Mitochondrial Fatty Acid Uptake Research Articles

Itaconate stabilizes CPT1a to enhance lipid utilization during inflammation

One primary metabolic manifestation of inflammation is the diversion of cis-aconitate within the tricarboxylic acid (TCA) cycle to synthesize the immunometabolite itaconate. Itaconate is well established to possess immunomodulatory and metabolic effects within myeloid cells and lymphocytes, however, its effects in other organ systems during sepsis remain less clear. Utilizing Acod1 knockout mice that are deficient in synthesizing itaconate, we aimed to understand the metabolic role of itaconate in the liver and systemically during sepsis. We find itaconate aids in lipid metabolism during sepsis. Specifically, Acod1 KO mice develop a heightened level of hepatic steatosis when induced with polymicrobial sepsis. Proteomics analysis reveals enhanced expression of enzymes involved in fatty acid oxidation in following 4-octyl itaconate (4-OI) treatment in vitro. Downstream analysis reveals itaconate stabilizes the expression of the mitochondrial fatty acid uptake enzyme CPT1a, mediated by its hypoubiquitination. Chemoproteomic analysis revealed itaconate interacts with proteins involved in protein ubiquitination as a potential mechanism underlying its stabilizing effect on CPT1a. From a systemic perspective, we find itaconate deficiency triggers a hypothermic response following endotoxin stimulation, potentially mediated by brown adipose tissue (BAT) dysfunction. Finally, by use of metabolic cage studies, we demonstrate Acod1 KO mice rely more heavily on carbohydrates versus fatty acid sources for systemic fuel utilization in response to endotoxin treatment. Our data reveal a novel metabolic role of itaconate in modulating fatty acid oxidation during polymicrobial sepsis.

Itaconate stabilizes CPT1a to enhance lipid utilization during inflammation.

One primary metabolic manifestation of inflammation is the diversion of cis-aconitate within the tricarboxylic acid (TCA) cycle to synthesize the immunometabolite itaconate. Itaconate is well established to possess immunomodulatory and metabolic effects within myeloid cells and lymphocytes, however, its effects in other organ systems during sepsis remain less clear. Utilizing Acod1 knockout mice that are deficient in synthesizing itaconate, we aimed to understand the metabolic role of itaconate in the liver and systemically during sepsis. We find itaconate aids in lipid metabolism during sepsis. Specifically, Acod1 KO mice develop a heightened level of hepatic steatosis when induced with polymicrobial sepsis. Proteomics analysis reveals enhanced expression of enzymes involved in fatty acid oxidation in following 4-octyl itaconate (4-OI) treatment in vitro. Downstream analysis reveals itaconate stabilizes the expression of the mitochondrial fatty acid uptake enzyme CPT1a, mediated by its hypoubiquitination. Chemoproteomic analysis revealed itaconate interacts with proteins involved in protein ubiquitination as a potential mechanism underlying its stabilizing effect on CPT1a. From a systemic perspective, we find itaconate deficiency triggers a hypothermic response following endotoxin stimulation, potentially mediated by brown adipose tissue (BAT) dysfunction. Finally, by use of metabolic cage studies, we demonstrate Acod1 KO mice rely more heavily on carbohydrates versus fatty acid sources for systemic fuel utilization in response to endotoxin treatment. Our data reveal a novel metabolic role of itaconate in modulating fatty acid oxidation during polymicrobial sepsis.

LCP1 knockdown in monocyte-derived macrophages: mitigating ischemic brain injury and shaping immune cell signaling and metabolism.

Rationale: Ischemic stroke poses a significant health burden with limited treatment options. Lymphocyte Cytosolic Protein 1 (LCP1) facilitates cell migration and immune responses by aiding in actin polymerization, cytoskeletal rearrangements, and phagocytosis. We have demonstrated that the long non-coding RNA (lncRNA) Maclpil silencing in monocyte-derived macrophages (MoDMs) led to LCP1 inhibition, reducing ischemic brain damage. However, the role of LCP1 of MoDMs in ischemic stroke remains unknown. Methods and Results: We investigated the impact of LCP1 on ischemic brain injury and immune cell signaling and metabolism. We found that knockdown of LCP1 in MoDMs demonstrated robust protection against ischemic infarction and improved neurological behaviors in mice. Utilizing the high-dimensional CyTOF technique, we demonstrated that knocking down LCP1 in MoDMs led to a reduction in neuroinflammation and attenuation of lymphopenia, which is linked to immunodepression. It also showed altered immune cell signaling by modulating the phosphorylation levels of key kinases and transcription factors, including p-PLCg2, p-ERK1/2, p-EGFR, p-AKT, and p4E-BP1 as well as transcription factors like p-STAT1, p-STAT3, and p-STAT4. Further bioinformatic analysis indicated that Akt and EGFR are particularly involved in fatty acid metabolism and glycolysis. Indeed, single-cell sequencing analysis confirmed that enrichment of fatty acid and glycolysis metabolism in Lcp1high monocytes/macrophages. Furthermore, Lcp1high cells exhibited enhanced oxidative phosphorylation, chemotaxis, migration, and ATP biosynthesis pathways. In vitro experiments confirmed the role of LCP1 in regulating mitochondrial function and fatty acid uptake. Conclusions: These findings contribute to a deeper understanding of LCP1 in the context of ischemic stroke and provide valuable insights into potential therapeutic strategies targeting LCP1 and metabolic pathways, aiming to attenuating neuroinflammation and lymphopenia.

