Mitochondrial Protein Content Research Articles

Dark-cutting beef mitochondrial proteomic signatures reveal increased biogenesis proteins and bioenergetics capabilities

Mitochondria remain active in postmortem muscles and can influence meat color via oxygen consumption. Previous studies have shown that dark-cutting compared with normal-pH beef has greater mitochondrial protein and DNA content per gram of muscle tissue. However, the mechanism regulating mitochondrial content in dark-cutting vs. normal-pH beef is still unknown. Therefore, the objective was to compare mitochondrial proteomes of dark-cutting vs. normal-pH beef using LC-MS/MS-based proteomics and mitochondrial respiratory capacity using a Clark oxygen electrode. Dark-cutting compared with normal-pH beef has up-regulation of proteins involved in mitochondrial biogenesis, oxidative phosphorylation, intracellular protein transport, and cellular calcium ion homeostasis. Mitochondria isolated from dark-cutting phenotypes showed greater mitochondrial complex II respiration and uncoupled oxidative phosphorylation. However, mitochondrial membrane integrity and respiration at complexes I and IV were not different between normal-pH and dark-cutting beef. These results indicate that dark-cutting beef has greater mitochondrial biogenesis proteins than normal-pH beef, increasing mitochondrial content and contributing to dark-cutting beef. SignificanceDefective glycogen metabolism resulting from chronic stress before slaughter coupled with the greater mitochondrial protein and DNA content per gram of muscle tissue promotes muscle darkening in dark-cutting phenotypes in beef. However, the mechanistic basis for this occurrence in dark-cutting phenotypes is still unknown. In this work, we show that dark-cutting beef phenotype is caused, in part, as a consequence of over-proliferation of mitochondria. This is supported by the up-regulation of proteins involved in mitochondrial biogenesis, mitochondrial electron transport, calcium homeostasis, and fatty acid metabolism. Hence, the study of mitochondrial proteome changes provides a set of mitochondrial biogenesis proteins that could be used as potential candidate markers for detecting changes in pre-slaughter developmental events contributing to dark-cutting phenotypes in beef.

Endosymbiotic Chlorella variabilis reduces mitochondrial number in the ciliate Paramecium bursaria

Extant symbioses illustrate endosymbiosis is a driving force for evolution and diversification. In the ciliate Paramecium bursaria, the endosymbiotic alga Chlorella variabilis in perialgal vacuole localizes beneath the host cell cortex by adhesion between the perialgal vacuole membrane and host mitochondria. We investigated whether host mitochondria are also affected by algal endosymbiosis. Transmission electron microscopy of host cells showed fewer mitochondria beneath the algae-bearing host cell cortex than that of alga-free cells. To compare the density and distribution of host mitochondria with or without symbiotic algae, we developed a monoclonal antibody against Paramecium mitochondria. Immunofluorescence microscopy with the monoclonal antibody showed that the mitochondrial density of the algae-bearing P. bursaria was significantly lower than that of the alga-free cells. The total cell protein concentration of alga-free P. bursaria cells was approximately 1.8-fold higher than that of algae-bearing cells, and the protein content of mitochondria was significantly higher in alga-free cells than that in the algae-bearing cells. These results corresponded with those obtained by transmission electron and immunofluorescence microscopies. This paper shows that endosymbiotic algae affect reduced mitochondrial number in the host P. bursaria significantly.

The combination of NRF1 and Nrf2 activators in myoblasts stimulate mechanisms of proteostasis without changes in mitochondrial respiration

