Mitochondrial Metabolic Function Research Articles

Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility

Even in stallions with sperm quality within normal reference ranges at ejaculation, subtle differences in sperm quality exist that in many cases lead to reduced time frames for conservation of the ejaculate and/or reduced fertility. The spermatozoon is a cell highly suitable for proteomics studies, and the use of this technique is allowing rapid advances in the understanding of sperm biology. The aim of the present study was to investigate differences among stallions of variable sperm quality (based on motility and sperm velocities), although all horses had sperm characteristics within normal ranges. The proteome was studied using UHPLC/MS/MS and posterior bioinformatic and enrichment analysis; data are available via ProteomeXchange with identifier PXD025807. Sperm motility, linear motility and circular, straight line and average velocities (VCL, VSL, VAP) were measured using computer assisted sperm analysis (CASA). In stallions showing better percentages of motility, circular and average velocity predominated mitochondrial proteins with roles in the Citric acid cycle, pyruvate metabolism and oxidative phosphorylation. Interestingly, in stallions with better percentages of total motility, sperm proteins were also enriched in proteins within the gene ontology (G0) terms, single fertilization (G0: 0007338), fertilization (G0: 0009566), and zona pellucida receptor complex (GO:0002199). The enrichment of this proteins in samples with better percentages of total motility may offer a molecular explanation for the link between this parameter and fertility. SignificanceProteomic analysis identified a high degree of specificity of stallion sperm proteins with discriminant power for motility, linear motility, and sperm velocities (VCL, VAP and VSL). These findings may represent an interesting outcome in relation to the molecular biology regulating the movement of the spermatozoa, and the biological meaning of the measurements that computer assisted sperm analysis (CASA) provide. Of a total of 903 proteins identified in stallion spermatozoa, 24 were related to the percentage of total motility in the sample; interestingly, gene ontology (G0) analysis revealed that these proteins were enriched in terms like single fertilization and fertilization, providing a molecular link between motility and fertility. Field studies indicate that the percentage of total motility is the CASA derived parameter with the best correlation with fertility in stallions.

Metabolic Defects of Peripheral T Cells in COVID-19 Patients.

The relatively low partial pressure of oxygen, reduced oxygen saturation, and aberrant plasma metabolites in COVID-19 may alter energy metabolism in peripheral immune cells. However, little is known regarding the immunometabolic defects of T cells in COVID-19 patients, which may contribute to the deregulated immune functions of these cells. In this study, we longitudinally characterized the metabolic profiles of resting and activated T cells from acutely infected and convalescent COVID-19 patients by flow cytometry and confirmed the metabolic profiles with a Seahorse analyzer. Non-COVID-19 and healthy subjects were enrolled as controls. We found that ex vivo T cells from acutely infected COVID-19 patients were highly activated and apoptotic and displayed more extensive mitochondrial metabolic dysfunction, especially cells in CD8+ T cell lineages, than those from convalescent COVID-19 patients or healthy controls, but slightly disturbed mitochondrial metabolic activity was observed in non-COVID-19 patients. Importantly, plasma IL-6 and C-reactive protein (CRP) levels positively correlated with mitochondrial mass and negatively correlated with fatty acid uptake in T cells from COVID-19 patients. Additionally, compared with those from healthy controls, in vitro-activated T cells from acutely infected COVID-19 patients showed signs of lower glycolysis, a reduced glycolytic capacity, and a decreased glycolytic reserve, accompanied by lower activation of mTOR signaling. Thus, newly identified defects in T cell mitochondrial metabolic functions and metabolic reprogramming upon activation might contribute to immune deficiency in COVID-19.

Mesenchymal stromal cell mitochondrial transfer to human induced T-regulatory cells mediates FOXP3 stability

The key obstacle to clinical application of human inducible regulatory T cells (iTreg) as an adoptive cell therapy in autoimmune disorders is loss of FOXP3 expression in an inflammatory milieu. Here we report human iTreg co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) during short-term ex vivo expansion enhances the stability of iTreg FOXP3 expression and suppressive function in vitro and in vivo, and further that a key mechanism of action is MSC mitochondrial (mt) transfer via tunneling nanotubules (TNT). MSC mt transfer is driven by mitochondrial metabolic function (CD39/CD73 signaling) in proliferating iTreg and promotes iTreg expression of FOXP3 stabilizing factors BACH2 and SENP3. These results elucidate cellular and molecular mechanisms underlying human MSC mt transfer to proliferating cells. MSC mt transfer stabilizes FOXP3 expression in iTregs, thereby enhancing and sustaining their suppressive function in inflammatory conditions in vitro and in vivo.

