Biology subjects

Kowaltowski, A. J.

Publications and source records attributed to Kowaltowski, A. J..

18 recordsLinked to original sources

bioRxiv2026

Mitochondrial Metabolism and Calcium Handling in Parkinsons Disease hiPSC-derived Astrocytes

Parkinsons disease (PD) is the second most common neurodegenerative disorder worldwide, and mutations in the LRRK2 and PRKN genes are among the most common familial causes of the disease. In neurodegenerative diseases such as PD, disturbances in Ca{superscript 2} homeostasis and cellular bioenergetics impair the function of neurons and glial cells, contributing to disease progression. These changes are not limited to neurons; mitochondrial dysfunction and disrupted Ca2+ homeostasis in astrocytes are increasingly recognized as key contributors to PD, impairing bioenergetics, redox balance, neuroinflammatory responses, and metabolic support essential for dopaminergic neuron survival. In this study, we investigated mitochondrial calcium homeostasis, mitochondrial oxidative phosphorylation, morphology and distribution in human induced pluripotent stem cell (hiPSC)-derived astrocytes with mutations in the PD genes LRRK2 (G2019S) and PRKN (c.155delA; Ex3-4del) and wild-type controls. Intracellular calcium dynamics were assessed using Fura-2 AM. Compared with control astrocytes, LRRK2-related PD patient-derived mutant astrocytes exhibited lower intracellular calcium levels, and slower calcium extrusion following stimulation with ATP. Mitochondrial morphology was analyzed using MitoTracker Deep Red, revealing increased mitochondrial fragmentation and redistribution of mitochondria toward the cell periphery in both PD mutant cell types. Because oxidative phosphorylation is tightly regulated by mitochondrial morphology and calcium homeostasis, we next assessed oxygen consumption rates using a continuous metabolic monitoring system (Resipher) and quantified the expression of genes (RT-qPCR) and proteins (capillary electrophoresis-based western detection) involved in mitochondrial calcium transport and bioenergetics. These analyses showed that PRKN mutant astrocytes exhibit a more oxidative bioenergetic phenotype than LRRK2 mutant astrocytes, while both mutant lines displayed altered phosphorylation of mitochondrial morphology regulator DRP1 as well as decreased levels of respiratory complexes relative to control astrocytes. In summary, this study identifies astrocyte-specific mitochondrial dysfunctions and calcium dysregulation as key features of LRRK2- and PRKN-related pathology, providing new insights into how glial metabolic alterations contribute to neurodegeneration in PD.

neuroscience↗

bioRxiv2026

Caloric Restriction Promotes Ischemia/Reperfusion Cardioprotection Through Increased Mitochondrial Na+/Ca2+ Exchange

Caloric restriction (CR) protects against cardiac ischemia/reperfusion (I/R) injury, but the underlying mechanisms remain incompletely understood. Since mitochondrial Ca2+ overload and redox imbalance is a major driver of cardiac damage during reperfusion, we investigated if enhanced mitochondrial Ca2+ efflux contributes toward CR-induced cardioprotection. Rats were subjected to 16 weeks of ad libitum (AL) feeding or 40% caloric restriction. CR significantly increased mitochondrial Ca2+ retention capacity when Na+ ions were present, reduced H2O2 release, and increased the expression of proteins related to mitochondrial Ca2+ extrusion. In cardiomyocytes exposed to serum from CR rats, Ca2+ retention capacity also markedly increased, as well as Ca2+ efflux and the expression of extrusion proteins NCLX and TMEM65. Following I/R, CR hearts exhibited improved functional recovery accompanied by enhanced retention and increased mitochondrial Ca2+ efflux activity, as well as reduced H2O2 release compared to AL controls. Inhibition of mitochondrial Na+/Ca2+ exchange abolished the mitochondrial adaptive effects of CR, eliminating its protection against damage in cardiomyocytes and perfused hearts. These findings demonstrate that CR protects the heart from I/R injury by enhancing CGP-sensitive Na+-dependent mitochondrial Ca2+ efflux, preserving mitochondrial function, limiting redox imbalance, and improving post-ischemic cardiac recovery.

biochemistry↗

bioRxiv2026

An Open-Source Code To Analyze Mitochondrial Intracellular Distribution From Fluorescence Microscopy Images