Activation of the integrated stress response rewires cardiac metabolism in Barth syndrome

Barth Syndrome (BTHS) is an inherited cardiomyopathy caused by defects in the mitochondrial transacylase TAFAZZIN (Taz), required for the synthesis of the phospholipid cardiolipin. BTHS is characterized by heart failure, increased propensity for arrhythmias and a blunted inotropic reserve. Defects in Ca2+-induced Krebs cycle activation contribute to these functional defects, but despite oxidation of pyridine nucleotides, no oxidative stress developed in the heart. Here, we investigated how retrograde signaling pathways orchestrate metabolic rewiring to compensate for mitochondrial defects. In mice with an inducible knockdown (KD) of TAFAZZIN, and in induced pluripotent stem cell-derived cardiac myocytes, mitochondrial uptake and oxidation of fatty acids was strongly decreased, while glucose uptake was increased. Unbiased transcriptomic analyses revealed that the activation of the eIF2α/ATF4 axis of the integrated stress response upregulates one-carbon metabolism, which diverts glycolytic intermediates towards the biosynthesis of serine and fuels the biosynthesis of glutathione. In addition, strong upregulation of the glutamate/cystine antiporter xCT increases cardiac cystine import required for glutathione synthesis. Increased glutamate uptake facilitates anaplerotic replenishment of the Krebs cycle, sustaining energy production and antioxidative pathways. These data indicate that ATF4-driven rewiring of metabolism compensates for defects in mitochondrial uptake of fatty acids to sustain energy production and antioxidation.

Abstract P3087: Macrophage Fatty Acid Metabolism Promotes Splenic Hematopoiesis Through Mitochondrial Signaling During Heart Failure With Preserved Ejection Fraction

Rationale: Diastolic dysfunction, including during heart failure with preserved ejection fraction (HFpEF), is a widespread morbidity with limited therapeutic options. Though heterogeneous in etiology, major risk factors include hyperlipidemia, hypertension, and inflammation. Recent studies have also shown significant changes in systemic metabolite availability during HFpEF, further suggesting that metabolic changes may be an important indicator of HFpEF pathophysiology. Macrophages, which have previously been implicated in the promotion of diastolic dysfunction, utilize specific metabolic pathways to drive inflammation or tissue repair. However, little is understood about myeloid metabolism during HFpEF. Objective: We set out to define the functional and metabolic heterogeneity of the cardiac immune response during diastolic dysfunction. Methods/Results: Diastolic dysfunction was induced by HFD and eNOS inhibition in mice. scRNAseq showed a specific induction of pro-fibrotic and lipid handling pathways in cardiac macrophages, including the fatty acid receptor Cd36. In live animals, myeloid deficiency of Cd36 prevented diastolic dysfunction and was associated with dampened cardiac inflammation. Investigating immune cell sourcing during HFpEF revealed that a splenic hematopoietic niche formed in wild-type mice. Further, mitochondrial metabolism in splenic macrophages was associated with increased expression of VCAM-1, the latter of which has been shown to play a role in retention of HSCs in the spleen. Indeed, blocking VCAM-1 or genetic deletion of VCAM-1 in myeloid cells was sufficient to disrupt retention of HSCs within the spleen during HFpEF. Culturing splenic macrophages with saturated fatty acids (SFAs) in vitro was sufficient to induce expression of Vcam1. Increased cellular citrate levels were correlated with increased histone acetylation at the Vcam1 promoter in splenic macrophages cultured with SFAs. Inhibition of mitochondrial uptake of fatty acids through CPT1a siRNA or inhibition of ACLY were sufficient to prevent this upregulation of Vcam1 expression. Conclusions: Our data suggest a critical role for myeloid metabolism in the sourcing of detrimental-monocyte-derived macrophages from the spleen to the heart.