Metabolic Reprogramming of the Retinal Pigment Epithelium Drives TGFβ2‐Induced Epithelial‐Mesenchymal Transition

Transforming growth factor-beta 2 (TGFβ2) is a key orchestrator of retinal wound healing through induction of epithelial-mesenchymal-transition (EMT) in retinal pigment epithelial cells (RPE). Here we describe a previously unrecognized function of TGFβ2 in modulating mitochondrial morphology and metabolic function in human RPE cells. Treating ARPE-19 (human RPE cell line) with TGFβ2 (10 ng/ml) induced defects in mitochondrial network integrity with increased sphericity and fragmentation. Correspondingly, TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial oxidative phosphorylation (OXPHOS) levels consistent with reduced expression of NDUFB5, a key gene of Complex I of the electron transport chain. The reduced mitochondrial respiration was associated with a compensatory increase in gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA) and glycolytic reserve. TGFβ2 induced a severe suppression of PGC-1α gene expression and treatment with the selective small molecule activator of PGC-1α, ZLN005, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a profound metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. The PGC-1α promoter, ZLN005, effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment for subretinal fibrosis.

Investigation of Mitochondrial Adaptations to Modulation of Carbohydrate Supply during Adipogenesis of 3T3-L1 Cells by Targeted 1H-NMR Spectroscopy.

(1) Background: White adipose tissue (WAT) is a dynamic and plastic tissue showing high sensitivity to carbohydrate supply. In such a context, the WAT may accordingly modulate its mitochondrial metabolic activity. We previously demonstrated that a partial replacement of glucose by galactose in a culture medium of 3T3-L1 cells leads to a poorer adipogenic yield and improved global mitochondrial health. In the present study, we investigate key mitochondrial metabolic actors reflecting mitochondrial adaptation in response to different carbohydrate supplies. (2) Methods: The metabolome of 3T3-L1 cells was investigated during the differentiation process using different glucose/galactose ratios and by a targeted approach using 1H-NMR (Proton nuclear magnetic resonance) spectroscopy; (3) Results: Our findings indicate a reduction of adipogenic and metabolic overload markers under the low glucose/galactose condition. In addition, a remodeling of the mitochondrial function triggers the secretion of metabolites with signaling and systemic energetical homeostasis functions. Finally, this study also sheds light on a new way to consider the mitochondrial metabolic function by considering noncarbohydrates related pathways reflecting both healthier cellular and mitochondrial adaptation mechanisms; (4) Conclusions: Different carbohydrates supplies induce deep mitochondrial metabolic and function adaptations leading to overall adipocytes function and profile remodeling during the adipogenesis.

POS-075 Decreased IFT88 causes cilia shortening and mitochondrial dysfunction in cisplatin-induced tubular injury

Renal primary cilia are associated with the pathogenesis of various diseases, including acute kidney injury (AKI). The length of the primary cilia dynamically change during the progression of diseases: tubular primary cilia shortened in cisplatin-induced AKI mouse model. However, its relevance in kidney disease and the underlying mechanism are largely unknown. Tubular damage in AKI closely links to mitochondrial dysfunction. Thus, we investigated the interaction between primary cilia and mitochondria in cisplatin-induced tubular injury. C57BL6 mice with cisplatin-induced AKI were euthanized at 72 h after disease induction to collect blood and kidney samples. In in vitro experiments, we used RPTEC/TERT1 cells, which are human proximal tubular epithelial cells that maintain the cilia length at high cell densities, and knocked down IFT88 (IFT88-KD), a cilia maintenance protein, by siRNA. Cisplatin-treated or IFT88-KD cells were assessed for the cellular phenotypic changes or mitochondrial metabolic function using the Flux Analyzer. We found that the expression of protein IFT88 was decreased in damaged tubules of cisplatin-induced AKI mice. These data were consistent with that IFT88 expression decreased in the cilia of cisplatin-treated RPTEC/TERT1 at mRNA and protein levels in association with shortening of the primary cilia, suggesting the pathogenic link between tubular damage and IFT88-mediated cilia alteration. Interestingly, IFT88-KD cells significantly exhibited shorter cilia as compared to control siRNA-transfected cells and showed downregulation of mitochondrial oxidative phosphorylation capacity and decreased ATP production, indicating the contribution of IFT88 to mitochondrial homeostasis. In addition, the knockdown of IFT88 decreases fatty acid oxidation (FAO) in the same direction as cisplatin treatment. Our findings suggest that tubular mitochondrial dysfunction in cisplatin-induced AKI is mediated by a decreased IFT88 with shortening cilia, at least in part. Tubular mitochondrial damage followed by tubular injury in AKI may occur by the alteration of IFT88 expression, and subsequent cilia shortening in the tubular epithelial cells.

Enhanced PRL-1 expression in placenta-derived mesenchymal stem cells accelerates hepatic function via mitochondrial dynamics in a cirrhotic rat model