Mitochondria have a plethora of roles in cells, many of which are related to dynamic changes in their size, shape, and intracellular location. Mitochondrial morphology is commonly assessed by microscopy with targeted fluorescent probes. However, tools to easily estimate mitochondrial localization within a cell are still lacking. A code was designed to estimate per-cell mitochondrial radial localization (perinuclear or peripheral) from fluorescence microscopy files in a variety of formats and using different mitochondrial markers (https://github.com/cavalcantegc/mito_localization.git). Three case studies with different cell types and stainings demonstrate that mitochondrial localization can be easily extracted and plotted with this code.

cell biology↗

bioRxiv2025

YME1L-dependent regulation of mitochondrial Ca2+ transport: a role for mitochondrial uptake protein 1 (MICU1) in nutrient sensing

Mitochondrial calcium uptake via the mitochondrial calcium uniporter complex (MCUc) is tightly regulated by gatekeeper proteins such as MICU1 and MICU2. While long-term nutritional interventions have been shown to remodel the MCUc, its short-term regulation during postprandial transitions and acute nutrient stress remains unclear. Here, we demonstrate that non-gated MCUc increases in the postprandial liver due to a transient loss of mature MICU1 (m-MICU1), accompanied by changes in its precursor form (p-MICU1). These changes are more pronounced and occur earlier than the general increase in mitochondrial mass and respiratory protein content, indicating that they are independent of mitochondrial remodelling during refeeding. In vitro, serum deprivation, insulin signalling, modulation of mTOR activity, and glutamine starvation all affect mature or precursor MICU1 protein levels, indicating that multiple nutritional changes promote MICU1 processing alterations, leading to the formation of a non-gated, MICU-deficient MCUc. Under these conditions, mitochondrial, but not cytosolic, Ca{superscript 2} levels increase in intact cells. Moreover, mitochondrial Ca{superscript 2} uptake rates are enhanced, which correlates with higher mitochondrial oxygen consumption, increased reactive oxygen species (ROS) production, and enhanced sensitivity to mitochondrial permeability transition. We identify the mitochondrial protease YME1L as essential for MICU1 turnover in response to nutrient availability. YME1L silencing prevents MICU1 degradation and suppresses the enhanced Ca{superscript 2} uptake induced by glutamine deprivation. Together, these results reveal nutrient-sensitive, YME1L-dependent remodelling of the MCU complex and establish MICU1 degradation as a key mechanism linking metabolic signals to mitochondrial Ca{superscript 2} transport.

physiology↗

bioRxiv2025

Mitochondrial ATP-Sensitive K+ Channels (MitoKATP) Regulate Brown Adipocyte Differentiation and Metabolism

Brown adipose tissue (BAT) plays a central role in mammalian non-shivering thermogenesis, dissipating mitochondrial membrane potentials through the activity of uncoupling protein UCP1 to release heat. Inner membranes of mitochondria are known to be permeable to potassium ions (K+), which enter the matrix either through ATP-sensitive channels (MitoKATP) or leakage across the bilayer driven by inner membrane potentials. Mitochondrial K+ influx is associated with increased osmotic pressure, promoting water influx and increasing matrix volume. Since BAT mitochondria have lower inner membrane potentials due to uncoupling protein 1 (UCP1) activity, we hypothesized this could involve compensatory changes in MitoKATP activity, and thus tested MitoKATP involvement in brown adipocyte activities under basal and stimulated conditions. We find that cold exposure and adrenergic stimulation in mice modulate BAT MitoK levels, the channel portion of MitoKATP. Genetic ablation of the gene that codes for the pore-forming subunit of MitoKATP in human pre-adipocytes decreased cellular respiration and proliferation, compromising differentiation into mature adipocytes. In mouse cell lines, the absence of the protein limited cellular oxygen consumption in the precursor stage, but not in mature adipocytes. Interestingly, inhibition of MitoKATP in mature adipocytes increased adrenergic-stimulated oxygen consumption, indicating that shutdown of this pathway is important for full BAT thermogenesis. Similarly, MitoKATP inhibition increased oxygen consumption in BAT mitochondria isolated from mice treated with beta 3 adrenergic receptor agonist CL316,243. Overall, our results suggest that the activity of MitoKATP regulates differentiation and metabolism of brown adipocytes, impacting on thermogenesis. New and NoteworthyBrown fat cells are important to maintain healthy body weight by promoting mitochondrial uncoupling. Here, we demonstrate that mitochondrial ATP-sensitive potassium channels (MitoKATP) have important roles both in the differentiation of brown fat cells and in the activation of energy-dissipating uncoupling in this tissue.

