpretations, and current understanding.

Lactate (La-) has long been at the center of controversy in research, clinical, and athletic settings. Since its discovery in 1780, La- has often been erroneously viewed as simply a hypoxic waste product with multiple deleterious effects. Not until the 1980s, with the introduction of the cell-to-cell lactate shuttle did a paradigm shift in our understanding of the role of La- in metabolism begin. The evidence for La- as a major player in the coordination of whole-body metabolism has since grown rapidly. La- is a readily combusted fuel that is shuttled throughout the body, and it is a potent signal for angiogenesis irrespective of oxygen tension. Despite this, many fundamental discoveries about La- are still working their way into mainstream research, clinical care, and practice. The purpose of this review is to synthesize current understanding of La- metabolism via an appraisal of its robust experimental history, particularly in exercise physiology. That La- production increases during dysoxia is beyond debate, but this condition is the exception rather than the rule. Fluctuations in blood [La-] in health and disease are not typically due to low oxygen tension, a principle first demonstrated with exercise and now understood to varying degrees across disciplines. From its role in coordinating whole-body metabolism as a fuel to its role as a signaling molecule in tumors, the study of La- metabolism continues to expand and holds potential for multiple clinical applications. This review highlights La-'s central role in metabolism and amplifies our understanding of past research.

Lactate is oxidized outside of the mitochondrial matrix in rodent brain.

The nature and existence of mitochondrial lactate oxidation is debated in the literature. Obscuring the issue are disparate findings in isolated mitochondria, as well as relatively low rates of lactate oxidation observed in permeabilized muscle fibres. However, respiration with lactate has yet to be directly assessed in brain tissue with the mitochondrial reticulum intact. To determine if lactate is oxidized in the matrix of brain mitochondria, oxygen consumption was measured in saponin-permeabilized mouse brain cortex samples, and rat prefrontal cortex and hippocampus (dorsal) subregions. While respiration in the presence of ADP and malate increased with the addition of lactate, respiration was maximized following the addition of exogenous NAD+, suggesting maximal lactate metabolism involves extra-matrix lactate dehydrogenase. This was further supported when NAD+-dependent lactate oxidation was significantly decreased with the addition of either low-concentration α-cyano-4-hydroxycinnamate or UK-5099, inhibitors of mitochondrial pyruvate transport. Mitochondrial respiration was comparable between glutamate, pyruvate, and NAD+-dependent lactate oxidation. Results from the current study demonstrate that permeabilized brain is a feasible model for assessing lactate oxidation, and support the interpretation that lactate oxidation occurs outside the mitochondrial matrix in rodent brain.

Autotaxin Is Regulated by Glucose and Insulin in Adipocytes.

Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid. Despite recent studies implicating adipose-derived ATX in metabolic disorders including obesity and insulin resistance, the nutritional and hormonal regulation of ATX in adipocytes remains unclear. The current study examined the regulation of ATX in adipocytes by glucose and insulin and the role of ATX in adipocyte metabolism. Induction of insulin resistance in adipocytes with high glucose and insulin concentrations increased ATX secretion, whereas coincubation with the insulin sensitizer, rosiglitazone, prevented this response. Moreover, glucose independently increased ATX messenger RNA (mRNA), protein, and activity in a time- and concentration-dependent manner. Glucose also acutely upregulated secreted ATX activity in subcutaneous adipose tissue explants. Insulin elicited a biphasic response. Acute insulin stimulation increased ATX activity in a PI3Kinase-dependent and mTORC1-independent manner, whereas chronic insulin stimulation decreased ATX mRNA, protein, and activity. To examine the metabolic role of ATX in 3T3-L1 adipocytes, we incubated cells with the ATX inhibitor, PF-8380, for 24 hours. Whereas ATX inhibition increased the expression of peroxisome proliferator-activated receptor-γ and its downstream targets, insulin signaling and mitochondrial respiration were unaffected. However, ATX inhibition enhanced mitochondrial H2O2 production. Taken together, this study suggests that ATX secretion from adipocytes is differentially regulated by glucose and insulin. This study also suggests that inhibition of autocrine/paracrine ATX-lysophosphatidic acid signaling does not influence insulin signaling or mitochondrial respiration, but increases reactive oxygen species production in adipocytes.