BackgroundPlacenta-derived mesenchymal stem cells (PD-MSCs) have been highlighted as an alternative cell therapy agent that has become a next-generation stem cell treatment. Phosphatase of regenerating liver-1 (PRL-1), an immediate early gene, plays a critical role during liver regeneration. Here, we generated enhanced PRL-1 in PD-MSCs (PD-MSCsPRL-1, PRL-1+) using lentiviral and nonviral gene delivery systems and investigated mitochondrial functions by PD-MSCPRL-1 transplantation for hepatic functions in a rat bile duct ligation (BDL) model.MethodsPD-MSCsPRL-1 were generated by lentiviral and nonviral AMAXA gene delivery systems and analyzed for their characteristics and mitochondrial metabolic functions. Liver cirrhosis was induced in Sprague-Dawley (SD) rats using common BDL for 10 days. PKH67+ naïve and PD-MSCsPRL-1 using a nonviral sysyem (2 × 106 cells/animal) were intravenously administered into cirrhotic rats. The animals were sacrificed at 1, 2, 3, and 5 weeks after transplantation and engraftment of stem cells, and histopathological analysis and hepatic mitochondrial functions were performed.ResultsPD-MSCsPRL-1 were successfully generated using lentiviral and nonviral AMAXA systems and maintained characteristics similar to those of naïve cells. Compared with naïve cells, PD-MSCsPRL-1 improved respirational metabolic states of mitochondria. In particular, mitochondria in PD-MSCsPRL-1 generated by the nonviral AMAXA system showed a significant increase in the respirational metabolic state, including ATP production and mitochondrial biogenesis (*p < 0.05). Furthermore, transplantation of PD-MSCsPRL-1 using a nonviral AMAXA system promoted engraftment into injured target liver tissues of a rat BDL cirrhotic model and enhanced the metabolism of mitochondria via increased mtDNA and ATP production, thereby improving therapeutic efficacy.ConclusionsOur findings will further our understanding of the therapeutic mechanism of enhanced MSCs and provide useful data for the development of next-generation MSC-based cell therapy and therapeutic strategies for regenerative medicine in liver disease.

Mitochondrial Fission Factor (MFF) Inhibits Mitochondrial Metabolism and Reduces Breast Cancer Stem Cell (CSC) Activity.

Elevated mitochondrial biogenesis and metabolism represent key features of breast cancer stem cells (CSCs), whose propagation is conducive to disease onset and progression. Therefore, interfering with mitochondria biology and function may be regarded as a useful approach to eradicate CSCs. Here, we used the breast cancer cell line MCF7 as a model system to interrogate how mitochondrial fission contributes to the development of mitochondrial dysfunction toward the inhibition of metabolic flux and stemness. We generated an isogenic MCF7 cell line transduced with Mitochondrial Fission Factor (MCF7-MFF), which is primarily involved in mitochondrial fission. We evaluated the biochemical, molecular and functional properties of MCF7-MFF cells, as compared to control MCF7 cells transduced with the empty vector (MCF7-Control). We observed that MFF over-expression reduces both mitochondrial mass and activity, as evaluated using the mitochondrial probes MitroTracker Red and MitoTracker Orange, respectively. The analysis of metabolic flux using the Seahorse XFe96 revealed the inhibition of OXPHOS and glycolysis in MCF7-MFF cells, suggesting that increased mitochondrial fission may impair the biochemical properties of these organelles. Notably, CSCs activity, assessed by 3D-tumorsphere assays, was reduced in MCF7-MFF cells. A similar trend was observed for the activity of ALDH, a well-established marker of stemness. We conclude that enhanced mitochondrial fission may compromise CSCs propagation, through the impairment of mitochondrial function, possibly leading to a quiescent cell phenotype. Unbiased proteomic analysis revealed that proteins involved in mitochondrial dysfunction, oxidative stress-response, fatty acid metabolism and hypoxia signaling are among the most highly up-regulated in MCF7-MFF cells. Of note, integrated analysis of top regulatory networks obtained from unbiased proteomics in MCF7-MFF cells predicts that this cell phenotype activates signaling systems and effectors involved in the inhibition of cell survival and adhesion, together with the activation of specific breast cancer cell death programs. Overall, our study shows that unbalanced and abnormal activation of mitochondrial fission may drive the impairment of mitochondrial metabolic function, leading to inhibition of CSC propagation, and the activation of quiescence programs. Exploiting the potential of mitochondria to control pivotal events in tumor biology may, therefore, represent a useful tool to prevent disease progression.

Cytoprotection by a naturally occurring variant of ATP5G1 in Arctic ground squirrel neural progenitor cells.

Many organisms in nature have evolved mechanisms to tolerate severe hypoxia or ischemia, including the hibernation-capable Arctic ground squirrel (AGS). Although hypoxic or ischemia tolerance in AGS involves physiological adaptations, little is known about the critical cellular mechanisms underlying intrinsic AGS cell resilience to metabolic stress. Through cell survival-based cDNA expression screens in neural progenitor cells, we identify a genetic variant of AGS Atp5g1 that confers cell resilience to metabolic stress. Atp5g1 encodes a subunit of the mitochondrial ATP synthase. Ectopic expression in mouse cells and CRISPR/Cas9 base editing of endogenous AGS loci revealed causal roles of one AGS-specific amino acid substitution in mediating cytoprotection by AGS ATP5G1. AGS ATP5G1 promotes metabolic stress resilience by modulating mitochondrial morphological change and metabolic functions. Our results identify a naturally occurring variant of ATP5G1 from a mammalian hibernator that critically contributes to intrinsic cytoprotection against metabolic stress.