physiology↗

bioRxiv2024

Mitochondrial Fatty Acid Oxidation is Stimulated by Red Light Irradiation

Keratinocytes are the primary constituents of sunlight-exposed epidermis. In these cells, UVA completely inhibited oxidative phosphorylation, while equivalent doses of blue and green light preserved metabolic fluxes, but reduced viability. In contrast, red light enhanced proliferation and elevated basal and maximal oxygen consumption rates for 48 h, without altering protein levels of the electron transport chain. Targeted flux analysis revealed that red light specifically activates AMPK-dependent mitochondrial fatty acid oxidation. This was accompanied by reduced the levels of free fatty acids and increased acetyl CoA carboxylase phosphorylation. Together, our results characterize wavelength- selective regulation of keratinocyte metabolism: UV/visible wavelengths induce damage, while red light triggers AMPK-dependent fatty acid oxidation, providing a mechanistic explanation for photobiomodulation in epidermal cells. HighlightsUVA (365 nm) abolishes oxidative phosphorylation, while blue/green light (450/517 nm) induce photosensitized toxicity, without metabolic disruption. Red light (660 nm) boosts keratinocyte proliferation and sustains elevated mitochondrial respiration for 48 h. Permeabilized cell electron transport remains unchanged, implicating cytosolic (not mitochondrial) regulation of fuel utilization. Red light activates AMPK and phosphorylates/inhibits acetyl CoA carboxylase (ACC). Photobiomodulation with red light occurs through AMPK/ACC-mediated lipid oxidation, not direct respiratory chain modulation.

biochemistry↗

bioRxiv2023

A practical and robust method to evaluate metabolic fluxes in pancreatic islets

Aims/hypothesisEfficient mitochondrial oxidative phosphorylation is essential for pancreatic beta cell responses to nutrient levels. Consequently, the evaluation of mitochondrial oxygen consumption and ATP production is important to investigate essential aspects of pancreatic islet pathophysiology. Currently, most studies use cell lines instead of primary islets due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables that are expensive, complicated to use, and poorly reproducible. The aim of this study is to establish a robust and practical method to assess primary islet metabolic fluxes using Extracellular Flux Technology and standard commercial consumables. MethodsPancreatic islets were isolated from 8 to 12-week-old mice and rats, and submitted to a dispersion protocol using trypsin. Dispersed islets were adhered overnight to pre-coated standard Seahorse microplates, and oxygen consumption rates were evaluated using a Seahorse Extracellular Flux Analyzer. We also validated the functionality of dispersed islets by analyzing glucose-stimulated insulin secretion (GSIS) and calcium (Ca2+) influx in response to different modulators by fluorescence microscopy. ResultsWe provide a detailed protocol with all steps necessary to optimize islet isolation and dispersion, in order to achieve a high yield of functional islets and perform metabolic flux analysis. With this method, which requires only a few islets per replicate, both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators (oligomycin, CCCP, antimycin and rotenone) and glucose addition. Both oligomycin and CCCP concentrations were titrated. Our method was also validated by other functional assays, which show these cells present conserved Ca2+ influx and insulin secretion in response to glucose. Conclusions/interpretationWe established a practical and robust method to assess ex vivo islet metabolic fluxes and oxidative phosphorylation. Our findings cover an important gap in primary islet physiology studies, providing a valuable tool we hope is useful to uncover basic beta cell metabolic mechanisms, as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols. Research in context What is already known about this subject?O_LIPancreatic beta cells efficiently couple oxidative phosphorylation and ATP production with insulin secretion; mitochondrial ATP production is crucial for proper insulin secretion. C_LIO_LIMost studies of beta cell respiration use cell lines instead of primary islets, which are a much more robust model to evaluate beta cell function. C_LIO_LIThe few works with primary islet respiration use specific equipment and consumables that are expensive, complicated, and poorly reproducible. C_LI What is the key question?O_LIIs it possible to develop a practical method to evaluate metabolic fluxes and ATP production in isolated islets, using the standard Seahorse Extracellular Flux Technology? C_LI What are the new findings?O_LIWe optimized rodent islet isolation and functional analysis protocols using standard extracellular flux analysis equipment and consumables. C_LIO_LIOur method allows for increased islet yield and robust islet respiration measurements. C_LI How might this impact on clinical practice in the foreseeable future?O_LIQuantitative measurements of metabolic fluxes and oxidative phosphorylation are the cornerstone of new discoveries in beta cells, and can contribute toward the establishment of new cellular protocols, such as for cell transplantation, as well as the development of new pharmacological agents targeted to these cells. C_LI