Soil Water Holding Capacity Mitigates Downside Risk and Volatility in US Rainfed Maize: Time to Invest in Soil Organic Matter?

Yield stability is fundamental to global food security in the face of climate change, and better strategies are needed for buffering crop yields against increased weather variability. Regional- scale analyses of yield stability can support robust inferences about buffering strategies for widely-grown staple crops, but have not been accomplished. We present a novel analytical approach, synthesizing 2000-2014 data on weather and soil factors to quantify their impact on county-level maize yield stability in four US states that vary widely in these factors (Illinois, Michigan, Minnesota and Pennsylvania). Yield stability is quantified as both 'downside risk' (minimum yield potential, MYP) and 'volatility' (temporal yield variability). We show that excessive heat and drought decreased mean yields and yield stability, while higher precipitation increased stability. Soil water holding capacity strongly affected yield volatility in all four states, either directly (Minnesota and Pennsylvania) or indirectly, via its effects on MYP (Illinois and Michigan). We infer that factors contributing to soil water holding capacity can help buffer maize yields against variable weather. Given that soil water holding capacity responds (within limits) to agronomic management, our analysis highlights broadly relevant management strategies for buffering crop yields against climate variability, and informs region-specific strategies.

A Systematic Review of Perennial Staple Crops Literature Using Topic Modeling and Bibliometric Analysis.

Research on perennial staple crops has increased in the past ten years due to their potential to improve ecosystem services in agricultural systems. However, multiple past breeding efforts as well as research on traditional ratoon systems mean there is already a broad body of literature on perennial crops. In this review, we compare the development of research on perennial staple crops, including wheat, rice, rye, sorghum, and pigeon pea. We utilized the advanced search capabilities of Web of Science, Scopus, ScienceDirect, and Agricola to gather a library of 914 articles published from 1930 to the present. We analyzed the metadata in the entire library and in collections of literature on each crop to understand trends in research and publishing. In addition, we applied topic modeling to the article abstracts, a type of text analysis that identifies frequently co-occurring terms and latent topics. We found: 1.) Research on perennials is increasing overall, but individual crops have each seen periods of heightened interest and research activity; 2.) Specialist journals play an important role in supporting early research efforts. Research often begins within communities of specialists or breeders for the individual crop before transitioning to a more general scientific audience; 3.) Existing perennial agricultural systems and their domesticated crop material, such as ratoon rice systems, can provide a useful foundation for breeding efforts, accelerating the development of truly perennial crops and farming systems; 4.) Primary research is lacking for crops that are produced on a smaller scale globally, such as pigeon pea and sorghum, and on the ecosystem service benefits of perennial agricultural systems.

Soil Functional Zone Management: A Vehicle for Enhancing Production and Soil Ecosystem Services in Row-Crop Agroecosystems.

There is increasing global demand for food, bioenergy feedstocks and a wide variety of bio-based products. In response, agriculture has advanced production, but is increasingly depleting soil regulating and supporting ecosystem services. New production systems have emerged, such as no-tillage, that can enhance soil services but may limit yields. Moving forward, agricultural systems must reduce trade-offs between production and soil services. Soil functional zone management (SFZM) is a novel strategy for developing sustainable production systems that attempts to integrate the benefits of conventional, intensive agriculture, and no-tillage. SFZM creates distinct functional zones within crop row and inter-row spaces. By incorporating decimeter-scale spatial and temporal heterogeneity, SFZM attempts to foster greater soil biodiversity and integrate complementary soil processes at the sub-field level. Such integration maximizes soil services by creating zones of 'active turnover', optimized for crop growth and yield (provisioning services); and adjacent zones of 'soil building', that promote soil structure development, carbon storage, and moisture regulation (regulating and supporting services). These zones allow SFZM to secure existing agricultural productivity while avoiding or minimizing trade-offs with soil ecosystem services. Moreover, the specific properties of SFZM may enable sustainable increases in provisioning services via temporal intensification (expanding the portion of the year during which harvestable crops are grown). We present a conceptual model of 'virtuous cycles', illustrating how increases in crop yields within SFZM systems could create self-reinforcing feedback processes with desirable effects, including mitigation of trade-offs between yield maximization and soil ecosystem services. Through the creation of functionally distinct but interacting zones, SFZM may provide a vehicle for optimizing the delivery of multiple goods and services in agricultural systems, allowing sustainable temporal intensification while protecting and enhancing soil functioning.

Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore.

The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) -sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix.

Mitochondrial Bioenergetics and Fiber Type Assessments in Microbiopsy vs. Bergstrom Percutaneous Sampling of Human Skeletal Muscle.

Microbiopsies of human skeletal muscle are increasingly adopted by physiologists for a variety of experimental assays given the reduced invasiveness of this procedure compared to the classic Bergstrom percutaneous biopsy technique. However, a recent report demonstrated lower mitochondrial respiration in saponin-permeabilized muscle fiber bundles (PmFB) prepared from microbiopsies vs. Bergstrom biopsies. We hypothesized that ADP-induced contraction (rigor) of smaller length microbiopsy PmFB causes a greater reduction in maximal respiration vs. Bergstrom, such that respiration could be increased by a myosin II ATPase-inhibitor (Blebbistatin; BLEB). Eleven males and females each received a 2 mm diameter percutaneous microbiopsy and a 5 mm diameter Bergstrom percutaneous biopsy in opposite legs. Glutamate/malate (5/0.5 mM)-supported respiration in microbiopsy PmFB was lower than Bergstrom at submaximal concentrations of ADP. 5 µM BLEB reduced this impairment such that there were no differences relative to Bergstrom ± BLEB. Surprisingly, pyruvate (5 mM)-supported respiration was not different between either biopsy technique ±BLEB, whereas BLEB increased succinate-supported respiration in Bergstrom only. H2O2 emission was lower in microbiopsy PmFB compared to Bergstrom PmFB in the presence of BLEB. Microbiopsies contained fewer type I fibers (37 vs. 47%) and more type IIX fibers (20 vs. 8%) compared to Bergstrom possibly due to sampling site depth and/or longitudinal location. These findings suggest that smaller diameter percutaneous biopsies yield lower glutamate-supported mitochondrial respiratory kinetics which is increased by preventing ADP-induced rigor with myosin inhibition. Microbiopsies of human skeletal muscle can be utilized for assessing mitochondrial respiratory kinetics in PmFB when assay conditions are supplemented with BLEB, but fiber type differences with this method should be considered.

Lactate oxidation at the mitochondria: a lactate-malate-aspartate shuttle at work.

Lactate, the conjugate base of lactic acid occurring in aqueous biological fluids, has been derided as a "dead-end" waste product of anaerobic metabolism. Catalyzed by the near-equilibrium enzyme lactate dehydrogenase (LDH), the reduction of pyruvate to lactate is thought to serve to regenerate the NAD(+) necessary for continued glycolytic flux. Reaction kinetics for LDH imply that lactate oxidation is rarely favored in the tissues of its own production. However, a substantial body of research directly contradicts any notion that LDH invariably operates unidirectionally in vivo. In the current Perspective, a model is forwarded in which the continuous formation and oxidation of lactate serves as a mitochondrial electron shuttle, whereby lactate generated in the cytosol of the cell is oxidized at the mitochondria of the same cell. From this perspective, an intracellular lactate shuttle operates much like the malate-aspartate shuttle (MAS); it is also proposed that the two shuttles are necessarily interconnected in a lactate-MAS. Among the requisite features of such a model, significant compartmentalization of LDH, much like the creatine kinase of the phosphocreatine shuttle, would facilitate net cellular lactate oxidation in a variety of cell types.

