A model for stimulation of enzyme activity by a competitive inhibitor based on the interaction of terazosin and phosphoglycerate kinase 1.

The drug terazosin (TZ) binds to and can enhance the activity of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and can increase ATP levels. That finding prompted studies of TZ in Parkinson's disease (PD) in which decreased neuronal energy metabolism is a hallmark feature. TZ was neuroprotective in cell-based and animal PD models and in large epidemiological studies of humans. However, how TZ might increase PGK1 activity has remained a perplexing question because structural data revealed that the site of TZ binding to PGK1 overlaps with the site of substrate binding, predicting that TZ would competitively inhibit activity. Functional data also indicate that TZ is a competitive inhibitor. To explore the paradoxical observation of a competitive inhibitor increasing enzyme activity under some conditions, we developed a mass action model of TZ and PGK1 interactions using published data on PGK1 kinetics and the effect of varying TZ concentrations. The model indicated that TZ-binding introduces a bypass pathway that accelerates product release. At low concentrations, TZ binding circumvents slow product release and increases the rate of enzymatic phosphotransfer. However, at high concentrations, TZ inhibits PGK1 activity. The model explains stimulation of enzyme activity by a competitive inhibitor and the biphasic dose-response relationship for TZ and PGK1 activity. By providing a plausible mechanism for interactions between TZ and PGK1, these findings may aid development of TZ or other agents as potential therapeutics for neurodegenerative diseases. The results may also have implications for agents that interact with the active site of other enzymes.

Dual-process brain mitochondria isolation preserves function and clarifies protein composition.

The brain requires continuously high energy production to maintain ion gradients and normal function. Mitochondria critically undergird brain energetics, and mitochondrial abnormalities feature prominently in neuropsychiatric disease. However, many unique aspects of brain mitochondria composition and function are poorly understood. Developing improved neuroprotective therapeutics thus requires more comprehensively understanding brain mitochondria, including accurately delineating protein composition and channel-transporter functional networks. However, obtaining pure mitochondria from the brain is especially challenging due to its distinctive lipid and cell structure properties. As a result, conflicting reports on protein localization to brain mitochondria abound. Here we illustrate this problem with the neuropsychiatric disease-associated L-type calcium channel Cav1.2α1 subunit previously observed in crude mitochondria. We applied a dual-process approach to obtain functionally intact versus compositionally pure brain mitochondria. One branch utilizes discontinuous density gradient centrifugation to isolate semipure mitochondria suitable for functional assays but unsuitable for protein localization because of endoplasmic reticulum (ER) contamination. The other branch utilizes self-forming density gradient ultracentrifugation to remove ER and yield ultrapure mitochondria that are suitable for investigating protein localization but functionally compromised. Through this process, we evaluated brain mitochondria protein content and observed the absence of Cav1.2α1 and other previously reported mitochondrial proteins, including the NMDA receptor, ryanodine receptor 1, monocarboxylate transporter 1, excitatory amino acid transporter 1, and glyceraldehyde 3-phosphate dehydrogenase. Conversely, we confirmed mitochondrial localization of several plasma membrane proteins previously reported to also localize to mitochondria. We expect this dual-process isolation procedure will enhance understanding of brain mitochondria in both health and disease.

Evaluating a species phylogeny using ddRAD SNPs: Cyto-nuclear discordance and introgression in the salmonid genus Thymallus (Salmonidae).

Hybridization and introgression are very common among freshwater fishes due to the dynamic nature of hydrological landscapes. Cyclic patterns of allopatry and secondary contact provide numerous opportunities for interspecific gene flow, which can lead to discordant paths of evolution for mitochondrial and nuclear genomes. Here, we used double digest restriction-site associated DNA sequencing (ddRADseq) to obtain a genome-wide single nucleotide polymorphism (SNP) dataset comprehensive for allThymallus (Salmonidae)species to infer phylogenetic relationships and evaluate potential recent and historical gene flow among species. The newly obtained nuclear phylogeny was largely concordant with a previously published mitogenome-based topology but revealed a few cyto-nuclear discordances. These incongruencies primarily involved the placement of internal nodes rather than the resolution of species, except for one European species where anthropogenic stock transfers are thought to be responsible for the observed pattern. The analysis of four contact zones where multiple species are found revealed a few cases of mitochondrial capture and limited signals of nuclear introgression. Interestingly, the mechanisms restricting interspecific gene flow might be distinct; while in zones of secondary contact, small-scale physical habitat separation appeared as a limiting factor, biologically based reinforcement mechanisms are presumed to be operative in areas where species presumably evolved in sympatry. Signals of historical introgression were largely congruent with the routes of species dispersal previously inferred from mitogenome data. Overall, the ddRADseq dataset provided a robust phylogenetic reconstruction of the genus Thymallus including new insights into historical hybridization and introgression, opening up new questions concerning their evolutionary history.

