Mitochondrial Substrate Utilization Research Articles

Intercellular Mitochondrial transfer: Therapeutic implications for energy metabolism in heart failure.

Heart failure (HF) remains one of the leading causes of high morbidity and mortality globally. Impaired cardiac energy metabolism plays a critical role in the pathological progression of HF. Various forms of HF exhibit marked differences in energy metabolism, particularly in mitochondrial function and substrate utilization. Recent studies have increasingly highlighted that improving energy metabolism in HF patients as a crucial treatment strategy. Mitochondrial transfer is emerging as a promising and precisely regulated therapeutic strategy for treating metabolic disorders. This paper specifically reviews the characteristics of mitochondrial energy metabolism across different types of HF and explores the modes and mechanisms of mitochondrial transfer between different cell types in the heart, such as cardiomyocytes, fibroblasts, and immune cells. We focused on the therapeutic potential of intercellular mitochondrial transfer in improving energy metabolism disorders in HF. We also discuss the role of signal transduction in mitochondrial transfer, highlighting that mitochondria not only function as energy factories but also play crucial roles in intercellular communication, metabolic regulation, and tissue repair. This study provides new insights into improving energy metabolism in heart failure patients and proposes promising new therapeutic strategies.

Abstract Tu126: Unveiling the Role of GRAF1 in Orchestrating Mitophagy and Metabolic Flexibility in Stressed Cardiomyocytes

Mitochondria are essential for cardiomyocyte contraction by generating ATP and orchestrating metabolic pathways. Understanding how mitophagy—selective removal of damaged mitochondria in lysosome—and metabolic flexibility are coordinated is crucial for cardiac function under physiological and pathological conditions. Our study explores the molecular mechanisms behind this coordination in cardiomyocytes. We identify GRAF1(Arhgap26) as a pivotal mediator in PINK1-Parkin dependent mitophagy, facilitating damaged mitochondria clearance and regulating mitochondrial substrate flexibility. Depletion of GRAF1 in neonatal rat ventricular cardiomyocytes (NRVCMs) compromises mitochondrial function, evidenced by reduced membrane potential, impaired oxidative phosphorylation, and elevated cleaved Caspase 3 levels. Moreover, GRAF1 depletion attenuates LC3II activation and increases mitochondrial mass following toxin-induced stress. Using tamoxifen-inducible cardiomyocyte-restricted GRAF1 knockout mice (GRAF1 CKO ), we observe normal cardiac function under basal conditions. However, upon isoproterenol (ISO) treatment, GRAF1 CKO mice display cardiac dysfunction and reduced mitophagy. Metabolomics analysis reveal distinct metabolic signatures between ISO-treated control and GRAF1 CKO mice, with impaired fuel flexibility observed in GRAF1 CKO mice, as highlighted by impaired glucose utilization relative to control mice. Mechanistically, phosphorylation of specific sites (S668, T670, and S671) within the proline-rich region of GRAF1 by PINK1 or PINK1-dependent kinases regulates its dual functions. This phosphorylation event releases the autoinhibitory state of the SH3 domain, enabling it to interact with ABI2 and WAVE2 complex for actin remodeling necessary for efficient mitochondria clearance. Additionally, phosphorylation enhances GRAF1’s affinity for Malonyl-CoA Decarboxylase (MCD), promoting MCD degradation and subsequent elevation of cellular malonyl-CoA levels. This rise in malonyl-CoA inhibits carnitine pamitoyl transferase-I (CPT-I), thereby suppressing fatty acid oxidation while promoting glucose oxidation in mitochondria, contributing to the mitochondrial metabolic flexibility. In summary, our findings elucidate GRAF1's role in orchestrating mitophagy and metabolic flexibility in stressed cardiomyocytes. GRAF1 phosphorylation serves as a molecular switch, ensuring efficient mitochondrial clearance and substrate utilization to maintain cardiac energetics and function.

A Lacticaseibacillus rhamnosus secretome induces immunoregulatory transcriptional, functional and immunometabolic signatures in human THP-1 monocytes