Persistent oxidative stress contributes to hallmarks of aging, including impaired proteostasis and mitochondrial dysfunction, while acute oxidative challenges resolved swiftly contribute to beneficial adaptations. Adaptive homeostasis is where acute exposures to sub‐toxic stimuli kindle transient expansion of responses necessary to reestablish homeostasis. Elucidating mechanisms underlying adaptive homeostasis will provide novel targets for healthspan extension. Nrf2 is a key regulator of cytoprotective gene transcription for redox homeostasis; NRF1 is a transcription factor that regulates expression of genes necessary for mitochondrial function. Regulation of both is compromised with advancing age. We hypothesized that NRF1 (NRF1a) and Nrf2 (Nrf2a) activators might improve adaptive homeostasis in response to an oxidative challenge by promoting mitochondrial proteome maintenance and function in C2C12 myoblasts. Using deuterium stable isotope tracing, we assessed protein synthesis over a 16‐hr treatment with a CON (DMSO), NRF1a, Nrf2a, or both, with and without a hydrogen peroxide (H2O2) stress. We assessed mitochondrial function using high‐resolution respirometry. Co‐treatment of NRF1a and Nrf2a under H2O2 stress favored proteostatic maintenance (p<0.05) through maintained protein synthesis (p>0.05), but a decrease in cell proliferation (p<0.05). H2O2 stress significantly decreased mitochondrial maximal respiration, but this decrease was not rescued by NRF1a/Nrf2a co‐treatment indicating the same ATP availability. Interestingly, there were no differences in submaximal ADP stimulated respiration or ADP sensitivity between CON, H2O2, or the NRF1a/Nrf2a co‐treatment (p>0.05). Further, there were no differences in endogenous redox capacity or mitochondrial protein content between treatment groups. These results suggest that simultaneously targeting NRF1 and Nrf2 may be a viable approach for reestablishing mitochondrial protein homeostasis following a stress, but that this adaptation may not improve respiratory capacity.

Mitochondrial respiratory chain protein co-regulation in the human brain

Mitochondrial respiratory chain (RC) function requires the stoichiometric interaction among dozens of proteins but their co-regulation has not been defined in the human brain. Here, using quantitative proteomics across three independent cohorts we systematically characterized the co-regulation patterns of mitochondrial RC proteins in the human dorsolateral prefrontal cortex (DLPFC). Whereas the abundance of RC protein subunits that physically assemble into stable complexes were correlated, indicating their co-regulation, RC assembly factors exhibited modest co-regulation. Within complex I, nuclear DNA-encoded subunits exhibited >2.5-times higher co-regulation than mitochondrial (mt)DNA-encoded subunits. Moreover, mtDNA copy number was unrelated to mtDNA-encoded subunits abundance, suggesting that mtDNA content is not limiting. Alzheimer's disease (AD) brains exhibited reduced abundance of complex I RC subunits, an effect largely driven by a 2–4% overall lower mitochondrial protein content. These findings provide foundational knowledge to identify molecular mechanisms contributing to age- and disease-related erosion of mitochondrial function in the human brain.

Factors affecting liver mitochondrial hydrogen peroxide emission.

Mitochondria are key cellular sources of reactive oxygen species (ROS) and contain at least 12 known sites on multiple enzymes that convert molecular oxygen to superoxide and hydrogen peroxide (H2O2). Quantitation of site-specific ROS emission is critical to understand the relative contribution of different sites and the pathophysiologic importance of mitochondrial ROS. However, factors that affect mitochondrial ROS emission are not well understood. We characterized and optimized conditions for maximal total and site-specific H2O2 emission during oxidation of standard substrates and probed the source of the high H2O2 emission in unenergized rainbow trout liver mitochondria. We found that mitochondrial H2O2 emission capacity depended on the substrate being oxidized, mitochondrial protein concentration, and composition of the ROS detection system. Contrary to our expectation, addition of exogenous superoxide dismutase reduced H2O2 emission. Titration of conventional mitochondrial electron transfer system (ETS) inhibitors over a range of conditions revealed that one size does not fit all; inhibitor concentrations evoking maximal responses varied with substrate and were moderated by the presence of other inhibitors. Moreover, the efficacy of suppressors of electron leak (S1QEL1.1 and S3QEL2) was low and depended on the substrate being oxidized. We found that H2O2 emission in unenergized rainbow trout liver mitochondria was suppressed by GKT136901 suggesting that it is associated with NADPH oxidase activity. We conclude that optimization of assay conditions is critical for quantitation of maximal H2O2 emission and would facilitate more valid comparisons of mitochondrial total and site-specific H2O2 emission capacities between studies, tissues, and species.

Mitochondrial and metabolic remodelling in human skin fibroblasts in response to glucose availability.

Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine, and the validation of ingredients for cosmetics. Given these cells' short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in normal human dermal fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level [low glucose medium (LGm)], or its complete removal and replacement by galactose [medium that forces oxidative phosphorylation (OXPHOSm)], always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm was it possible to observe the selective inhibition of mitochondrial adenosine triphosphate (ATP) production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate and extracellular acidification rate, oxidation of citric acid cycle substrates, fatty acids, lactate, and other substrates, increased mitochondrial network extension and polarization, the increased protein content of voltage-dependent anion channel (VDAC) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha and changes in several key transcripts related to energy metabolism. LGm did not promote significant metabolic changes in NHDF, although mitochondrial network extension and VDAC protein content were increased compared to HGm-cultured cells. Our results indicate that short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.