Freshwater adaptation in prickly sculpin (Pisces: Cottidae): intraspecific comparisons reveal evidence for water pH and Na+ concentration driving diversity in gill H+-ATPase and ion regulation.

Phenotypic divergence is a hallmark of adaptive radiation. One example involves differentiation in physiological traits involved in ion regulation among species with contrasting lifestyles and living in distinct environments. Differentiation in ion regulation and its ecological implications among populations within species are, however, less well understood. To address this knowledge gap, we collected prickly sculpin (Cottus asper) from distinct habitat types including coastal rivers connected to estuaries, coastal lakes and interior lakes, all from British Columbia, Canada. We tested for differences in plasma Na+ and Cl-, gill Na+/K+-ATPase and H+-ATPase activity and protein abundance as well as changes in body mass and arterial blood pH in fish sampled from the field and acclimated to two different freshwater conditions in the laboratory: artificial lake water (ALW) and ion-poor water (IPW). We also tested for links between environmental water chemistry and the physiological characteristics associated with ion regulation. Transfer to IPW resulted in upregulation of gill Na+/K+-ATPase and H+-ATPase activity as well as increases in gill H+-ATPase protein expression level in each habitat compared with that in the common ALW treatment. Despite the presence of population-within-habitat-type differences, significant habitat-type effects were revealed in most of the ion regulation characteristics examined under different acclimation conditions. Significantly lower plasma Cl- was detected in fish from coastal rivers than in fish from the other two habitat types during the IPW treatment, which was also significantly lower compared with that in ALW. Similarly, gill Na+/K+-ATPase activity was lower in the coastal river populations in IPW than in fish from coastal and interior lakes, which was not in accordance with the protein expression in the gill. For gill H+-ATPase, fish from interior lake populations had the highest level of activity across all habitat types under all conditions, which was related to the protein levels in the gill. The activity of gill H+-ATPase was positively correlated with the combined effect of water Na+ and pH under the ALW treatment. Our results suggest that variation in habitat may be an important factor driving differences in gill Na+/K+-ATPase and H+-ATPase activity across populations of C. asper. Further, the combined effect of water Na+ and pH may have played a key role in physiological adaptation in C. asper during post-glacial freshwater colonization and dispersal.

Assessing conservation status with extensive but low-resolution data: Application of frequentist and Bayesian models to endangered Athabasca River rainbow trout.

Use of extensive but low-resolution abundance data is common in the assessment of species at-risk status based on quantitative decline criteria under International Union for Conservation of Nature (IUCN) and national endangered species legislation. Such data can be problematic for 3 reasons. First, statistical power to reject the null hypothesis of no change is often low because of small sample size and high sampling uncertainty leading to a high frequency of type II errors. Second, range-wide assessments composed of multiple site-specific observations do not effectively weight site-specific trends into global trends. Third, uncertainty in site-specific temporal trends and relative abundance are not propagated at the appropriate spatial scale. A common result is the propensity to underestimate the magnitude of declines and therefore fail to identify the appropriate at-risk status for a species. We used 3 statistical approaches, from simple to more complex, to estimate temporal decline rates for a designatable unit (DU) of rainbow trout in the Athabasca River watershed in western Canada. This DU is considered a native species for purposes of listing because of its genetic composition characterized as >0.95 indigenous origin in the face of continuing introgressive hybridization with introduced populations in the watershed. Analysis of abundance trends from 57 time series with a fixed-effects model identified 33 sites with negative trends, but only 2 were statistically significant. By contrast, a hierarchical linear mixed model weighted by site-specific abundance provided a DU-wide decline estimate of 16.4% per year and a 3-generation decline of 93.2%. A hierarchical Bayesian mixed model yielded a similar 3-generation decline trend of 91.3% and the posterior distribution showed that the estimate had a >99% probability of exceeding thresholds for an endangered listing. We conclude that the Bayesian approach was the most useful because it provided a probabilistic statement of threshold exceedance in support of an at-risk status recommendation.