Macrophage responses to activation are fluid and dynamic in their ability to respond appropriately to challenges, a role integral to host defence. While bacteria can influence macrophage differentiation and polarization into pro-inflammatory and alternatively activated phenotypes through direct interactions, many questions surround indirect communication mechanisms mediated through secretomes derived from gut bacteria, such as lactobacilli. We examined effects of secretome-mediated conditioning on THP-1 human monocytes, focusing on the ability of the Lacticaseibacillus rhamnosus R0011 secretome (LrS) to drive macrophage differentiation and polarization and prime immune responses to subsequent challenge with lipopolysaccharide (LPS). Genome-wide transcriptional profiling revealed increased M2-associated gene transcription in response to LrS conditioning in THP-1 cells. Cytokine and chemokine profiling confirmed these results, indicating increased M2-associated chemokine and cytokine production (IL-1Ra, IL-10). These cells had increased cell-surface marker expression of CD11b, CD86, and CX3CR1, coupled with reduced expression of the M1 macrophage-associated marker CD64. Mitochondrial substrate utilization assays indicated diminished reliance on glycolytic substrates, coupled with increased utilization of citric acid cycle intermediates, characteristics of functional M2 activity. LPS challenge of LrS-conditioned THP-1s revealed heightened responsiveness, indicative of innate immune priming. Resting stage THP-1 macrophages co-conditioned with LrS and retinoic acid also displayed an immunoregulatory phenotype with expression of CD83, CD11c and CD103 and production of regulatory cytokines. Secretome-mediated conditioning of macrophages into an immunoregulatory phenotype is an uncharacterized and potentially important route through which lactic acid bacteria and the gut microbiota may train and shape innate immunity at the gut-mucosal interface.

Smoking primes the metabolomic response in trauma.

Smoking is a public health threat because of its well-described link to increased oxidative stress-related diseases including peripheral vascular disease and coronary artery disease. Tobacco use has been linked to risk of inpatient trauma morbidity including acute respiratory distress syndrome; however, its mechanistic effect on comprehensive metabolic heterogeneity has yet to be examined. Plasma was obtained on arrival from injured patients at a Level 1 trauma center and analyzed with modern mass spectrometry-based metabolomics. Patients were stratified by nonsmoker, passive smoker, and active smoker by lower, interquartile, and upper quartile ranges of cotinine intensity peaks. Patients were substratified by high injury/high shock (Injury Severity Score, ≥15; base excess, <-6) and compared with healthy controls. p Value of <0.05 following false discovery rate correction of t test was considered significant. Forty-eight patients with high injury/high shock (7 nonsmokers [15%], 25 passive smokers [52%], and 16 active smokers [33%]) and 95 healthy patients who served as controls (30 nonsmokers [32%], 43 passive smokers [45%], and 22 active smokers [23%]) were included. Elevated metabolites in our controls who were active smokers include enrichment in chronic inflammatory and oxidative processes. Elevated metabolites in active smokers in high injury/high shock include enrichment in the malate-aspartate shuttle, tyrosine metabolism, carnitine synthesis, and oxidation of very long-chain fatty acids. Smoking promotes a state of oxidative stress leading to mitochondrial dysfunction, which is additive to the inflammatory milieu of trauma. Smoking is associated with impaired mitochondrial substrate utilization of long-chain fatty acids, aspartate, and tyrosine, all of which accentuate oxidative stress following injury. This altered expression represents an ideal target for therapies to reduce oxidative damage toward the goal of personalized treatment of trauma patients. Prognostic and Epidemiological; Level IV.

Novel approach for evaluation of mitochondrial substrate utilization in fibroblasts from patients with Friedreich's ataxia

Novel approach for evaluation of mitochondrial substrate utilization in fibroblasts from patients with Friedreich's ataxia

Mitochondrial dynamics and myocardial metabolism alterations during anthracycline-cardiotoxicity

Abstract Background Despite the increased effectiveness of chemotherapeutic agents, cancer represents a lethal illness worldwide and anticancer strategies still present severe adverse effects. Cardiac dysfunction is a dose-dependent common affliction of anthracyclines treatment. Anthracycline-induced cardiotoxicity (AIC) and consequent heart failure has recently been suggested to be mediated by mitochondrial dysfunction; however, the molecular mechanisms driving this connection are incompletely understood. Purpose To study the early molecular changes (before onset of overt heart failure phenotype) related to mitochondrial biology and cardiac metabolism in a mouse model of AIC. Methods CD1 mice underwent 5 weekly i.p. injections (5 mg/kg) of doxorubicin. Cardiac anatomic and functional changes were evaluated by echocardiography at 1, 9 and 15 weeks-after last doxorubicin injection. At each time-point, some animals were sacrificed and hearts were collected for characterisation of mitochondrial dynamics and metabolic substrate utilization by western blot, PCR, immunocytochemistry, high-resolution respirometry, transmission electron microscopy and PET/CT. Results Doxorubicin administration resulted in an early reduction in left ventricle (LV) mass which was the result of marked cardiomyocyte atrophy as early as 1 week after doxorubicin completion. Cardiac atrophy preceded functional abnormalities, which were apparent in the form of reduction in LV ejection fraction at 9 weeks. Mitochondrial dynamics (fusion/fission) and membranes were altered as early as 1 week after doxorubicin completion (i.e. long before changes in LV function). Moreover, mitochondrial respiratory rate was decreased in these animals, together with a decrease in mitochondrial DNA content and biogenesis at early timepoints. Early (1 week) mitochondrial molecular changes were accompanied by altered cardiac metabolism (i.e. switch in substrate utilization to glucose). Conclusions Here we have characterized the dynamics of molecular, anatomical and functional changes during AIC. LV systolic dysfunction is a late phenomenon happening long after molecular and metabolic changes in the heart. Cardiomyocyte atrophy, altered mitochondrial dynamics and respiration, and metabolic switching occur long before overt heart failure phenotype. These early changes appear as promising therapeutic targets to prevent overt AIC and heart failure.