Elovl2-Ablation Leads to Mitochondrial Membrane Fatty Acid Remodeling and Reduced Efficiency in Mouse Liver Mitochondria

The fatty acid elongase elongation of very long-chain fatty acids protein 2 (ELOVL2) controls the elongation of polyunsaturated fatty acids (PUFA) producing precursors for omega-3, docosahexaenoic acid (DHA), and omega-6, docosapentaenoic acid (DPAn-6) in vivo. Expectedly, Elovl2-ablation drastically reduced the DHA and DPAn-6 in liver mitochondrial membranes. Unexpectedly, however, total PUFAs levels decreased further than could be explained by Elovl2 ablation. The lipid peroxidation process was not involved in PUFAs reduction since malondialdehyde-lysine (MDAL) and other oxidative stress biomarkers were not enhanced. The content of mitochondrial respiratory chain proteins remained unchanged. Still, membrane remodeling was associated with the high voltage-dependent anion channel (VDAC) and adenine nucleotide translocase 2 (ANT2), a possible reflection of the increased demand on phospholipid transport to the mitochondria. Mitochondrial function was impaired despite preserved content of the respiratory chain proteins and the absence of oxidative damage. Oligomycin-insensitive oxygen consumption increased, and coefficients of respiratory control were reduced by 50%. The mitochondria became very sensitive to fatty acid-induced uncoupling and permeabilization, where ANT2 is involved. Mitochondrial volume and number of peroxisomes increased as revealed by transmission electron microscopy. In conclusion, the results imply that endogenous DHA production is vital for the normal function of mouse liver mitochondria and could be relevant not only for mice but also for human metabolism.

PEG35 as a Preconditioning Agent against Hypoxia/Reoxygenation Injury.

Pharmacological conditioning is a protective strategy against ischemia/reperfusion injury, which occurs during liver resection and transplantation. Polyethylene glycols have shown multiple benefits in cell and organ preservation, including antioxidant capacity, edema prevention and membrane stabilization. Recently, polyethylene glycol 35 kDa (PEG35) preconditioning resulted in decreased hepatic injury and protected the mitochondria in a rat model of cold ischemia. Thus, the study aimed to decipher the mechanisms underlying PEG35 preconditioning-induced protection against ischemia/reperfusion injury. A hypoxia/reoxygenation model using HepG2 cells was established to evaluate the effects of PEG35 preconditioning. Several parameters were assessed, including cell viability, mitochondrial membrane potential, ROS production, ATP levels, protein content and gene expression to investigate autophagy, mitochondrial biogenesis and dynamics. PEG35 preconditioning preserved the mitochondrial function by decreasing the excessive production of ROS and subsequent ATP depletion, as well as by recovering the membrane potential. Furthermore, PEG35 increased levels of autophagy-related proteins and the expression of genes involved in mitochondrial biogenesis and fusion. In conclusion, PEG35 preconditioning effectively ameliorates hepatic hypoxia/reoxygenation injury through the enhancement of autophagy and mitochondrial quality control. Therefore, PEG35 could be useful as a potential pharmacological tool for attenuating hepatic ischemia/reperfusion injury in clinical practice.

Dysfunctional mitochondrial translation and combined oxidative phosphorylation deficiency in a mouse model of hepatoencephalopathy due to Gfm1 mutations.

Hepatoencephalopathy due to combined oxidative phosphorylation deficiency type 1 (COXPD1) is a recessive mitochondrial translation disorder caused by mutations in GFM1, a nuclear gene encoding mitochondrial elongation factor G1 (EFG1). Patients with COXPD1 typically present hepatoencephalopathy early after birth with rapid disease progression, and usually die within the first few weeks or years of life. We have generated two different mouse models: a Gfm1knock-in (KI) harboring the p.R671C missense mutation, found in at least 10 patients who survived more than 1year, and a Gfm1knock-out (KO) model. Homozygous KO mice (Gfm1-/- ) were embryonically lethal, whereas homozygous KI (Gfm1R671C/R671C ) mice were viable and showed normal growth. R671C mutation in Gfm1 caused drastic reductions in the mitochondrial EFG1 protein content in different organs. Six- to eight-week-old Gfm1R671C/R671C mice showed partial reductions of in organello mitochondrial translation and respiratory complex IV enzyme activity in the liver. Compound heterozygous Gfm1R671C/- showed a more pronounced decrease of EFG1 protein in liver and brain mitochondria, as compared with Gfm1R671C/R671C mice. At 8weeks of age, their mitochondrial translation rates were significantly reduced in both tissues. Additionally, Gfm1R671C/- mice showed combined oxidative phosphorylation deficiency (reduced complex I and IV enzyme activities in liver and brain), and blue native polyacrylamide gel electrophoresis analysis revealed lower amounts of both affected complexes. We conclude that the compound heterozygous Gfm1R671C/- mouse presents a clear dysfunctional molecular phenotype, showing impaired mitochondrial translation and combined respiratory chain dysfunction, making it a suitable animal model for the study of COXPD1.