Effects of Fluorescent Diamond Particles FDP-NV-800nm on Essential Biochemical Functions of Primary Human Umbilical Vein Cells and Human Hepatic Cell Line, HepG-2 in vitro (Part VI): Acute Biocompatibility Studies.

BackgroundRecently, we reported the safety and biocompatibility of fluorescent diamond particles, FDP-NV-Z-800nm (FDP-NV) injected intravenously into rats, where no morbidity and mortality were noted over a period of 3 months. The acute effects of FDP-NV-800nm particles on cultured human endothelial and hepatic cells remain unexplored.PurposeIn this study, we aimed to explore select cellular and biochemical functions in cultured human umbilical endothelial cells (HUVEC) and a human hepatic cancer cell line (HepG-2) exposed to FDP-NV-800 in vitro at exposure levels within the pharmacokinetics (Cmax and the nadir) previously reported in vivo.MethodsDiverse cellular and biochemical functions were monitored, which cumulatively can provide insights into some vital cellular functions. Cell proliferation and migration were assessed by quantitative microscopy. Mitochondrial metabolic functions were tested by the MTT assay, and cytosolic esterase activity was studied by the calcein AM assay. Chaperons (CHOP), BiP and apoptosis (caspase-3 activation) were monitored by using Western blot (WB). MAPK Erk1/2 signaling was assessed by the detection of the phosphorylated form of the protein (P-Erk 1/2) and its translocation into the cell nucleus.ResultsAt all concentrations tested (0.001–0.1mg/mL), FDP-NV did not affect any of the biomarkers of cell integrity of HepG2 cells. In contrast, the proliferation of HUVEC was affected at the highest concentration tested (0.1mg/mL, Cmax). Exposure of HUVEC to (0.01 mg/mL) FDP-NV had a mild-moderate effect on cell proliferation as evident in the MTT assay and was absent when proliferation was assessed by direct cell counting or by using the calcein AM assays. In both cell types, exposure to the highest concentration (0.1 mg/mL) of FDP-NV did neither affect FBS-stimulated cell signaling (MAPK Erk1/2 phosphorylation) nor did it activate of Caspase 3.ConclusionOur data suggest that FDP-NV-800nm are largely biocompatible with HepG-2 cells proliferation within the pharmacokinetic data reported previously. In contrast, HUVEC proliferation at the highest exposure dose (0.1 mg/mL) responded adversely with respect to several biomarkers of cell integrity. However, since the Cmax levels are very short-living, the risk for endothelial injury is likely minimal for slow rate cell proliferation such as endothelial cells.

Autofagia e Câncer: uma revisão da literatura

Introdução: A autofagia, ou macroautofagia, é um fenômeno de degradação e reciclagem celular que ocorre constitutivamente em baixos níveis nas células eucarióticas. Tem ganhado destaque como via de mobilização de substratos metabólicos que permitem células cancerosas sobreviverem a microambientes com baixa oferta de nutrientes ou em condições de estresse oxidativo, onde o papel de proteínas como o Beclin-1 e LC3B tem sido amplamente pesquisado em diversos tipos de câncer. Objetivo: O objetivo do presente estudo foi fazer uma revisão de literatura acerca da atuação do processo de autofagia e sua relação com o desenvolvimento de câncer através da autofagia tumoral. Metodologia: Para concepção deste artigo foi realizado um levantamento bibliográfico que abrangeu o período de 1998 a 2019. Foram utilizados os portais de pesquisa Google Acadêmico, Scielo, LIlacs e Medline. Resultados e Conclusão: Nesta revisão tem como resultado que a autofagia tem papel dual no câncer pois pode prevenir a iniciação tumoral através da supressão de danos crônicos, inflamação, acúmulo de organelas danificadas e instabilidade genômica, porém a autofagia também mantém as funções metabólicas mitocondriais que fornece nutrientes para o crescimento das células tumorais, criando canceres agressivos.