Impaired cardiac BCAA catabolism associated with impaired myocardial efficiency during exercise in a porcine model with multiple risk factors

Abstract Background Diabetes mellitus (DM), chronic kidney disease (CKD) and consumption of a high-fat diet (HFD), are well known risk factors for diastolic heart failure. We recently developed a multiple comorbidity swine model (MCS), with DM (streptozotocin), HFD and CKD (kidney embolization), and showed that 6 months of sustained hyperglycemia, hypercholesterolemia and kidney dysfunction resulted in inflammation and oxidative stress associated with coronary microvascular dysfunction and left ventricular diastolic dysfunction. Additionally, MCS animals showed impaired myocardial efficiency during exercise as demonstrated by increased myocardial oxygen consumption (Fig. A), suggesting an impairment in myocardial mitochondrial function. Purpose Here we used a combined omics (single nuclei RNA sequencing and proteomics) approach to study the molecular pathways involved in mitochondrial dysfunction. Methods 15 MCS and 10 healthy control female swine were included in the study. Proteome analysis and single nuclei RNA sequencing, was performed on frozen left ventricle myocardial samples. Results Proteome analysis of MCS hearts showed reduced expression of branched-chain amino acid (BCAA) catabolism proteins (Fig. B), most notably BCAT2, MCCC1 and MCCC2. Single nuclei RNA sequencing of a subgroup of the same animals demonstrated a downregulation of these genes specifically in cardiomyocyte subpopulations. Besides being a source of ATP, via oxidative phosphorylation in mitochondria, BCAAs, encompassing essential amino acids (leucine, valine and isoleucine), act as signaling molecules. Thus, increased BCAA levels reduce insulin sensitivity, modulate mitochondrial substrate utilization and impair mitochondrial function, resulting in increased mitochondrial production of reactive oxygen species (ROS). These findings are consistent with our data in the MCS swine indicating that impaired BCAA metabolism was found in the myocardium of animals with comorbidities and were associated with increased ROS levels (8-isoprostane 12.9±0.8 pg/mg protein in MCS vs 10.3±0.5 in healthy animals, P=0.02 by T-test) and impaired myocardial efficiency during exercise (Fig. A). Conclusion Taken together, these findings suggest an essential role of altered BCAA cardiomyocyte metabolism resulting in mitochondrial dysfunction, increased oxidative stress and subsequent alterations in the myocardial function.Fig.

MCUb is an inducible regulator of calcium-dependent mitochondrial metabolism and substrate utilization in muscle

Mitochondria use the electron transport chain to generate high-energy phosphate from oxidative phosphorylation, a process also regulated by the mitochondrial Ca2+ uniporter (MCU) and Ca2+ levels. Here, we show that MCUb, an inhibitor of MCU-mediated Ca2+ influx, is induced by caloric restriction, where it increases mitochondrial fatty acid utilization. To mimic the fasted state with reduced mitochondrial Ca2+ influx, we generated genetically altered mice with skeletal muscle-specific MCUb expression that showed greater fatty acid usage, less fat accumulation, and lower body weight. In contrast, mice lacking Mcub in skeletal muscle showed increased pyruvate dehydrogenase activity, increased muscle malonyl coenzyme A (CoA), reduced fatty acid utilization, glucose intolerance, and increased adiposity. Mechanistically, pyruvate dehydrogenase kinase 4 (PDK4) overexpression in muscle of Mcub-deleted mice abolished altered substrate preference. Thus, MCUb is an inducible control point in regulating skeletal muscle mitochondrial Ca2+ levels and substrate utilization that impacts total metabolic balance.

Age-related changes of skeletal muscle metabolic response to contraction are also sex-dependent.