biochemistry↗

bioRxiv2023

Autophagy is Regulated by Mitochondrial Calcium Transporters NCLX and MCU

Mitochondria shape intracellular Ca2+ signaling through the concerted activity of Ca2+ uptake via mitochondrial calcium uniporter, and efflux from by Na+/Ca2+ exchangers (NCLX). Here, we describe a novel relationship between NCLX, intracellular Ca2+, and autophagic activity. Conditions that stimulate autophagy in vivo and in vitro, such as caloric restriction and nutrient deprivation, upregulate NCLX expression in hepatic tissue and cells. Conversely, knockdown of NCLX impairs basal and starvation-induced autophagy. Similarly, acute inhibition of NCLX activity by CGP 37157 affects bulk and endoplasmic reticulum autophagy (ER-phagy) without significant impacts on mitophagy. Mechanistically, CGP 37157 inhibited the formation of FIP200 puncta and downstream autophagosome biogenesis. Inhibition of NCLX caused decreased cytosolic Ca2+ levels, and intracellular Ca2+ chelation similarly suppressed autophagy. Furthermore, chelation did not exhibit an additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+ efflux regulates autophagy through the modulation of Ca2+ signaling. Collectively, our results show that the mitochondrial Ca2+ extrusion pathway through NCLX is an important regulatory node linking nutrient restriction and autophagy regulation.

cell biology↗

bioRxiv2022

Mitochondrial sodium/calcium exchanger NCLX regulates glycolysis in astrocytes, impacting on cognitive performance

Intracellular Ca2+ concentrations are strictly controlled by plasma membrane transporters, the endoplasmic reticulum, and mitochondria, in which Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUc), while efflux occurs mainly through the mitochondrial Na+/Ca2+ exchanger (NCLX). RNAseq database repository searches led us to identify the Nclx transcript as highly enriched in astrocytes when compared to neurons. To assess the role of NCLX in mouse primary culture astrocytes, we inhibited its function both pharmacologically or genetically. This resulted in re-shaping of cytosolic Ca2+ signaling and a metabolic shift that increased glycolytic flux and lactate secretion in a Ca2+-dependent manner. Interestingly, in vivo genetic deletion of NCLX in hippocampal astrocytes improved cognitive performance in behavioral tasks, whereas hippocampal neuron-specific deletion of NCLX impaired cognitive performance. These results unveil a role for NCLX as a novel modulator of astrocytic glucose metabolism, impacting on cognition.

biochemistry↗

bioRxiv2022

Adiponectin Promotes Glucose-Sensitive Insulin Secretion and Prevents β-Cell Damage by Obesity