El uso de datos extensivos, pero de baja resolución, de la abundancia es una práctica común en la evaluación del estado de riesgo de una especie con base en los criterios cuantitativos de declinación establecidos por la Unión Internacional para la Conservación de la Naturaleza (UICN) y la legislación nacional sobre especies en peligro extinción. Dicha información puede ser problemática por tres razones: primero, el poder estadístico para rechazar la hipótesis nula de ningún cambio es frecuentemente bajo debido a un tamaño pequeño de la muestra y a la elevada incertidumbre del muestreo, lo que resulta en una frecuencia elevada de errores de tipo II; segundo, las evaluaciones de amplia variedad compuestas de varias observaciones específicas de sitio no sopesan efectivamente las tendencias específicas de sitio dentro de las tendencias globales; y tercero, la incertidumbre en las tendencias temporales específicas de sitio y en la abundancia relativa no se propagan a la escala espacial apropiada. Un resultado común del uso de esta información es la propensión a subestimar la magnitud de las declinaciones, y por lo tanto equivocarse en la identificación del estado de riesgo apropiado para la especie. Usamos tres estrategias estadísticas, de simples a más complejas, para estimar las tasas de declinación temporal para una unidad designable (UD) de trucha arcoíris en la cuenca del río Athabasca al oeste de Canadá. Esta UD es considerada una especie nativa por razones de listado debido a su composición genética, caracterizada como >0-95 de origen nativo de frente a la continua hibridación introgresiva con poblaciones introducidas a la cuenca. El análisis de las tendencias de abundancia de 57 series de tiempo con un modelo de efectos fijos identificó 33 sitios con tendencias negativas, pero sólo dos fueron estadísticamente significativas. En contraste, un modelo lineal mixto de jerarquías sopesado por abundancia específica de sitio proporcionó una estimación de declinación en toda la UD de 16.4% año−1 y una declinación a tres generaciones de 93.2%. Un modelo bayesiano de jerarquías produjo una tendencia de declinación a tres generaciones de 91.3% y la distribución posterior mostró que el estimado tuvo una probabilidad >99% de exceder los umbrales para la categorización como especie en peligro. Concluimos que la estrategia bayesiana fue la más útil porque proporcionó una afirmación probabilística de la superación del umbral a favor de una recomendación de categorizar el estado como en riesgo.

Impact of short- and long-term electrically induced muscle exercise on gene signaling pathways, gene expression, and PGC1a methylation in men with spinal cord injury.

Exercise attenuates the development of chronic noncommunicable diseases (NCDs). Gene signaling pathway analysis offers an opportunity to discover if electrically induced muscle exercise regulates key pathways among people living with spinal cord injury (SCI). We examined short-term and long-term durations of electrically induced skeletal muscle exercise on complex gene signaling pathways, specific gene regulation, and epigenetic tagging of PGC1a, a major transcription factor in skeletal muscle of men with SCI. After short- or long-term electrically induced exercise training, participants underwent biopsies of the trained and untrained muscles. RNA was hybridized to an exon microarray and analyzed by a gene set enrichment analysis. We discovered that long-term exercise training regulated the Reactome gene sets for metabolism (38 gene sets), cell cycle (36 gene sets), disease (27 gene sets), gene expression and transcription (22 gene sets), organelle biogenesis (4 gene sets), cellular response to stimuli (8 gene sets), immune system (8 gene sets), vesicle-mediated transport (4 gene sets), and transport of small molecules (3 gene sets). Specific gene expression included: oxidative catabolism of glucose including PDHB (P < 0.001), PDHX (P < 0.001), MPC1 (P < 0.009), and MPC2 (P < 0.007); Oxidative phosphorylation genes including SDHA (P < 0.006), SDHB (P < 0.001), NDUFB1 (P < 0.002), NDUFA2 (P < 0.001); transcription genes including PGC1α (P < 0.030) and PRKAB2 (P < 0.011); hypertrophy gene MSTN (P < 0.001); and the myokine generating FNDC5 gene (P < 0.008). Long-term electrically induced exercise demethylated the major transcription factor PGC1a. Taken together, these findings support that long-term electrically induced muscle activity regulates key pathways associated with muscle health and systemic metabolism.