Abstract B19: Neurofibromin 2 regulates metabolism in the heart

Abstract Introduction: Neurofibromin 2 (NF2) is a tumor suppressor that can engage the Hippo signaling pathway and modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and injury caused by acute myocardial infarction (MI) through inhibition of the Hippo target YAP. However, the function of NF2 in the heart remains largely uncharacterized. Our current study sought to determine whether NF2 contributes to cardiac remodeling, dysfunction, and heart failure due to chronic stress. Methods and Results: We used a transverse aortic constriction (TAC) model in WT C57BL/6J mice to generate chronic pressure overload (PO) stress, which elicits cardiac hypertrophy, remodeling, and eventually heart failure. We found that NF2 is transiently upregulated in mouse myocardium in response to PO stress. To investigate if increased NF2 contributes to pathology, we used cardiomyocyte-specific NF2 knockout (cKO) mice. At baseline, adult NF2 cKO mice had normal cardiac morphology and function. However, following TAC, NF2 cKO hearts unexpectedly showed worsened cardiac function, assessed by echocardiography and hemodynamic analysis, compared to littermate controls. Interestingly, no differences in the extent of TAC-induced cardiac hypertrophy or cardiomyocyte apoptosis were observed between NF2 cKO and control mice. To investigate the mechanism underlying the NF2 cKO phenotype, we performed RNAseq. Our analysis indicated downregulation of several metabolic pathways including oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) in unstressed NF2 cKO hearts, which was confirmed by qPCR. We also determined that ATP content in NF2 cKO hearts was significantly reduced at baseline compared to control mice. Estrogen-related receptors (ERRs) are important regulators of mitochondrial biogenesis and metabolic function. RNAseq revealed the downregulation of ERRβ and ERRγ in NF2 cKO hearts, which was confirmed by qPCR. Experiments in neonatal cardiomyocytes confirmed that regulation of ERRβ and ERRγ expression by NF2 is cell autonomous. Luciferase reporter assays demonstrated that modulation of NF2 regulates ERR function in cardiomyocytes. We also found that YAP activation is enhanced in NF2 cKO hearts and cardiomyocytes deficient for NF2. Co-IP analysis indicated that YAP associates with components of the nucleosome remodeling and deacetylase (NuRD) complex in cardiomyocytes, which may mediate targeted gene suppression. Conclusion: Myocardial NF2 expression is upregulated and may be compensatory during PO stress through preservation of metabolic gene expression and energy content in the heart. Citation Format: Yu Zhang, Wataru Mizushima, Shinichi Oka, Peiyong Zhai, Dominic Del Re. Neurofibromin 2 regulates metabolism in the heart [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B19.

Protective effect of Argan oil on mitochondrial function and oxidative stress against acrylamide-induced liver and kidney injury in rats

Context Acrylamide (ACR) is now a risk for general public health. Argan oil (AO) is harvested from the fruits of Argania spinosa and its rich source of antioxidant and phenolic compounds. Objective The aim of present study was to investigate the protective effect of AO against ACR-induced liver and kidney injury in rats. Materials and methods Rats were exposed to ACR (50 mg/kg/day three times per week), AO (6 ml/kg/day per day) and ACR together with AO for 30 days. Oxidative status and mitochondrial functions were evaluated in liver and kidney. Results Although ALT, AST, urea and creatine levels in serum, myeloperoxidase and total nitrite (NOx) levels in the tissues, lipid peroxidation and protein carbonyls levels were increased in the ACR-treated rats, cytosolic glucose-6-phosphate dehydrogenase and glutathione-S-transferase activities, mitochondrial antioxidant enzyme activities, glutathione levels, oxidative phosphorylation enzymes, TCA cycle enzymes, mitochondrial metabolic function and ATP level were decreased. The administration of ACR together with AO normalised almost all these parameters. Conclusion Over recent years, compounds that specifically target mitochondria have emerged as promising therapeutic options for patients with hepatic and renal diseases. We think that AO oil is one of these compounds due to its unique content.

S100A6 is a critical regulator of hematopoietic stem cells

The fate options of hematopoietic stem cells (HSCs) include self-renewal, differentiation, migration, and apoptosis. HSCs self-renewal divisions in stem cells are required for rapid regeneration during tissue damage and stress, but how precisely intracellular calcium signals are regulated to maintain fate options in normal hematopoiesis is unclear. S100A6 knockout (KO) HSCs have reduced total cell numbers in the HSC compartment, decreased myeloid output, and increased apoptotic HSC numbers in steady state. S100A6KO HSCs had impaired self-renewal and regenerative capacity, not responding to 5-Fluorouracil. Our transcriptomic and proteomic profiling suggested that S100A6 is a critical HSC regulator. Intriguingly, S100A6KO HSCs showed decreased levels of phosphorylated Akt (p-Akt) and Hsp90, with an impairment of mitochondrial respiratory capacity and a reduction of mitochondrial calcium levels. We showed that S100A6 regulates intracellular and mitochondria calcium buffering of HSC upon cytokine stimulation and have demonstrated that Akt activator SC79 reverts the levels of intracellular and mitochondrial calcium in HSC. Hematopoietic colony-forming activity and the Hsp90 activity of S100A6KO are restored through activation of the Akt pathway. We show that p-Akt is the prime downstream mechanism of S100A6 in the regulation of HSC self-renewal by specifically governing mitochondrial metabolic function and Hsp90 protein quality.