Mitochondria adapt to increased energy demands during muscle contraction by acutely altering metabolite fluxes and substrate oxidation. With age, an impaired mitochondrial metabolic response may contribute to reduced exercise tolerance and decreased skeletal muscle mass, specific force, increased overall fatty depositions in the skeletal muscle, frailty and depressed energy maintenance. We hypothesized that elevated energy stress in mitochondria with age alters the capacity of mitochondria to utilize different substrates following muscle contraction. To test this hypothesis, we used in vivo electrical stimulation to simulate high-intensity intervals (HII) or low intensity steady-state (LISS) exercise in young (5-7months) and aged (27-29months) male and female mice to characterize effects of age and sex on mitochondrial substrate utilization in skeletal muscle following contraction. Mitochondrial respiration using glutamate decreased in aged males following HII and glutamate oxidation was inhibited following HII in both the contracted and non-stimulated muscle of aged female muscle. Analyses of the muscle metabolome of female mice indicated that changes in metabolic pathways induced by HII and LISS contractions in young muscle are absent in aged muscle. To test improved mitochondrial function on substrate utilization following HII, we treated aged females with elamipretide (ELAM), a mitochondrially-targeted peptide shown to improve mitochondrial bioenergetics and restore redox status in aged muscle. ELAM removed inhibition of glutamate oxidation and showed increased metabolic pathway changes following HII, suggesting rescuing redox status and improving bioenergetic function in mitochondria from aged muscle increases glutamate utilization and enhances the metabolic response to muscle contraction in aged muscle. KEY POINTS: Acute local contraction of gastrocnemius can systemically alter mitochondrial respiration in non-stimulated muscle. Age-related changes in mitochondrial respiration using glutamate or palmitoyl carnitine following contraction are sex-dependent. Respiration using glutamate after high-intensity contraction is inhibited in aged female muscle. Metabolite level and pathway changes following muscle contraction decrease with age in female mice. Treatment with the mitochondrially-targeted peptide elamipretide can partially rescue metabolite response to muscle contraction.

Proteomic analysis of murine kidney proximal tubule sub-segment derived cell lines reveals preferences in mitochondrial pathway activity

The proximal tubule (PT) is a nephron segment that is responsible for the majority of solute and water reabsorption in the kidney. Each of its sub-segments have specialized functions; however, little is known about the genes and proteins that determine the oxidative phosphorylation capacity of the PT sub-segments. This information is critical to understanding kidney function and will provide a comprehensive landscape of renal cell adaptations to injury, physiologic stressors, and development. This study analyzed three immortalized murine renal cell lines (PT S1, S2, and S3 segments) for protein content and compared them to a murine fibroblast cell line. All three proximal tubule cell lines generate ATP predominantly by oxidative phosphorylation while the fibroblast cell line is glycolytic. The proteomic data demonstrates that the most significant difference in proteomic signatures between the cell lines are proteins known to be localized in the nucleus followed by mitochondrial proteins. Mitochondrial metabolic substrate utilization assays were performed using the proximal tubule cell lines to determine substrate utilization kinetics thereby providing a physiologic context to the proteomic dataset. This data will allow researchers to study differences in nephron-specific cell lines, between epithelial and fibroblast cells, and between actively respiring cells and glycolytic cells. SignificanceProteomic analysis of proteins expressed in immortalized murine renal proximal tubule cells was compared to a murine fibroblast cell line proteome. The proximal tubule segment specific cell lines: S1, S2 and S3 are all grown under conditions whereby the cells generate ATP by oxidative phosphorylation while the fibroblast cell line utilizes anaerobic glycolysis for ATP generation. The proteomic studies allow for the following queries: 1) comparisons between the proximal tubule segment specific cell lines, 2) comparisons between polarized epithelia and fibroblasts, 3) comparison between cells employing oxidative phosphorylation versus anaerobic glycolysis and 4) comparisons between cells grown on clear versus opaque membrane supports. The data finds major differences in nuclear protein expression and mitochondrial proteins. This proteomic data set will be an important baseline dataset for investigators who need immortalized renal proximal tubule epithelial cells for their research.

Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

ObjectiveMitophagy removes damaged mitochondria to maintain cellular homeostasis. Aryl hydrocarbon receptor (AhR) expression in the liver plays a crucial role in supporting normal liver functions, but its impact on mitochondrial function is unclear. Here, we identified a new role of AhR in the regulation of mitophagy to control hepatic energy homeostasis. MethodsIn this study, we utilized primary hepatocytes from AhR knockout (KO) mice and AhR knockdown AML12 hepatocytes. An endogenous AhR ligand, kynurenine (Kyn), was used to activate AhR in AML12 hepatocytes. Mitochondrial function and mitophagy process were comprehensively assessed by MitoSOX and mt-Keima fluorescence imaging, Seahorse XF-based oxygen consumption rate measurement, and Mitoplate S-1 mitochondrial substrate utilization analysis. ResultsTranscriptomic analysis indicated that mitochondria-related gene sets were dysregulated in AhR KO liver. In both primary mouse hepatocytes and AML12 hepatocyte cell lines, AhR inhibition strongly suppressed mitochondrial respiration rate and substrate utilization. AhR inhibition also blunted the fasting response of several essential autophagy genes and the mitophagy process. We further identified BCL2 interacting protein 3 (BNIP3), a mitophagy receptor that senses nutrient stress, as an AhR target gene. AhR is directly recruited to the Bnip3 genomic locus, and Bnip3 transcription was enhanced by AhR endogenous ligand treatment in wild-type liver and abolished entirely in AhR KO liver. Mechanistically, overexpression of Bnip3 in AhR knockdown cells mitigated the production of mitochondrial reactive oxygen species (ROS) and restored functional mitophagy. ConclusionsAhR regulation of the mitophagy receptor BNIP3 coordinates hepatic mitochondrial function. Loss of AhR induces mitochondrial ROS production and impairs mitochondrial respiration. These findings provide new insight into how endogenous AhR governs hepatic mitochondrial homeostasis.