Oxaloacetic acid mediates ADP-dependent inhibition of mitochondrial complex II-driven respiration.

We recently reported a previously unrecognized mitochondrial respiratory phenomenon. When [ADP] was held constant ("clamped") at sequentially increasing concentrations in succinate-energized muscle mitochondria in the absence of rotenone (commonly used to block complex I), we observed a biphasic, increasing then decreasing, respiratory response. Here we investigated the mechanism. We confirmed decades-old reports that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH). We then used an NMR method to assess OAA concentrations (known as difficult to measure by MS) as well as those of malate, fumarate, and citrate in isolated succinate-respiring mitochondria. When these mitochondria were incubated at varying clamped ADP concentrations, respiration increased at low [ADP] as expected given the concurrent reduction in membrane potential. With further increments in [ADP], respiration decreased associated with accumulation of OAA. Moreover, a low pyruvate concentration, that alone was not enough to drive respiration, was sufficient to metabolize OAA to citrate and completely reverse the loss of succinate-supported respiration at high [ADP]. Further, chemical or genetic inhibition of pyruvate uptake prevented OAA clearance and preserved respiration. In addition, we measured the effects of incremental [ADP] on NADH, superoxide, and H2O2 (a marker of reverse electron transport from complex II to I). In summary, our findings, taken together, support a mechanism (detailed within) wherein succinate-energized respiration as a function of increasing [ADP] is initially increased by [ADP]-dependent effects on membrane potential but subsequently decreased at higher [ADP] by inhibition of succinate dehydrogenase by OAA. The physiologic relevance is discussed.

Parallel introgression and selection on introduced alleles in a native species.

As humans cause the redistribution of species ranges, hybridization between previously allopatric species is on the rise. Such hybridization can have complex effects on overall fitness of native species as new allelic combinations are tested. Widespread species introductions provide a unique opportunity to study selection on introgressed alleles in independent, replicated populations. We examined selection on alleles that repeatedly introgressed from introduced rainbow trout (Oncorhynchus mykiss) into native westslope cutthroat trout (Oncorhynchus clarkii lewisi) populations in western Canada. We found that the degree of introgression of individual single nucleotide polymorphisms from the invasive species into the native is correlated between independent watersheds. A number of rainbow trout alleles have repeatedly swept to high frequency in native populations, suggesting parallel adaptive advantages. Using simulations, we estimated large selection coefficients up to 0.05 favoring several rainbow trout alleles in the native background. Although previous studies have found reduced hybrid fitness and genome-wide resistance to introgression in westslope cutthroat trout, our results suggest that some introduced genomic regions are strongly favored by selection. Our study demonstrates the utility of replicated introductions as case studies for understanding parallel adaptation and the interactions between selection and introgression across the genome. We suggest that understanding this variation, including consideration of beneficial alleles, can inform management strategies for hybridizing species.

Inferring phylogenetic structure, hybridization and divergence times within Salmoninae (Teleostei: Salmonidae) using RAD-sequencing.