Mitochondrial fission mediates ceramide-induced metabolic disruption in skeletal muscle.

Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress-induced pathways. Ceramide has long been known to affect the mitochondria, altering both morphology and physiology. We sought to assess the impact of ceramide on skeletal muscle mitochondrial structure and function. A primary observation was the rapid and dramatic division of mitochondria in ceramide-treated cells. This effect is likely to be a result of increased Drp1 (dynamin-related protein 1) action, as ceramide increased Drp1 expression and Drp1 inhibition prevented ceramide-induced mitochondrial fission. Further, we found that ceramide treatment reduced mitochondrial O2 consumption (i.e. respiration) in cultured myotubes and permeabilized red gastrocnemius muscle fibre bundles. Ceramide treatment also increased H2O2 levels and reduced Akt/PKB (protein kinase B) phosphorylation in myotubes. However, inhibition of mitochondrial fission via Drp1 knockdown completely protected the myotubes and fibre bundles from ceramide-induced metabolic disruption, including maintained mitochondrial respiration, reduced H2O2 levels and unaffected insulin signalling. These data suggest that the forced and sustained mitochondrial fission that results from ceramide accrual may alter metabolic function in skeletal muscle, which is a prominent site not only of energy demand (via the mitochondria), but also of ceramide accrual with weight gain.

Calculation of ion currents across the inner membrane of functionally intact mitochondria.

Mitochondrial ion transport systems play a central role in cell physiology. Rates of Ca (2+) and K(+) transport across the inner mitochondrial membrane have been derived from the measurement of ion accumulation over time within functional isolated mitochondria or mitochondria of cultured cells. Alternatively, the electrical currents generated by ionic flux have been directly measured in purified and swollen mitochondrial samples (mitoplasts) or reconstituted channels, and typically range from 1 pA to several 100s pA. However, the direct electrophysiological approach necessarily requires extensive processing of the mitochondria prior to measurement, which can only be performed on isolated mitoplasts. To compare rates of mitochondrial ion transport measured in electrophysiological experiments to those measured in intact mitochondria and cells, we converted published rates of mitochondrial ion uptake into units of ionic current. We estimate that for monovalent ions, uptake by intact mitochondria at the rate of 1 nmol ∙ mg(-1) protein ∙ min(-1) is equivalent to 0.2 fA of current per whole single mitochondrion (0.4 fA for divalent ions). In intact mitochondria, estimated rates of electrogenic cation uptake are limited to 1-100 fA of integral current per single mitochondrion. These estimates are orders of magnitude lower than the currents through mitochondrial channels directly measured via patch-clamp or artificial lipid bilayer approaches.

Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria.

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD(+) significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD(+)-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

Methods for assessing mitochondrial function in diabetes.

A growing body of research is investigating the potential contribution of mitochondrial function to the etiology of type 2 diabetes. Numerous in vitro, in situ, and in vivo methodologies are available to examine various aspects of mitochondrial function, each requiring an understanding of their principles, advantages, and limitations. This review provides investigators with a critical overview of the strengths, limitations and critical experimental parameters to consider when selecting and conducting studies on mitochondrial function. In vitro (isolated mitochondria) and in situ (permeabilized cells/tissue) approaches provide direct access to the mitochondria, allowing for study of mitochondrial bioenergetics and redox function under defined substrate conditions. Several experimental parameters must be tightly controlled, including assay media, temperature, oxygen concentration, and in the case of permeabilized skeletal muscle, the contractile state of the fibers. Recently developed technology now offers the opportunity to measure oxygen consumption in intact cultured cells. Magnetic resonance spectroscopy provides the most direct way of assessing mitochondrial function in vivo with interpretations based on specific modeling approaches. The continuing rapid evolution of these technologies offers new and exciting opportunities for deciphering the potential role of mitochondrial function in the etiology and treatment of diabetes.

Mitochondrial creatine kinase activity and phosphate shuttling are acutely regulated by exercise in human skeletal muscle.