Myoblast Mitochondrial Adaptation to Chronic Alcohol Administration

Skeletal muscle adaptation to chronic alcohol consumption involves disruption of anabolic, catabolic, and oxidative metabolism pathways and is associated with decreased muscle mass and impaired metabolic function. These maladaptive responses may contribute to increased risk of metabolic disorders, particularly in people living with human immunodeficiency virus (PLWH) on combination antiretroviral therapy (ART). Aging, increased survival of PLWH on ART, and lifestyle choices including unhealthy alcohol use are complicated by metabolic comorbidities. We tested the hypothesis that mitochondrial homeostasis is critically impacted by chronic binge alcohol (CBA) administration. Specifically, we hypothesized that CBA impairs skeletal muscle expression of genes underlying mitochondrial metabolic function and alters myoblast function in SIV‐infected, ART‐treated rhesus macaques. Female rhesus macaques (n=10) were assigned to receive either CBA (50–60 mM peak blood alcohol/day, 5 days/week) or isovolumetric water (VEH) for 3 months before SIVmac251 infection. After 2.5 months, ART was initiated, and skeletal muscle samples were collected from all animals at study endpoint (~12 months post‐SIV infection). mRNA was extracted and analyzed for genes related to mitochondrial biogenesis and metabolic function [peroxisome proliferator‐activated receptor‐gamma coactivator (PGC)‐1α, PGC‐1β, mitochondrial transcription factor A (TFAM), estrogen related receptor (ERR)α, nuclear respiratory factor (NRF)1 and 2]. Naïve (no alcohol or SIV) primary myoblasts were isolated from skeletal muscle and incubated with alcohol (50 mM) or control (media only) for three days. To assess mitochondrial metabolic function, oxygen consumption rate was determined at baseline in alcohol‐treated and control myoblasts after the addition of oligomycin, carbonyl cyanide‐4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone and antimycin A using Agilent Seahorse technology. CBA reduced expression of PGC‐1β, TFAM, ERRα, NRF1, and NRF2, but not PGC‐1α mRNA in skeletal muscle from SIV‐infected, ART‐treated female rhesus macaques. In vitro, alcohol treatment reduced basal and maximal oxygen consumption rate of naïve myoblasts. Together, our findings demonstrate maladaptive changes in myoblast mitochondrial gene expression and function. These results identified potential therapeutic targets to improve skeletal muscle metabolic health in PLWH. Specifically, the role of PGC‐1β as a potential mechanism by which alcohol impairs skeletal muscle mitochondrial function warrants further investigation on its responsiveness to pharmacotherapeutic intervention to restore its levels and improve mitochondrial metabolic health in PLWH with unhealthy alcohol use.Support or Funding InformationThis research was supported by grants from the National Institute on Alcohol Abuse and Alcoholism, 1F32AA027982‐01A1, 5P60AA009803‐25, K01AA024494‐03, and 5T32AA007577‐20.

Ca2+‐induced Complex I Inactivity: A Model for Early Mitochondrial Dysfunction Following Volumetric Muscle Loss Injury

Calcium (Ca2+) is a secondary messenger, responsible for the regulation of a plethora of metabolic pathways that must be tightly regulated to avoid cytotoxicity 1. During early stage volumetric muscle loss (VML) injuries, damaged muscle fibers are open to the extracellular environment until the myofiber can be sealed, a process that may take hours 2– 3. This allows for free Ca2+ exchange between intra‐ and extracellular environments to concentrations beyond those appropriate for cell homeostasis. The purpose of this study was to model the changes in mitochondrial metabolism of early VML injuries as a response to a drastic increase of intracellular Ca2+.C57BL6 mice underwent unilateral surgery to create a VML defect in the gastrocnemius muscle, as approved by the University of Georgia’s Institutional Animal Care and Use Committee. Animals were sacrificed via CO2 inhalation, 18 hrs. following surgery. Total [Ca2+] per kg of tissue ([Ca2+]TWM) was measured using a BAPTA complexation assay. Gastrocnemius muscles from naïve mice were dissected into individual fiber bundles, permeabilized, and washed in either normal isotonic or Ca2+‐supplemented medium. Mitochondrial metabolic function and mitochondrial membrane potential were measured through oxygen consumption and Safranin‐O fluorescence rates, respectively, in a High‐Resolution Fluoro‐respirometer.[Ca2+]TWM was elevated in VML‐injured tissue, as expected (n=3, p&lt; 0.05). Acute Ca2+ exposure negatively affected Complex I (C‐I) activity of the electron transport chain. Specifically, C‐I activity was targeted by the addition of glutamate and malate (Glu‐Mal) and confirmed by the sequential addition of C‐I inhibitor Rotenone (Rot). In normal fibers, respiration increased after Glu‐Mal addition and subsequently decreased significantly following Rot, while Ca2+‐incubated fibers exhibited no change in oxygen consumption rate. Succinate (Succ) was used to rescue respiratory function by bypassing C‐I. Succ addition significantly increased oxygen consumption in both normal and Ca2+‐incubated fibers (n=3, p&lt;0.01), confirming a C‐I dysfunction in Ca2+‐treated fibers. Safranin fluorescence is sensitive to changes in mitochondrial membrane potential, decreasing with complex activity and increasing upon inhibition. In normal fibers, Safranin signal decreased with Glu‐Mal and Succ additions and increased with Rot, as expected. However, Ca2+‐treated fibers were only sensitive to Succ treatment, further supporting C‐I dysfunction (n=3, p&lt; 0.01).Herein, we show that an acute increase in intracellular Ca2+ negatively affects the process of oxidative phosphorylation within the mitochondria by disturbing the electron potential and inactivating Complex I. This alteration in membrane potential and decrease in complex activity may be the first step in a systematic shut down of mitochondrial metabolic function that leaves the muscle remaining after injury beyond the hope of effective repair.Support or Funding InformationFunded through the Department of Defense Neuromusculoskeletal Injuries Rehabilitation Research Award W81XWH‐18‐1‐0710 to SMG (UMinn) and JAC (UGA)A‐ Quantification of internal [Ca2+] of VML‐injured and control gastrocnemius muscles using BAPTA absorbance (n=3, p &lt; 0.05). B‐ Oxygen consumption rates of control and Ca2+‐treated muscle fibers after Complex I activation, inhibition, and bypass, respectively (n=3 in duplicate, p &lt; 0.05). C‐ Safranin‐O signal fluctuation in control and Ca2+‐treated muscle fibers after Complex I activation, inhibition, and bypass, respectively (n=3, p&lt; 0.05).Figure 1