Obesity significantly decreases life expectancy and increases the incidence of age-related dysfunctions, including {beta}-cell dysregulation leading to inadequate insulin secretion. Here, we show that diluted plasma from obese human donors acutely impairs {beta}-cell integrity and insulin secretion relative to plasma from lean subjects. Similar results were observed with diluted sera from obese rats fed ad libitum, when compared to sera from lean, calorically-restricted, animals. The damaging effects of obese circulating factors on {beta}-cells occurs in the absence of nutrient overload, and mechanistically involves mitochondrial dysfunction, limiting glucose-supported oxidative phosphorylation and ATP production. We demonstrate that increased levels of adiponectin, as found in lean plasma, are the protective characteristic preserving {beta}-cell function; indeed, sera from adiponectin knockout mice limits {beta}-cell metabolic fluxes relative to controls. Furthermore, oxidative phosphorylation and glucose-sensitive insulin secretion, which are completely abrogated in the absence of this hormone, are restored by the presence of adiponectin alone, surprisingly even in the absence of other serological components, for both the insulin-secreting INS1 cell line and primary islets. The addition of adiponectin to cells treated with plasma from obese donors completely restored {beta}-cell functional integrity, indicating the lack of this hormone was causative of the dysfunction. Overall, our results demonstrate that low circulating adiponectin is a key damaging element for {beta}-cells, and suggest strong therapeutic potential for the modulation of the adiponectin signaling pathway in the prevention of age-related {beta}-cell dysfunction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/501128v3_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1df5041org.highwire.dtl.DTLVardef@e066borg.highwire.dtl.DTLVardef@c79132org.highwire.dtl.DTLVardef@d89d1_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: Incubation of -cells with sera or plasma from obese rats and humans hampers mitochondrial oxidative phosphorylation and glucose-stimulated insulin secretion (GSIS) relative to sera and plasma from lean rats and humans. Adiponectin, found at elevated levels in lean subjects, supports -cell function on its own, in the absence of sera, and also reverses the effects of obese plasma. Prepared using Biorender.com. C_FIG

biochemistry↗

bioRxiv2022

Goldilocks calcium and the mitochondrial respiratory chain: too much, too little, just right

Calcium (Ca2+) is a key regulator in diverse intracellular signaling pathways, and has long been implicated in metabolic control and mitochondrial function. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture, and cell death. Moderate Ca2+ within the organelle, on the other hand, can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. Here, we aimed to determine in a quantitative manner if extra or intramitochondrial Ca2+ modulate oxidative phosphorylation in mouse liver mitochondria and intact hepatocyte cell lines. To do so, we monitored the effects of more modest versus supra-physiological increases in cytosolic and mitochondrial Ca2+ on oxygen consumption rates. Isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low (2.4 {+/-} 0.6 M) and medium (22.0 {+/-} 2.4 M) Ca2+ concentrations in the presence of complex I-linked substrates pyruvate plus malate and -ketoglutarate, respectively, but not complex II-linked succinate. In intact cells, both low and high cytosolic Ca2+ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca2+ decreased mitochondrial respiration in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific "just right" concentrations that activate oxidative phosphorylation.

biochemistry↗

bioRxiv2021

Regulation of Kidney Mitochondrial Function by Caloric Restriction

Caloric restriction (CR) prevents obesity, promotes healthy aging, and increases resilience against several pathological stimuli in laboratory rodents. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and capacities, avoiding Ca2+-induced mitochondrial permeability transition. Dietary restriction has also been shown to increase kidney resistance against damaging stimuli such as ischemia/reperfusion, but if these effects are related to similar mitochondrial adaptations had not yet been uncovered. Here, we characterized changes in mitochondrial function in response to six months of CR in rats, measuring bioenergetic parameters, redox balance and calcium homeostasis. CR promoted an increase in mitochondrial oxygen consumption rates under non-phosphorylating and uncoupled conditions. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2O2 release was enhanced, although levels of carbonylated proteins and methionine sulfoxide were unchanged. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+-induced mitochondrial permeability transition. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials or the amounts of the mitochondrial calcium uniporter (MCU). Instead, increased mitochondrial calcium uptake rates in CR kidneys correlate with a loss of MICU2, an MCU modulator. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet-sensitive regulatory role controlling mitochondrial calcium homeostasis. Together, our results highlight the organ-specific bioenergetic, redox, and ionic transport effects of CR. Specifically, we describe the regulation of the expression of MICU2 and its effects on mitochondrial calcium transport as a novel and interesting aspect of the metabolic responses to dietary interventions.