Phylogenetic studies focusing on Salmonidae have revealed significant obstacles in trying to clarify some interspecific relationships within the Salmoninae subfamily, due to a limited number of markers typed, conflicting phylogenetic signals and ancient hybridization events. To infer reliable phylogenetic relationships, evaluate several putative scenarios of ancient hybridization, and estimate divergence times within Salmoninae, we applied restriction-site associated DNA sequencing (RAD-seq) to 43 samples, including 26 genetic lineages across 21 species, largely representing the subfamily, with an emphasis on the genus Salvelinus. We identified 28,402 loci and 28,363 putatively unlinked SNPs, which were used in downstream analyses. Using an iterative k-means partitioned dataset and a Maximum Likelihood approach; we generated a well-supported phylogeny, providing clear answers to several previous phylogenetic uncertainties. We detected several significant introgression signals, presumably ancient, in the genus Salvelinus. The most recent common ancestor of Salmonidae dates back to approximately 58.9MY ago (50.8-64 MY) and the crown age of Salmoninae was estimated to be 37.7 MY (35.2-40.8 MY) using a Bayesian molecular dating analysis with a relaxed molecular clock. The divergence among genera of the subfamily occurred between the late Eocene and middle of the Miocene (≈38-11 MY) such as the divergence between the genus Oncorhynchus and Salvelinus, which we estimated to 21.2 MY ago (95% HPD: 19.8-23.0 MY), while species diversification took place mainly during the Neogene (≈22-1.5 MY), with more than half of these events occurring in the last 10 MY.

A stress-responsive system for mitochondrial protein degradation.

We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive--Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.

A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity.

The discovery that theMPC1andMPC2genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [(14)C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPCKm(28.0 ± 3.9 µm) andVmax(1.08 ± 0.05 nmol/min/mg), which have not previously been reported.KmandVmaxof the rat liver MPC were found to be 71.2 ± 17 µmand 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role.

The beneficial metabolic effects of insulin sensitizers are not attenuated by mitochondrial pyruvate carrier 2 hypomorphism.

NEW FINDINGS: What is the central question of this study? The antidiabetic effects of thiazolidinedione (TZD) drugs may be mediated in part by a molecular interaction with the constituent proteins of the mitochondrial pyruvate carrier complex (MPC1 and MPC2). We examined the ability of a mutant mouse strain expressing an N-terminal truncation of MPC2 (Mpc2Δ16 mice) to respond to TZD treatment. What is the main finding and its importance? The response of Mpc2Δ16 mice to TZD treatment was not significantly different from that of wild-type C57BL6/J control animals, suggesting that the 16 N-terminal amino acids of MPC2 are dispensable for the effects of TZD treatment. Rosiglitazone and pioglitazone are thiazolidinedione (TZD) compounds that have been used clinically as insulin-sensitizing drugs and are generally believed to mediate their effects via activation of the peroxisome proliferator-activated receptor Î³ (PPARγ). Recent work has shown that it is possible to synthesize TZD compounds with potent insulin-sensitizing effects and markedly diminished affinity for PPARγ. Both clinically used TZDs and investigational PPARγ-sparing TZDs, such as MSDC-0602, interact with the mitochondrial pyruvate carrier (MPC) and inhibit its activity. The MPC complex is composed of two proteins, MPC1 and MPC2. Herein, we used mice expressing a hypomorphic MPC2 protein missing 16 amino acids in the N-terminus (Mpc2Δ16 mice) to determine the effects of these residues in mediating the insulin-sensitizing effects of TZDs in diet-induced obese mice. We found that both pioglitazone and MSDC-0602 elicited their beneficial metabolic effects, including improvement in glucose tolerance, attenuation of hepatic steatosis, reduction of adipose tissue inflammation and stimulation of adipocyte browning, in both wild-type and Mpc2Δ16 mice after high-fat diet feeding. In addition, truncation of MPC2 failed to attenuate the interaction between TZDs and the MPC in a bioluminescence resonance energy transfer-based assay or to affect the suppression of pyruvate-stimulated respiration in cells. Collectively, these data suggest that the interaction between TZDs and MPC2 is not affected by loss of the N-terminal 16 amino acids nor are these residues required for the insulin-sensitizing effects of these compounds.

Regulation of pyruvate metabolism and human disease.