PGC1α protects against cisplatin-induced skeletal muscle dysfunction

Background and Hypothesis: We and others have shown that chemotherapy promotes skeletal muscle wasting and weakness (i.e., cachexia) by disrupting mitochondrial homeostasis and causing oxidative stress. Peroxisome proliferative-activated receptor gamma coactivator 1-alpha (PGC1α) is a pivotal regulator of mitochondrial biogenesis and is involved in reducing oxidative damage in skeletal muscle. Hence, in the present study we investigated whether overexpression of skeletal muscle PGC1α (mPGC1α) was sufficient to preserve skeletal muscle mass and function in young and old mice treated with cisplatin.
 Experimental Design or Project Methods: Young (2-month; n = 5) and old (18-month; n = 5-8) male wild type (WT) or mPGC1α transgenic mice were treated with cisplatin (2.5mg/kg), while age-matched WT mice received vehicle for 2 weeks. Animals were assessed for muscle force and motor unit number estimation (MUNE). Skeletal muscles were weighed and processed for molecular analyses, including assessment of mitochondrial protein content.
 Results: Young WT mice exposed to cisplatin showed evidence of cachexia, as indicated by reduced gastrocnemius size (-16%), plantarflexion force (-8%) and MUNE (-56%), whereas mPGC1α mice were only partially protected. Interestingly, despite exacerbated cachexia in aged WT mice treated with chemotherapy, as demonstrated by markedly decreased gastrocnemius size (-22%), plantarflexion force (-18%) and MUNE (-80%) compared to untreated WT, muscle mass, strength and innervation were fully preserved in age-matched mPGC1α mice. Follow-up molecular analyses revealed that WT animals exposed to chemotherapy present loss of muscle mitochondrial proteins PGC1α, OPA1 and CytochromeC, whereas their levels in mPGC1α mice were robustly increased.
 Conclusion and Potential Impact: Altogether, our data suggest that PGC1α plays a pivotal role in preserving skeletal muscle mass and function, usually impaired by anticancer treatments. These findings enforce developing mitochondria-targeting therapeutics to combat the negative consequences that chemotherapy has on skeletal muscle.

Isolation and Measurement of Respiration and Structural Studies of Purified Mitochondria from Heterotrophic Plant Tissues.

Mitochondria are the power houses of eukaryotic cells. These organelles contain various oxidoreductase complexes. Electron transfer from different reducing equivalents channeled via these complexes drives proton translocation across the inner mitochondrial membrane, leading to ATP generation. Plant mitochondria contain alternative NAD(P)H dehydrogenases, alternative oxidase, and uncoupling protein, and TCA cycle enzymes are located in their matrix. Apart from ATP production, mitochondria are also involved in synthesis of vitamins and cofactors and participate in fatty acid, nucleotide, photorespiratory, and antioxidant metabolism. Recent emerging evidence suggests that mitochondria play a role in redox signaling and generation of reactive oxygen and nitrogen species. For mitochondrial studies, it is essential to isolate physiologically active mitochondria with good structural integrity. In this article, we explain a detailed procedure for isolation of mitochondria from various heterotrophic tissues, such as germinating chickpea seeds, potato tubers, and cauliflower florets. This procedure requires discontinuous Percoll gradient centrifugation and can give a good yield of mitochondria, in the range of 4 to 8 mg per 50 g tissue with active respiratory capacity. After MitoTracker staining, isolated mitochondria can be visualized by using a confocal microscope. The structure of mitochondria can be monitored by scanning electron microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mitochondria from germinating chickpea seeds, potato tubers, and cauliflower florets Basic Protocol 2: Quantification of mitochondrial protein concentration by Bradford assay Basic Protocol 3: Quantification of mitochondrial respiration using single-channel free-radical analyzer Basic Protocol 4: Staining of mitochondria and confocal imaging Basic Protocol 5: Visualization of isolated mitochondria under scanning electron microscope.