Changes in Metabolism and Mitochondrial Bioenergetics during Polyethylene-Induced Osteoclastogenesis.

Changes in mitochondrial bioenergetics are believed to take place during osteoclastogenesis. This study aims to assess changes in mitochondrial bioenergetics and reactive oxygen species (ROS) levels during polyethylene (PE)-induced osteoclastogenesis in vitro. For this purpose, RAW264.7 cells were cultured for nine days and allowed to differentiate into osteoclasts in the presence of PE and RANKL. The total TRAP-positive cells, resorption activity, expression of osteoclast marker genes, ROS level, mitochondrial bioenergetics, glycolysis, and substrate utilization were measured. The effect of tocotrienols-rich fraction (TRF) treatment (50 ng/mL) on those parameters during PE-induced osteoclastogenesis was also studied. During PE-induced osteoclastogenesis, as depicted by an increase in TRAP-positive cells and gene expression of osteoclast-related markers, higher proton leak, higher extracellular acidification rate (ECAR), as well as higher levels of ROS and NADPH oxidases (NOXs) were observed in the differentiated cells. The oxidation level of some substrates in the differentiated group was higher than in other groups. TRF treatment significantly reduced the number of TRAP-positive osteoclasts, bone resorption activity, and ROS levels, as well as modulating the gene expression of antioxidant-related genes and mitochondrial function. In conclusion, changes in mitochondrial bioenergetics and substrate utilization were observed during PE-induced osteoclastogenesis, while TRF treatment modulated these changes.

Mitochondrial function in spinal cord injury and regeneration.

Many people around the world suffer from some form of paralysis caused by spinal cord injury (SCI), which has an impact on quality and life expectancy. The spinal cord is part of the central nervous system (CNS), which in mammals is unable to regenerate, and to date, there is a lack of full functional recovery therapies for SCI. These injuries start with a rapid and mechanical insult, followed by a secondary phase leading progressively to greater damage. This secondary phase can be potentially modifiable through targeted therapies. The growing literature, derived from mammalian and regenerative model studies, supports a leading role for mitochondria in every cellular response after SCI: mitochondrial dysfunction is the common event of different triggers leading to cell death, cellular metabolism regulates the immune response, mitochondrial number and localization correlate with axon regenerative capacity, while mitochondrial abundance and substrate utilization regulate neural stem progenitor cells self-renewal and differentiation. Herein, we present a comprehensive review of the cellular responses during the secondary phase of SCI, the mitochondrial contribution to each of them, as well as evidence of mitochondrial involvement in spinal cord regeneration, suggesting that a more in-depth study of mitochondrial function and regulation is needed to identify potential targets for SCI therapeutic intervention.

HMTM-Mediated Enhancement of Brain Bioenergetics in a Mouse Tauopathy Model Is Blocked by Chronic Administration of Rivastigmine

The tau protein aggregation inhibitor hydromethylthionine mesylate (HMTM) was shown recently to have concentration-dependent pharmacological activity in delaying cognitive decline and brain atrophy in phase 3 Alzheimer’s disease (AD) clinical trials; the activity was reduced in patients receiving symptomatic therapies. The methylthionine (MT) moiety has been reported to increase the clearance of pathological tau and to enhance mitochondrial activity, which is impaired in AD patients. In line 1 (L1) mice (a model of AD), HMTM (5/15 mg/kg) was administered either as a monotherapy or as an add-on to a chronic administration with the cholinesterase inhibitor rivastigmine (0.1/0.5 mg/kg) to explore mitochondrial function and energy substrate utilization as potential targets of drug interference. Compared with wild-type NMRI mice, the L1 mice accumulated greater levels of l-lactate and of the LDH-A subunit responsible for the conversion of pyruvate into l-lactate. In contrast, the levels of LDH-B and mitochondrial ETC subunits and the activity of complexes I and IV was not altered in the L1 mice. The activity of complex I and complex IV tended to increase with the HMTM dosing, in turn decreasing l-lactate accumulation in the brains of the L1 mice, despite increasing the levels of LDH-A. The chronic pre-dosing of the L1 mice with rivastigmine partially prevented the enhancement of the activity of complexes I and IV by HMTM and the increase in the levels of LDH-A while further reducing the levels of l-lactate. Thus, HMTM in combination with rivastigmine leads to a depletion in the energy substrate l-lactate, despite bioenergetic production not being favoured. In this study, the changes in l-lactate appear to be regulated by LDH-A, since neither of the experimental conditions affected the levels of LDH-B. The data show that HMTM monotherapy facilitates the use of substrates for energy production, particularly l-lactate, which is provided by astrocytes, additionally demonstrating that a chronic pre-treatment with rivastigmine prevented most of the HMTM-associated effects.