biochemistry↗

bioRxiv2021

MS-driven metabolic alterations are recapitulated in iPSC-derived astrocytes

ObjectiveAstrocytes play a significant role in the pathology of Multiple Sclerosis (MS). Nevertheless, for ethical reasons, most of the studies in these cells were performed on the Experimental Autoimmune Encephalomyelitis model. As there are significant differences between human and mouse cells, we aimed here to better characterize astrocytes from patients with MS (PwMS), focusing mainly on mitochondrial function and cell metabolism. MethodsWe obtained and characterized induced pluripotent stem cell (iPSC)-derived astrocytes from three PwMS and three unaffected controls and performed functional assays including electron microscopy, flow cytometry, cytokine measurement, gene expression, in situ respiration, and metabolomics. ResultsWe detected several differences in MS astrocytes including: (i) enrichment of genes associated with mitophagy and neurodegeneration, (ii) increased mitochondrial fission and decreased mitochondrial to nuclear DNA ratio, indicating disruption of mitochondrial content, (iii) increased production of superoxide and MS-related proinflammatory chemokines, (iv) increased electron transport capacity and proton leak, in line with the increased oxidative stress, and (v) a distinct metabolic profile, with a deficiency in amino acid catabolism and increased sphingolipid metabolism, which have already been linked to MS. InterpretationTo our knowledge, this is the first study thoroughly describing the metabolic profile of iPSC-derived astrocytes from PwMS, and validating this model as a powerful tool to study disease mechanisms and to perform non-invasive drug targeting assays in vitro. Our findings recapitulate several disease features described in patients and provide new mechanistic insights into the metabolic rewiring of astrocytes in MS, which could be targeted in future therapeutic studies.

neuroscience↗

bioRxiv2021

Calorie Restriction Maintains Mitochondrial Function and Redox Balance Avoiding Lipidomic Reprogramming during Isoproterenol-Induced Cardiac Hypertrophy

Cardiac hypertrophy induces a metabolic shift, leading to a preferential consumption of glucose (over fatty acids) to support the high energetic demand. Typically, health cardiac tissue utilizes more fat than any other organ. Calorie restriction is a dietary procedure that induces health benefits and lifespan extension in many organisms. Given the beneficial effects of calorie restriction and the metabolic dysregulation seen during cardiac hypertrophy, we hypothesized that calorie restriction prevents cardiac hypertrophy, lipid, mitochondrial, and redox dysregulations. Strikingly, calorie restriction reversed isoproterenol-induced cardiac hypertrophy, lowered succinate driven mitochondrial H2O2 production, improved mitochondrial function (indicated as a higher Respiratory Control Ratio - RCR) and avoided mitochondrial superoxide dismutase (MnSOD) and glutathione peroxidase (GPX) repression. To gain insight into how calorie restriction could interfere with the metabolic changes induced by cardiac hypertrophy, we performed lipidomic profiling. Calorie restriction protected against the consumption of several triglycerides (TG) linked to unsaturated fatty acids, and the accumulation of TGs containing saturated fatty acids observed in hypertrophic samples. Cardiac hypertrophy induced an increase in ceramides, phosphoethanolamines and acylcarnitines (12:0, 14:0, 16:0 and 18:0) that were also reversed by calorie restriction. Altogether, our data demonstrate that hypertrophy changes the cardiac lipidome, causes mitochondrial disturbances and oxidative stress. All these changes are prevented by calorie restriction intervention in vivo. This study uncovers calorie restriction as a resource protect cardiac tissue and prevent cardiac hypertrophy-induced lipidomic remodeling.

biochemistry↗

bioRxiv2020

Increased glycolysis is an early outcome of palmitate-mediated lipotoxicity

Palmitic acid is the most abundant saturated fatty acid in human serum. In cell culture systems, palmitate overload is considered a toxic stimulus, and promotes lipid accumulation, insulin resistance, endoplasmic reticulum stress, oxidative stress, as well as cell death. An increased supply of fatty acids has also been shown to change the predominant form of the mitochondrial network, although the metabolic effects of this change are still unclear. Here, we aimed to uncover the early bioenergetic outcomes of lipotoxicity. We incubated hepatic PLC/PRF/5 cells with palmitate conjugated to BSA and followed real-time oxygen consumption and extracellular acidification for 6 hours. Palmitate increased glycolysis as soon as 1 hour after the stimulus, while oxygen consumption was not disturbed, despite overt mitochondrial fragmentation and cellular reductive imbalance. Palmitate only induced mitochondrial fragmentation if glucose and glutamine were available, while glycolytic enhancement did not require glutamine, showing it is not dependent on morphological changes. NAD(P)H levels were significantly abrogated in palmitate-treated cells. Knockdown of the mitochondrial NAD(P) transhydrogenase or addition of the mitochondrial oxidant-generator menadione in control cells modulated ATP production from glycolysis. Indeed, using selective inhibitors, we found that the production of superoxide/hydrogen peroxide at the IQ site of electron transport chain complex I is associated with the metabolic rewiring promoted by palmitate, while not changing mitochondrial oxygen consumption. In conclusion, we demonstrate that increased glycolytic flux linked to mitochondrially-generated redox imbalance is an early bioenergetic result of palmitate overload and lipotoxicity.