Exercise Enhanced Cardiac Function in Mice With Radiation-Induced Heart Disease via the FNDC5/Irisin-Dependent Mitochondrial Turnover Pathway.

Background: Despite the development of radiation therapy (RT) techniques, concern regarding the serious and irreversible heart injury induced by RT has grown due to the lack of early intervention measures. Although exercise can act as an effective and economic nonpharmacologic strategy to combat fatigue and improve quality of life for cancer survivors, limited data on its application in radiation-induced heart disease (RIHD) and the underlying molecular mechanism are available.Methods: Fifteen young adult male mice were enrolled in this study and divided into 3 groups (including exercised RIHD group, sedentary RIHD group, and controls; n =5 samples/group). While the mice in the control group were kept in cages without irradiation, those in the exercised RIHD group underwent 3weeks of aerobic exercise on the treadmill after radiotherapy. At the end of the 3rd week following RT, FNDC5/irisin expression, cardiac function, aerobic fitness, cardiomyocyte apoptosis, mitochondrial function, and mitochondrial turnover in the myocardium were assessed to identify the protective role of exercise in RIHD and investigate the potential mechanism.Results: While sedentary RIHD group had impaired cardiac function and aerobic fitness than controls, the exercised RIHD mice had improved cardiac function and aerobic fitness, elevated ATP production and the mitochondrial protein content, decreased mitochondrial length, and increased formation of mitophagosomes compared with sedentary RIHD mice. These changes were accompanied by the elevated expression of FNDC5/irisin, a fission marker (DRP1) and mitophagy markers (PINK1 and LC3B) in exercised RIHD group than that of sedentary RIHD group, but the expression of biogenesis (TFAM) and fusion (MFN2) markers was not significantly changed.Conclusion: Exercise could enhance cardiac function and aerobic fitness in RIHD mice partly through an autocrine mechanism via FNDC5/irisin, in which autophagy was selectively activated, suggesting that FNDC5/irisin may act as an intervening target to prevent the development of RIHD.

Characterization of mitochondrial DNA quantity and quality in the human aged and Alzheimer\u2019s disease brain

BackgroundMitochondrial dysfunction is a feature of neurodegenerative diseases, including Alzheimer’s disease (AD). Changes in the mitochondrial DNA copy number (mtDNAcn) and increased mitochondrial DNA mutation burden have both been associated with neurodegenerative diseases and cognitive decline. This study aims to systematically identify which common brain pathologies in the aged human brain are associated with mitochondrial recalibrations and to disentangle the relationship between these pathologies, mtDNAcn, mtDNA heteroplasmy, aging, neuronal loss, and cognitive function.MethodsWhole-genome sequencing data from n = 1361 human brain samples from 5 different regions were used to quantify mtDNAcn as well as heteroplasmic mtDNA point mutations and small indels. Brain samples were assessed for 10 common pathologies. Annual cognitive test results were used to assess cognitive function proximal to death. For a subset of samples, neuronal proportions were estimated from RNA-seq profiles, and mass spectrometry was used to quantify the mitochondrial protein content of the tissue.ResultsmtDNAcn was 7–14% lower in AD relative to control participants. When accounting for all 10 common neuropathologies, only tau was significantly associated with lower mtDNAcn in the dorsolateral prefrontal cortex. In the posterior cingulate cortex, TDP-43 pathology demonstrated a distinct association with mtDNAcn. No changes were observed in the cerebellum, which is affected late by pathologies. Neither age nor gender was associated with mtDNAcn in the studied brain regions when adjusting for pathologies. Mitochondrial content and mtDNAcn independently explained variance in cognitive function unaccounted by pathologies, implicating complex mitochondrial recalibrations in cognitive decline. In contrast, mtDNA heteroplasmy levels increased by 1.5% per year of life in the cortical regions, but displayed no association with any of the pathologies or cognitive function.ConclusionsWe studied mtDNA quantity and quality in relation to mixed pathologies of aging and showed that tau and not amyloid-β is primarily associated with reduced mtDNAcn. In the posterior cingulate cortex, the association of TDP-43 with low mtDNAcn points to a vulnerability of this region in limbic-predominant age-related TDP-43 encephalopathy. While we found low mtDNAcn in brain regions affected by pathologies, the absence of associations with mtDNA heteroplasmy burden indicates that mtDNA point mutations and small indels are unlikely to be involved in the pathogenesis of late-onset neurodegenerative diseases.