ApoE4 confers mitochondrial substrate oxidation defects that can be bioenergetically compensated by a ketogenic substrate beta-hydroxy butyrate (BHB).

Mitochondria are at the center of neural biogenergetics, and ApoE4 is the single most impactful risk factor for AD. We investigated the impact of ApoE on insulin sensitivity, on mitochondrial substrate utilization and bioenergetics. Persons with ApoE4 have reduced brain carbohydrate metabolism. To test for ApoE4 conferred neural mitochondrial metabolic differences, we constructed a novel stable-ApoE 2,3 and 4 N2a cell model, and tested ApoE's effects on Insulin sensitivity, and mitochondrial glucose, lipid and ketone oxidation. Binding of ApoE isoforms E2, E3 and E4 to Insulin Receptor (IR) was measured by BLI and Co-IP, the impact of ApoE isoforms on mitochondrial glucose and lipid oxidation was measured by Seahorse. ApoE3 was found to sensitize to insulin about 2-fold more potently than ApoE4. ApoE isoforms directly bind Insulin Receptor; the binding constants was in the range 200-300nM. Consistent with the previous insulin-sensitivity finding, ApoE3 caused a significant increase of the glycolytic rate and glucose oxidation relative to ApoE4. As there was no difference in oxidation of TCA cycle intermediates substrates in permeabilized cells, we infer ApoE3's glucose advantage is the result of increased insulin sensitivity. ApoE4 contributed a significant palmitate oxidation defect relative to ApoE2 and ApoE3. As this palmitate oxidation defect was observed in both mitochondria and cells it is likely to occur at or within mitochondria. We observed that the relative defect in ApoE4-dependent glucose and palmitate oxidation can be overcome by 5mM BHB. Thus, at the neural cell level, the metabolic defects contributed by ApoE 4 appear to be rescued by a ketogenic molecule, BHB, that requires neither insulin nor apolipoprotein particle to reach neural mitochondria and provide alternative metabolic support. ApoE4 confers 'double trouble' in mitochondrial glucose and lipid oxidation. ApoE4 confers a defect in mitochondrial lipid oxidation relative to all other isoforms. Simultaneously, ApoE4 lacks the benefit in glucose oxidation conferred by ApoE3, which appears to be driven by the reduced insulin sensitization potency of ApoE4. We also find that BHB can be an alternative source of neural bioenergy that enters mitochondria directly and thus is not affected by ApoE4 'double trouble'.

Human myocardial mitochondrial oxidative capacity is impaired in mild acute heart transplant rejection.