biochemistry↗

bioRxiv2020

Functional Changes Induced by Short Term Caloric Restriction in Cardiac and Skeletal Muscle Mitochondria

Caloric restriction (CR) is widely known to increase life span and resistance against different types of injuries in several organisms. We have previously shown that mitochondria from livers or brains of CR animals exhibit higher calcium uptake rates and lower sensitivity to calcium-induced mitochondrial permeability transition (mPT), an event related to the resilient phenotype exhibited by these organs. Given the importance of calcium in metabolic control and cell homeostasis, we aimed here to uncover possible changes in mitochondrial calcium handling, redox balance and bioenergetics in cardiac and skeletal muscle mitochondria. Unexpectedly, we found that CR does not alter the susceptibility to mPT in muscle (cardiac or skeletal), nor calcium uptake rates. Despite the lack in changes in calcium transport properties, CR consistently decreased respiration in the presence of ATP synthesis in heart and soleus muscle. In heart, such changes were accompanied by a decrease in respiration in the absence of ATP synthesis, lower maximal respiratory rates and a reduced rate of hydrogen peroxide release. Hydrogen peroxide release was unaltered by CR in skeletal muscle. No changes were observed in inner membrane potentials and respiratory control ratios. Together, these results highlight the tissue-specific bioenergetic and ion transport effects induced by CR, demonstrating that resilience against calcium-induced mPT is not present in all tissues.

biochemistry↗

bioRxiv2019

Adult Muscle Stem Cell Self-Renewal Induced by Endurance Exercise is Mediated by Inhibition of Mitochondrial Oxygen Consumption

Skeletal muscle stem cells (satellite cells) are well known to participate in regeneration and maintenance of the tissue over time. Studies have shown increases in the number of satellite cells after exercise, but their functional role in endurance training remains unexplored. Here, we found that injured muscles from endurance-exercised mice display improved regenerative capacity, demonstrated through higher densities of newly formed myofibers compared to controls, as well as lower inflammation and fibrosis. Enhanced myogenic function was accompanied by an increased fraction of satellite cells expressing self-renewal markers. Control satellite cells had morphologies suggestive of early differentiation, while endurance exercise enhanced myogenic colony formation. The beneficial effects of endurance exercise were associated with satellite cell metabolic reprogramming, including reduced mitochondrial respiration (O2 consumption) under resting conditions (absence of muscle injury) and increased stemness. During proliferation or activated states (three days after injury), O2 consumption was equal in control and exercised cells. Surprisingly, inhibition of mitochondrial O2 consumption was sufficient to enhance muscle stem cell self-renewal characteristics in vitro. Moreover, transplanted muscle satellite cells from exercised mice or cells with reduced mitochondrial respiration promoted a significant reduction in inflammation compared to controls. We propose that endurance exercise promotes self-renewal and inhibits differentiation in satellite cells, an effect promoted by metabolic reprogramming and respiratory inhibition, and which is associated with a more favorable muscular response to injury.

biochemistry↗

bioRxiv2019

Mitochondrial Morphology Regulates Organellar Ca2+ Uptake and Changes Cellular Ca2+ Homeostasis

Changes in mitochondrial size and shape have been implicated in several physiological processes, but their role in mitochondrial Ca2+ uptake regulation and overall cellular Ca2+ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics towards increased fusion through expression of a dominant negative form of the fission protein DRP1 (DRP1-DN) markedly increased both mitochondrial Ca2+ retention capacity and Ca2+ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion-promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin 2 (MFN2) strongly reduced mitochondrial Ca2+ uptake speed and capacity in these cells. These changes were not dependent on modifications in inner membrane potentials or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as measured by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium-replenishing process of store-operated calcium entry (SOCE) was impaired in MFN2 knockdown cells, while DRP1-DN-promoted fusion resulted in faster cytosolic Ca2+ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca2+ uptake, which impacts on cellular Ca2+ homeostasis.

cell biology↗