Protective effects of extra virgin olive oil and exercise training on rat skeletal muscle against high-fat diet feeding

A diet high in saturated fat leads to skeletal muscle deteriorations including insulin resistance, mitochondrial dysfunction and muscle fiber atrophy. Consumption of long-chain polyunsaturated fatty acids and exercise have shown promise in ameliorating high-fat diet (HFD)-induced oxidative stress and inflammation. However, the impact of extra virgin olive oil (EVOO) on mitochondrial homeostasis in muscle is largely unknown. This study aimed to investigate whether 12 wks of EVOO feeding alone and in conjunction with endurance training could protect against metabolic and mitochondrial dysfunction rat muscle with HFD. Female Sprague-Dawley rats were divided into 4 groups fed a control diet (C), HFD, EVOO diet, and EVOO diet with training (EVOO+T). Mitochondrial enzyme activity and protein content decreased with HFD compared to C, but were restored with EVOO and EVOO+T. EVOO+T elevated muscle cytochrome c and PGC-1α levels. HFD increased muscle proteolytic markers and protein ubiquitination, whereas these effects were not seen in EVOO and EVOO+T. HFD suppressed mitochondrial fusion protein level while increasing fission protein levels, but were restored with EVOO and EVOO+T. Mitophagy marker PINK1 content decreased with HFD, but was unchanged in EVOO and EVOO+T. EVOO+T upregulated autophagy markers, along with decreased phosphorylated/dephosphorylated FoxO3 ratio. Antioxidants enzyme levels were upregulated by EVOO and EVOO+T, and EVOO+T reduced HFD-induced lipid peroxidation. In conclusion, HFD impaired muscle oxidative capacity, promoted protein ubiquitination and mitochondrial fission, and upregulated autophagy markers. Replacement of HFD with EVOO corrected the observed adverse effects, while exercise training in conjunction with EVOO provided additional protection to the muscle.

Exercise prevents fatty liver by modifying the compensatory response of mitochondrial metabolism to excess substrate availability

ObjectiveLiver mitochondria adapt to high-calorie intake. We investigated how exercise alters the early compensatory response of mitochondria, thus preventing fatty liver disease as a long-term consequence of overnutrition. MethodsWe compared the effects of a steatogenic high-energy diet (HED) for six weeks on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome, and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high-resolution respirometry. ResultsHED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism, and lipogenesis. Training also counteracted the HED-induced glucose intolerance, the increase in fasting insulin, and in liver fat by lowering diacylglycerol species and c-Jun N-terminal kinase (JNK) phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content. ConclusionWe provide a comprehensive insight into the early adaptations of mitochondria in the liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity.

Fission accomplished: Uncovering the role of Drp1 in regulating mitochondrial dysfunction and age-related muscle atrophy.

Fission accomplished: Uncovering the role of Drp1 in regulating mitochondrial dysfunction and age-related muscle atrophy.

Lactate administration does not affect denervation-induced loss of mitochondrial content and muscle mass in mice.

Lactate is considered to be a signaling molecule that induces mitochondrial adaptation and muscle hypertrophy. The purpose of this study was to examine whether lactate administration attenuates denervation‐induced loss of mitochondrial content and muscle mass. Eight‐week‐old male Institute of Cancer Research mice underwent unilateral sciatic nerve transection surgery. The contralateral hindlimb served as a sham‐operated control. From the day of surgery, mice were injected intraperitoneally with PBS or sodium lactate (equivalent to 1 g·kg−1 body weight) once daily for 9 days. After 10 days of denervation, gastrocnemius muscle weight decreased to a similar extent in both the PBS‐ and lactate‐injected groups. Denervation significantly decreased mitochondrial enzyme activity, protein content, and MCT4 protein content in the gastrocnemius muscle. However, lactate administration did not have any significant effects. The current observations suggest that daily lactate administration for 9 days does not affect denervation‐induced loss of mitochondrial content and muscle mass.

Skeletal Muscle Mitochondrial Physiology in Children With Cerebral Palsy: Considerations for Healthy Aging.