Energy transfer between mitochondrial and cytosolic compartments is predominantly achieved by creatine-dependent phosphate shuttling (PCr/Cr) involving mitochondrial creatine kinase (miCK). However, ADP/ATP diffusion through adenine nucleotide translocase (ANT) and voltage-dependent anion carriers (VDACs) is also involved in this process. To determine if exercise alters the regulation of this system, ADP-stimulated mitochondrial respiratory kinetics were assessed in permeabilized muscle fibre bundles (PmFBs) taken from biopsies before and after 2 h of cycling exercise (60% ) in nine lean males. Concentrations of creatine (Cr) and phosphocreatine (PCr) as well as the contractile state of PmFBs were manipulated in situ. In the absence of contractile signals (relaxed PmFBs) and miCK activity (no Cr), post-exercise respiratory sensitivity to ADP was reduced in situ (up to 126% higher apparent K(m) to ADP) suggesting inhibition of ADP/ATP diffusion between matrix and cytosolic compartments (possibly ANT and VDACs). However this effect was masked in the presence of saturating Cr (no effect of exercise on ADP sensitivity). Given that the role of ANT is thought to be independent of Cr, these findings suggest ADP/ATP, but not PCr/Cr, cycling through the outer mitochondrial membrane (VDACs) may be attenuated in resting muscle after exercise. In contrast, in contracted PmFBs, post-exercise respiratory sensitivity to ADP increased with miCK activation (saturating Cr; 33% lower apparent K(m) to ADP), suggesting prior exercise increases miCK sensitivity in situ. These observations demonstrate that exercise increases miCK-dependent respiratory sensitivity to ADP, promoting mitochondrial-cytosolic energy exchange via PCr/Cr cycling, possibly through VDACs. This effect may mask an underlying inhibition of Cr-independent ADP/ATP diffusion. This enhanced regulation of miCK-dependent phosphate shuttling may improve energy homeostasis through more efficient coupling of oxidative phosphorylation to perturbations in cellular energy charge during subsequent bouts of contraction.

Simvastatin impairs ADP-stimulated respiration and increases mitochondrial oxidative stress in primary human skeletal myotubes.

Statins, the widely prescribed cholesterol-lowering drugs for the treatment of cardiovascular disease, cause adverse skeletal muscle side effects ranging from fatigue to fatal rhabdomyolysis. The purpose of this study was to determine the effects of simvastatin on mitochondrial respiration, oxidative stress, and cell death in differentiated primary human skeletal muscle cells (i.e., myotubes). Simvastatin induced a dose-dependent decrease in viability of proliferating and differentiating primary human muscle precursor cells, and a similar dose-dependent effect was noted in differentiated myoblasts and myotubes. Additionally, there were decreases in myotube number and size following 48 h of simvastatin treatment (5 µM). In permeabilized myotubes, maximal ADP-stimulated oxygen consumption, supported by palmitoylcarnitine+malate (PCM, complex I and II substrates) and glutamate+malate (GM, complex I substrates), was 32-37% lower (P<0.05) in simvastatin-treated (5 µM) vs control myotubes, providing evidence of impaired respiration at complex I. Mitochondrial superoxide and hydrogen peroxide generation were significantly greater in the simvastatin-treated human skeletal myotube cultures compared to control. In addition, simvastatin markedly increased protein levels of Bax (proapoptotic, +53%) and Bcl-2 (antiapoptotic, +100%, P<0.05), mitochondrial PTP opening (+44%, P<0.05), and TUNEL-positive nuclei in human skeletal myotubes, demonstrating up-regulation of mitochondrial-mediated myonuclear apoptotic mechanisms. These data demonstrate that simvastatin induces myotube atrophy and cell loss associated with impaired ADP-stimulated maximal mitochondrial respiratory capacity, mitochondrial oxidative stress, and apoptosis in primary human skeletal myotubes, suggesting that mitochondrial dysfunction may underlie human statin-induced myopathy.