AimsAcute cellular rejection (ACR) following heart transplantation (HTX) is associated with long‐term graft loss and increased mortality. Disturbed mitochondrial bioenergetics have been identified as pathophysiological drivers in heart failure, but their role in ACR remains unclear. We aimed to prove functional disturbances of myocardial bioenergetics in human heart transplant recipients with mild ACR by assessing myocardial mitochondrial respiration using high‐resolution respirometry, digital image analysis of myocardial inflammatory cell infiltration, and clinical assessment of HTX patients. We hypothesized that (i) mild ACR is associated with impaired myocardial mitochondrial respiration and (ii) myocardial inflammation, systemic oxidative stress, and myocardial oedema relate to impaired mitochondrial respiration and myocardial dysfunction.Methods and resultsWe classified 35 HTX recipients undergoing endomyocardial biopsy according International Society for Heart and Lung Transplantation criteria to have no (0R) or mild (1R) ACR. Additionally, we quantified immune cell infiltration by immunohistochemistry and digital image analysis. We analysed mitochondrial substrate utilization in myocardial fibres by high‐resolution respirometry and performed cardiovascular magnetic resonance (CMR). ACR (1R) was diagnosed in 12 patients (34%), while the remaining 23 patients revealed no signs of ACR (0R). Underlying cardiomyopathies (dilated cardiomyopathy 50% vs. 65%; P = 0.77), comorbidities (type 2 diabetes mellitus: 50% vs. 35%, P = 0.57; chronic kidney disease stage 5: 8% vs. 9%, P > 0.99; arterial hypertension: 59% vs. 30%, P = 0.35), medications (tacrolimus: 100% vs. 91%, P = 0.54; mycophenolate mofetil: 92% vs. 91%, P > 0.99; prednisolone: 92% vs. 96%, P > 0.99) and time post‐transplantation (21.5 ± 26.0 months vs. 29.4 ± 26.4 months, P = 0.40) were similar between groups. Mitochondrial respiration was reduced by 40% in ACR (1R) compared with ACR (0R) (77.8 ± 23.0 vs. 128.0 ± 33.0; P < 0.0001). Quantitative assessment of myocardial CD3+‐lymphocyte infiltration identified ACR (1R) with a cut‐off of >14 CD3+‐lymphocytes/mm2 (100% sensitivity, 82% specificity; P < 0.0001). Myocardial CD3+ infiltration (r = −0.41, P < 0.05), systemic oxidative stress (thiobarbituric acid reactive substances; r = −0.42, P < 0.01) and myocardial oedema depicted by global CMR derived T2 time (r = −0.62, P < 0.01) correlated with lower oxidative capacity and overt cardiac dysfunction (global longitudinal strain; r = −0.63, P < 0.01).ConclusionsMild ACR with inflammatory cell infiltration associates with impaired mitochondrial bioenergetics in cardiomyocytes. Our findings may help to identify novel checkpoints in cardiac immune metabolism as potential therapeutic targets in post‐transplant care.

Abstract P430: VSMCs Increase Glutamine Utilization For Energy Metabolism During Obesity

Obesity increases the risk of developing cardiovascular disease through vascular remodeling though the underlying mechanisms are not entirely understood. However, metabolic fuel partitioning and mitochondrial flexibility during energy metabolism may play a critical role. We demonstrated serum and glucocorticoid-inducible kinase 1 (SGK-1) is up-regulated in the vasculature of diet-induced obese mice and that SGK-1 deletion is protective against obesity-induced vascular remodeling by metabolically reprogramming vascular smooth muscle cell (VSMC) energy metabolism towards oxidative phosphorylation (OXPHOS) and away from glycolysis. Mitochondrial substrate availability and utilization of the primary metabolic fuels glucose, long chain fatty acids (LCFAs) and glutamine can drive metabolic reprogramming. Therefore, alterations in fuel utilization may contribute to vascular remodeling during obesity. The purpose of this study was to examine SGK-1’s role in 1) fuel dependency: a cell’s reliance for a specific fuel and 2) fuel capacity: a cell’s ability to oxidize a specific fuel to meet cellular energy demand under low-fat and high-fat diet-induced obesity. Using the MitoXpress Oxygen Consumption assay which measures OXPHOS, primary VSMCs isolated from wildtype (WT) and SMC-specific SGK-1 knockout (smSGK-1 KO) mice fed a 10% kcal low-fat or 45% kcal high-fat diet for eight weeks were seeded in a 96-well plate at a density of 6x10 4 cells/well in culture medium. To assess fuel dependency, cells were treated with fuel pathway inhibitors UK5099, Etomoxir or BPTES to block glucose, LCFA or glutamine oxidation, respectively. To measure fuel capacity, VSMCs were treated with a combination of two pathway inhibitors simultaneously. Next, samples were overlaid with a fluorescent extracellular oxygen consumption reagent, sealed with high-sensitivity mineral oil, then signals were read at 1.5-minute intervals for 2 hours at Ex/Em= 380/650 nm. Our results show WT VSMCs are exclusively glucose-dependent for OXPHOS regardless of dietary conditions. However, SGK-1 deletion induces a dependency for all three fuels for OXPHOS in VSMCs under low- and high-fat conditions. Even though WT and smSGK-1 KO VSMCs preferentially oxidized glucose for OXPHOS under low-fat conditions; SGK-1 deletion resulted in a 2.2-fold increase in glutamine capacity. Alternatively, WT VSMCs exposed to obesogenic conditions preferentially oxidized glutamine whereas SGK-1 deletion induced a nearly equal partitioning of all three fuels during obesity suggesting elevated mitochondrial flexibility. Overall, this study suggests SGK-1 increases glucose dependency for energy metabolism under physiological and obesogenic conditions. Also, increased glutamine utilization for OXPHOS during obesity may be an underlying cause of VSMC dysfunction and subsequent vascular impairment.