Methylmalonic Acid Compromises Energy Metabolism and Decreases the Expression of Neuronal Differentiation Genes in SH‐SY5Y Human Neuroblastoma Cells

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L‐methylmalonyl‐CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and symptoms during metabolic decompensation commonly appear in newborns. Considering that the mechanisms underlying neurological alterations of this disorder are not fully understood, this study aimed to investigate the toxic effects of MMA in a neuroblastoma cell line presenting neuronal features. Undifferentiated and retinoic acid (RA)‐differentiated human neuroblastoma SH‐SY5Y cells were exposed to MMA (1–10mM) for 24 or 48 hours and cell viability, glucose consumption and mitochondrial metabolic function were analyzed. Additionally, the expression of some molecular markers of neuronal differentiation was also quantified after co‐exposition with 10 μM RA and 1 mM of MMA for 3 and 7 days.MMA significantly reduced cell viability after 48‐hour period exposition in undifferentiated and RA differentiated SH‐SY5Y cells. It was also observed an increase in glucose consumption in cells exposed to MMA. Regarding oxygen consumption, there was an increase in respiratory parameters in RA differentiated SH‐SY5Y cells when compared to undifferentiated cells. MMA significantly reduced basal, ATP‐linked and maximal respiration, as well as the reserve respiratory capacity of cells after 48‐hour exposition in both undifferentiated and RA differentiated SH‐SY5Y cells. Regarding the effects of MMA in the neuronal differentiation process, it was observed that co‐exposition to RA and MMA significantly decreased the expression of enolase 2 (ENO2) and synaptophysin (SYP) genes after 7 days of exposition.In summary, MMA cell viability and oxygen consumption in SH‐SY5Y neuronal cells, suggesting that this organic acid compromises cell respiration, at resting and reserve capacity states, which might influence the cell capacity to meet energetic demands. In addition, glucose uptake was increased after exposition to MMA, corroborating that aerobic metabolism is compromised. The expression of mature neuronal genes was also reduced, suggesting that MMA possibly compromises neuronal differentiation, which can be involved in symptoms presented by affected patients during brain development. Finally, our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease.Support or Funding InformationThis study was supported by grants from FAPESP (2015/25541‐0), CAPES and UFABC.

Paradoxical Effect of TNFα on Mitochondrial Function and Metabolic Activity in the Retinal Pigment Epithelium (RPE)

The retinal pigment epithelium (RPE) acts as a metabolic gatekeeper between photoreceptors and the choroidal vasculature to maintain healthy retinal function. RPE dysfunction is a key feature of age‐related macular degeneration (AMD), the leading cause of blindness in developed countries. Tumor necrosis factor‐alpha (TNFα), a potent pro‐inflammatory cytokine, has been implicated in the pathogenesis of AMD. Growing evidence supports metabolic dysfunction as another key mechanism driving AMD. To date, there is no literature on the metabolic effects of TNFα on RPE and thus, this study sheds light on the impact of TNFα on mitochondrial morphology and metabolic function in RPE. Matured ARPE‐19 (human RPE cell line) were treated with TNFα (10 ng/ml) for up to 72h. TNFα induced ARPE‐19 to elongate into spindle‐shaped cells, reminiscent of epithelial‐mesenchymal transition (EMT). However, qPCR showed that TNFα reduced the expression of EMT genes (α‐SMA, Col1A1, fibronectin, MMP2, CTGF) indicating that the elongated cells were not mesenchymal in nature. To explore the effects of TNFα on metabolism, two major energy‐generating pathways, oxidative phosphorylation (OXPHOS) and glycolysis were assessed by Seahorse high‐resolution respirometry and qPCR. The Seahorse Mito Stress Test revealed an increase in basal respiration but reduced spare respiratory capacity following TNFα treatment. TNFα increased glycolytic reserve and capacity. Despite the increased OXPHOS and glycolysis, qPCR showed a significant reduction in expression of OXPHOS (ATP5O, COX4I1, COX5B, NDUFB5) and glycolysis (G6P, PFKL, PFKFB3, PGK1, LDHA) genes with TNFα treatment. ARPE‐19 were transfected with mitochondria‐tagged GFP and imaged using confocal microscopy. Parameters on mitochondrial morphology were extracted from the confocal images using automated image segmentation and revealed that TNFα disrupted mitochondrial network integrity. This was supported by qPCR data showing significantly reduced expression of genes regulating mitochondrial function (PGC‐1α, TFAM, POLG, MFN1, MFN2, FIS1, OPA1). TNFα significantly elevated SOD2, a mitochondrial antioxidant enzyme, but not SOD1. Taken together, we find that TNFα robustly disrupts mitochondrial function and morphology in RPE, although shifting the bioenergetic profile in a paradoxical manner, i.e. TNFα raised the levels of basal respiration and glycolysis despite the suppression of genes regulating OXPHOS and glycolysis. These findings highlight the potential of targeting metabolic pathways in RPE as a promising therapeutic avenue for AMD. Further research is required to elucidate the mechanisms underlying these intriguing TNFα‐driven metabolic changes.Support or Funding InformationGrimshaw‐Gudewicz Charitable Foundation; National Council of Scientific and Technological Development—Brazil (PDE 210474/2014‐9); The Iraty Award; NEI Core Grant P30EYE003790; Department of Defense, Spinal Vision Research Program under Award no. VR180132