Pyruvate is a keystone molecule critical for numerous aspects of eukaryotic and human metabolism. Pyruvate is the end-product of glycolysis, is derived from additional sources in the cellular cytoplasm, and is ultimately destined for transport into mitochondria as a master fuel input undergirding citric acid cycle carbon flux. In mitochondria, pyruvate drives ATP production by oxidative phosphorylation and multiple biosynthetic pathways intersecting the citric acid cycle. Mitochondrial pyruvate metabolism is regulated by many enzymes, including the recently discovered mitochondria pyruvate carrier, pyruvate dehydrogenase, and pyruvate carboxylase, to modulate overall pyruvate carbon flux. Mutations in any of the genes encoding for proteins regulating pyruvate metabolism may lead to disease. Numerous cases have been described. Aberrant pyruvate metabolism plays an especially prominent role in cancer, heart failure, and neurodegeneration. Because most major diseases involve aberrant metabolism, understanding and exploiting pyruvate carbon flux may yield novel treatments that enhance human health.

Tubular mitochondrial pyruvate carrier disruption elicits redox adaptations that protect from acute kidney injury.

OBJECTIVE: Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Although tubular metabolism changes markedly following acute kidney injury (AKI), it remains unclear which metabolic alterations are beneficial or detrimental. By analyzing large-scale, publicly available datasets, we observed that AKI consistently leads to downregulation of the mitochondrial pyruvate carrier (MPC). This investigation aimed to understand the contribution of the tubular MPC to kidney function, metabolism, and acute injury severity. METHODS: We generated tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice and employed renal function tests, in vivo renal 13C-glucose tracing, mechanistic enzyme activity assays, and tests of injury and survival in an established rhabdomyolysis model of AKI. RESULTS: MPC TubKO mice retained normal kidney function, displayed unchanged markers of kidney injury, but exhibited coordinately increased enzyme activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, compared to WT control mice, MPC TubKO mice showed increased glycolysis, decreased kidney injury and oxidative stress markers, and strikingly increased survival. CONCLUSIONS: Our findings suggest that decreased renal tubular mitochondrial pyruvate uptake hormetically upregulates oxidant defense systems before AKI and is a beneficial adaptive response after rhabdomyolysis-induced AKI. This raises the possibility of therapeutically modulating the MPC to attenuate AKI severity.

A pilot dose-finding study of Terazosin in humans.

Background: Parkinson's disease (PD) is a prevalent neurodegenerative disorder where progressive neuron loss is driven by impaired brain bioenergetics, particularly mitochondrial dysfunction and disrupted cellular respiration. Terazosin (TZ), an α-1 adrenergic receptor antagonist with a known efficacy in treating benign prostatic hypertrophy and hypertension, has shown potential in addressing energy metabolism deficits associated with PD due to its action on phosphoglycerate kinase 1 (PGK1). This study aimed to investigate the safety, tolerability, bioenergetic target engagement, and optimal dose of TZ in neurologically healthy subjects. Methods: Eighteen healthy men and women (60 - 85 years old) were stratified into two cohorts based on maximum TZ dosages (5 mg and 10 mg daily). Methods included plasma and cerebrospinal fluid TZ concentration measurements, whole blood ATP levels, 31 Phosphorous magnetic resonance spectroscopy for brain ATP levels, 18 F-FDG PET imaging for cerebral metabolic activity, and plasma metabolomics. Results: Our results indicated that a 5 mg/day dose of TZ significantly increased whole blood ATP levels and reduced global cerebral 18 F-FDG PET uptake without significant side effects or orthostatic hypotension. These effects were consistent across sexes. Higher doses did not result in additional benefits and showed a potential biphasic dose-response. Conclusions: TZ at a dosage of 5 mg/day engages its metabolic targets effectively in both sexes without inducing significant adverse effects and provides a promising therapeutic avenue for mitigating energetic deficiencies. Further investigation via clinical trials to validate TZ's efficacy and safety in neurodegenerative (i.e., PD) contexts is warranted.

Sedentary behavior in mice induces metabolic inflexibility by suppressing skeletal muscle pyruvate metabolism.

Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation. Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here, we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness, small mouse cage (SMC), that, unlike other classic models of disuse in mice, faithfully recapitulated metabolic responses that occur in humans. Bioenergetic phenotyping of skeletal muscle mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in the absence of a change in palmitate-stimulated JO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by heterozygous deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE in C2C12 myotubes was sufficient to increase glucose flux toward lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility in mice.

Metabolic control of transcription.

Light alters histone methylation in plants via nuclear α-ketoglutarate dehydrogenase.