Skeletal muscle contractile proteins require a constant supply of energy to produce force needed for movement. Energy (ATP) is primarily produced by mitochondrial organelles, located within and around muscle fibers, by oxidative phosphorylation that couples electron flux through the electron transport chain to create a proton gradient across the inner mitochondrial membrane that is in turn used by the ATP synthase. Mitochondrial networks increase in size by biogenesis to increase mitochondrial abundance and activity in response to endurance exercise, while their function and content reduce with constant inactivity, such as during muscle atrophy. During healthy aging, there is an overall decline in mitochondrial activity and abundance, increase in mitochondrial DNA mutations, potential increase in oxidative stress, and reduction in overall muscular capacity. Many of these alterations can be attenuated by consistent endurance exercise. Children with cerebral palsy (CP) have significantly increased energetics of movement, reduced endurance capacity, and increased perceived effort. Recent work in leg muscles in ambulatory children with CP show a marked reduction in mitochondrial function. Arm muscles show that mitochondrial protein content and mitochondria DNA copy number are lower, suggesting a reduction in mitochondrial abundance, along with a reduction in markers for mitochondrial biogenesis. Gene expression networks are reduced for glycolytic and mitochondrial pathways and share similarities with gene networks with aging and chronic inactivity. Given the importance of mitochondria for energy production and changes with aging, future work needs to assess changes in mitochondria across the lifespan in people with CP and the effect of exercise on promoting metabolic health.

A novel integrated approach to estimate the mitochondrial content of neuronal cells and brain tissues

BackgroundMitochondria and their dynamics fuel most cellular processes both in physiological and pathological conditions. In the central nervous system, mitochondria sustain synaptic transmission and plasticity via multiple mechanisms which include their redistribution and/or expansion to higher energy demanding sites, sustaining activity changes and promoting morphological circuit adaptations. New MethodTo be able to evaluate changes in mitochondrial number and protein phenotype, we propose a novel methodological approach where the simultaneous analysis of both mitochondrial DNA and protein content is performed on each individual microsample, avoiding non-homogeneous loss of material. ResultsWe validated this method on neuronal-like cells and tissue samples and obtained estimates for the mitochondrial/genomic DNA ratio as well as for the abundance of protein counterparts. When the mitochondrial content per cell was evaluated in different brain areas, our results matched the known regional variation in aerobic-anaerobic metabolism. When long-term potentiation (LTP) was induced on hippocampal neurons, we detected increases in the abundance of mitochondria that correlated with the degree of synaptic enhancement. ConclusionsOur approach can be effectively used to study the mitochondrial content andits changes in different brain cells and tissues.

Acetate Promotes a Differential Energy Metabolic Response in Human HCT 116 and COLO 205 Colon Cancer Cells Impacting Cancer Cell Growth and Invasiveness.

Under dysbiosis, a gut metabolic disorder, short-chain carboxylic acids (SCCAs) are secreted to the lumen, affecting colorectal cancer (CRC) development. Butyrate and propionate act as CRC growth inhibitors, but they might also serve as carbon source. In turn, the roles of acetate as metabolic fuel and protein acetylation promoter have not been clearly elucidated. To assess whether acetate favors CRC growth through active mitochondrial catabolism, a systematic study evaluating acetate thiokinase (AcK), energy metabolism, cell proliferation, and invasiveness was performed in two CRC cell lines incubated with physiological SCCAs concentrations. In COLO 205, acetate (+glucose) increased the cell density (50%), mitochondrial protein content (3–10 times), 2-OGDH acetylation, and oxidative phosphorylation (OxPhos) flux (36%), whereas glycolysis remained unchanged vs. glucose-cultured cells; the acetate-induced OxPhos activation correlated with a high AcK activity, content, and acetylation (1.5–6-fold). In contrast, acetate showed no effect on HCT116 cell growth, OxPhos, AcK activity, protein content, and acetylation. However, a substantial increment in the HIF-1α content, HIF-1α-glycolytic protein targets (1–2.3 times), and glycolytic flux (64%) was observed. Butyrate and propionate decreased the growth of both CRC cells by impairing OxPhos flux through mitophagy and mitochondrial fragmentation activation. It is described, for the first time, the role of acetate as metabolic fuel for ATP supply in CRC COLO 205 cells to sustain proliferation, aside from its well-known role as protein epigenetic regulator. The level of AcK determined in COLO 205 cells was similar to that found in human CRC biopsies, showing its potential role as metabolic marker.