ONC201 and Its Potent Analogues Disrupt Mitochondrial Metabolic Function in Triple Negative Breast Cancer

BackgroundONC201 is an imipridone compound in clinical trials for multiple cancer types, including triple negative breast cancer (TNBC). ONC201 is a promising anticancer therapy whose pharmacological target and function are not clearly defined. We have demonstrated that the target of ONC201 and its potent analogues (TR compounds) is the mitochondrial protease ClpP, consistent with previous reports of ONC201‐induced mitochondrial stress and depolarization in TNBC cells.Study ObjectiveThe objective of this study is to determine if ONC201 and TR compound treatment result in a loss of mitochondrial protein through activation of ClpP, leading to metabolic disruption in glutamine utilization and mitochondrial function.MethodsTo determine if ONC201 and TR compounds disrupt aerobic respiration, galactose was substituted for glucose in cell culture media. We used galactose‐containing cell culture media to shift cells from anaerobic to aerobic respiration, as galactose is utilized through aerobic respiration. The IC50 of ONC201 and TR compounds was measured with glucose‐ or galactose‐containing media by MTS. MitoPlate assays (Biolog) were performed to identify perturbations in the utilization of 30 different substrates in TNBC cells treated with ONC201 or TR compounds. Proteomic analysis of ONC201 or TR compound‐treated cells was conducted using LC‐MS.ResultsProteomic analysis revealed a large decline in mitochondrial peptides following ONC201 or TR compound treatment. We also demonstrated that substitution of glucose with galactose reduced the IC50 of ONC201 and TR compounds in TNBC cells. In addition, MitoPlate assays suggest a reduced utilization of TCA cycle substrates and L‐glutamine.ConclusionsAddition of galactose to cell culture media improved efficacy of ONC201 and TR compounds in TNBC cells. Sensitization and loss of mitochondrial peptides suggest that ONC201 and TR compounds cause loss of mitochondrial protein and reduction metabolic activity. Consistent with sensitization results, MitoPlate data demonstrates a loss of TCA cycle substrate and L‐glutamine utilization following ONC201 or TR compound treatment. Glutamine is known to facilitate both cell proliferation and energy production in cancer cells through protein biosynthesis and the TCA cycle. Proteomic analysis is being used to identify ClpP‐dependent protein loss. Uniformly 13C‐labeled substrates will be used to identify perturbations in energetic and biosynthetic pathways following ONC201 and TR compound treatment. Loss of mitochondrial metabolic activity and glutamine utilization may play a role in ONC201 and TR compound anticancer activity.Support or Funding InformationNIH Pharmacological Sciences T32 (GM007040), University Cancer Research Fund

Inflammation, Lipid (Per)oxidation, and Redox Regulation

Significance: Inflammation increases during the aging process. It is linked to mitochondrial dysfunction and increased reactive oxygen species (ROS) production. Mitochondrial macromolecules are critical targets of oxidative damage; they contribute to respiratory uncoupling with increased ROS production, redox stress, and a cycle of senescence, cytokine production, and impaired oxidative phosphorylation. Targeting the formation or accumulation of oxidized biomolecules, particularly oxidized lipids, in immune cells and mitochondria could be beneficial for age-related inflammation and comorbidities. Recent Advances: Inflammation is central to age-related decline in health and exhibits a complex relationship with mitochondrial redox state and metabolic function. Improvements in mass spectrometric methods have led to the identification of families of oxidized phospholipids (OxPLs), cholesterols, and fatty acids that increase during inflammation and which modulate nuclear factor erythroid 2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor gamma (PPARγ), activator protein 1 (AP1), and NF-κB redox-sensitive transcription factor activity. Critical Issues: The kinetic and spatial resolution of the modified lipidome has profound and sometimes opposing effects on inflammation, promoting initiation at high concentration and resolution at low concentration of OxPLs. Future Directions: There is an emerging opportunity to prevent or delay age-related inflammation and vascular comorbidity through a resolving (oxy)lipidome that is dependent on improving mitochondrial quality control and restoring redox homeostasis.