Endurance Is Improved in Female Rats After Living High-Training High Despite Alterations in Skeletal Muscle

Altitude camps are used during the preparation of endurance athletes to improve performance based on the stimulation of erythropoiesis by living at high altitude. In addition to such whole-body adaptations, studies have suggested that high-altitude training increases mitochondrial mass, but this has been challenged by later studies. Here, we hypothesized that living and training at high altitude (LHTH) improves mitochondrial efficiency and/or substrate utilization. Female rats were exposed and trained in hypoxia (simulated 3,200 m) for 5 weeks (LHTH) and compared to sedentary rats living in hypoxia (LH) or normoxia (LL) or those that trained in normoxia (LLTL). Maximal aerobic velocity (MAV) improved with training, independently of hypoxia, whereas the time to exhaustion, performed at 65% of MAV, increased both with training (P = 0.009) and hypoxia (P = 0.015), with an additive effect of the two conditions. The distance run was 7.98 ± 0.57 km in LHTH vs. 6.94 ± 0.51 in LLTL (+15%, ns). The hematocrit increased >20% with hypoxia (P < 0.001). The increases in mitochondrial mass and maximal oxidative capacity with endurance training were blunted by combination with hypoxia (−30% for citrate synthase, P < 0.01, and −23% for Vmax glut−succ, P < 0.001 between LHTH and LLTL). A similar reduction between the LHTH and LLTL groups was found for maximal respiration with pyruvate (−29%, P < 0.001), for acceptor-control ratio (−36%, hypoxia effect, P < 0.001), and for creatine kinase efficiency (−48%, P < 0.01). 3-hydroxyl acyl coenzyme A dehydrogenase was not altered by hypoxia, whereas maximal respiration with Palmitoyl-CoA specifically decreased. Overall, our results show that mitochondrial adaptations are not involved in the improvement of submaximal aerobic performance after LHTH, suggesting that the benefits of altitude camps in females relies essentially on other factors, such as the transitory elevation of hematocrit, and should be planned a few weeks before competition and not several months.

Perinatally acquired HIV infection is associated with abnormal blood mitochondrial function during childhood/adolescence.

We assessed differences in mitochondrial function between youth living with perinatal HIV (YPHIV) and youth perinatally HIV-exposed but uninfected (YPHEU). Cross-sectional analysis. We measured lactate and pyruvate values, as well as mitochondrial Complex I and Complex IV activity in peripheral blood mononuclear cells. Logistic or linear regression models were fit, as appropriate, to assess the association between PHIV status and each mitochondrial parameter, adjusted for confounders. We introduced interaction terms to assess effect modification of PHIV status on the relationship between anthropometric factors and each mitochondrial parameter. Among YPHIV, similar regression models were fit to assess the relationship between HIV-associated factors and each mitochondrial outcome. A total of 243 YPHIV and 118 YPHEU were compared. On average, YPHIV had higher lactate/pyruvate ratio (β: 7.511, 95% confidence interval [95% CI]: 0.402, 14.620) and Complex IV activity (β: 0.037, 95% CI: 0.002, 0.072) compared to YPHEU, adjusted for confounders. Among YPHIV, body mass index Z score (BMIZ) and Complex I activity were inversely associated, whereas, among YPHEU, there was a positive association (β for interaction: -0.048, P = 0.003). Among YPHIV, current (β: -0.789, 95% CI: -1.174, -0.404) and nadir CD4+% (β: -0.605, 95% CI: -1.086, -0.125) were inversely associated with lactate/pyruvate ratio; higher current (4.491, 95% CI: 0.754, 8.229) and peak (7.978, 95% CI: 1.499, 14.457) HIV RNA levels were positively associated with lactate/pyruvate ratio in adjusted models. Mitochondrial function and substrate utilization appear perturbed in YPHIV compared to YPHEU. Increasing immunosuppression and viremia are associated with mitochondrial dysfunction among YPHIV.

A Toolbox to Profile Immunometabolism Tested in Macrophages

Macrophages are highly plastic immune cells that can adopt several activation states. Often, macrophages are stimulated in vitro with lipopolysaccharide (LPS) to elicit pro-inflammatory macrophages or interleukin (IL)-4 to obtain a homeostatic phenotype. Fundamental to these functional activation states is the regulation of cellular metabolic processes. The metabolic alterations underlying LPS- and IL-4-induced phenotypes have been described thoroughly with several -omics techniques. However, methods allowing for time- and cost-effective screening of cellular metabolism are rather limited. Here, we establish a comprehensive toolbox to assess cellular metabolism in a semi-high-throughput 96-well-plate-based format. We validated the approach on LPS- and IL-4-activated mouse and human macrophages by measuring extracellular flux, SCENITH, mitochondrial mass and membrane potential, glucose and lipid uptake, and mitochondrial substrate utilization. By discussing the experienced strengths, limitations and complementarities of the distinct techniques, we guide readers for efficient application of the toolbox in their immune subsets of interest.