Abstract GS.04: Myocardial Tricarboxylic Acid Cycle Suppression Disrupts The Epigenetic Landscape In Human Heart Failure With Preserved Ejection Fraction

Heart Failure with Preserved Ejection Fraction (HFpEF) represents the greatest unmet therapeutic need in cardiology and is intimately related to obesity and metabolic syndrome. Using a bedside-to-bench approach, we investigated the hypothesis that suppressed tricarboxylic acid cycle (TCA) enzymes and metabolic intermediates in HFpEF influence epigenetic histone post-translational modifications (PTMs) that regulate genes involved in mitochondrial OxPhos and fatty acid metabolism. Endomyocardial biopsies were obtained from human HFpEF patients (n=32) and donor controls (n=12) and integrated proteomics, metabolomics, ChIP-seq, and RNAseq analyses were performed. Small molecule histone PTM modulators were screened in vitro in neonatal rat ventricular cardiomyocytes (NRVMs) to identify potential therapeutic targets. Proteomics demonstrates reduced TCA cycle enzyme abundance consistent with our recently published metabolomics data showing reduced succinate, fumarate, and malate. Histone PTM profiling found H3K27ac and H3K36me3 upregulated (p<0.001) and inversely correlated with TCA cycle intermediates succinate and fumerate. ChIP-seq promoter gene ontology analysis revealed enrichment of mitochondrial metabolism and fatty acid uptake ontologies (CD36), as well as major cardiac transcription factors such as GATA4. Small-molecule inhibitor proteomics screens of histone writers in neonatal rat ventricular myocytes showed that H3K27ac inhibition upregulates all TCA cycle enzymes 1.5-4 fold and may rescue TCA cycle function. Our multi-omics approach suggests an association between H3K27ac, H3K36me3, and TCA cycle intermediates. Inhibition of myocardial H3K27ac deposition represents a novel therapeutic target for HFpEF.

Endogenous Energy Stores Maintain a High ATP Concentration for Hours in Glucose-Depleted Cultured Primary Rat Astrocytes

Adenosine triphosphate (ATP) is the central energy currency of all cells. Cultured primary rat astrocytes contain a specific cellular ATP content of 27.9 ± 4.7 nmol/mg. During incubation in a glucose- and amino acid-free incubation buffer, this high cellular ATP content was maintained for at least 6 h, while within 24 h the levels of ATP declined to around 30% of the initial value without compromising cell viability. In contrast, cells exposed to 1 mM and 5 mM glucose maintained the initial high cellular ATP content for 24 and 72 h, respectively. The loss in cellular ATP content observed during a 24 h glucose-deprivation was fully prevented by the presence of glucose, fructose or mannose as well as by the mitochondrial substrates lactate, pyruvate, β-hydroxybutyrate or acetate. The high initial specific ATP content in glucose-starved astrocytes, was almost completely abolished within 30 min after application of the respiratory chain inhibitor antimycin A or the mitochondrial uncoupler BAM-15, while these inhibitors lowered in glucose-fed cells the ATP content only to 60% (BAM-15) and 40% (antimycin A) within 5 h. Inhibition of the mitochondrial pyruvate carrier by UK5099 alone or of mitochondrial fatty acid uptake by etomoxir alone hardly affected the high ATP content of glucose-deprived astrocytes during an incubation for 8 h, while the co-application of both inhibitors depleted cellular ATP levels almost completely within 5 h. These data underline the importance of mitochondrial metabolism for the ATP regeneration of astrocytes and demonstrate that the mitochondrial oxidation of pyruvate and fatty acids strongly contributes to the maintenance of a high ATP concentration in glucose-deprived astrocytes.

Combined metabolic activators improve cognitive functions in Alzheimer’s disease patients: a randomised, double-blinded, placebo-controlled phase-II trial

BackgroundAlzheimer’s disease (AD) is associated with metabolic abnormalities linked to critical elements of neurodegeneration. We recently administered combined metabolic activators (CMA) to the AD rat model and observed that CMA improves the AD-associated histological parameters in the animals. CMA promotes mitochondrial fatty acid uptake from the cytosol, facilitates fatty acid oxidation in the mitochondria, and alleviates oxidative stress.MethodsHere, we designed a randomised, double-blinded, placebo-controlled phase-II clinical trial and studied the effect of CMA administration on the global metabolism of AD patients. One-dose CMA included 12.35 g L-serine (61.75%), 1 g nicotinamide riboside (5%), 2.55 g N-acetyl-L-cysteine (12.75%), and 3.73 g L-carnitine tartrate (18.65%). AD patients received one dose of CMA or placebo daily during the first 28 days and twice daily between day 28 and day 84. The primary endpoint was the difference in the cognitive function and daily living activity scores between the placebo and the treatment arms. The secondary aim of this study was to evaluate the safety and tolerability of CMA. A comprehensive plasma metabolome and proteome analysis was also performed to evaluate the efficacy of the CMA in AD patients.ResultsWe showed a significant decrease of AD Assessment Scale-cognitive subscale (ADAS-Cog) score on day 84 vs day 0 (P = 0.00001, 29% improvement) in the CMA group. Moreover, there was a significant decline (P = 0.0073) in ADAS-Cog scores (improvement of cognitive functions) in the CMA compared to the placebo group in patients with higher ADAS-Cog scores. Improved cognitive functions in AD patients were supported by the relevant alterations in the hippocampal volumes and cortical thickness based on imaging analysis. Moreover, the plasma levels of proteins and metabolites associated with NAD + and glutathione metabolism were significantly improved after CMA treatment.ConclusionOur results indicate that treatment of AD patients with CMA can lead to enhanced cognitive functions and improved clinical parameters associated with phenomics, metabolomics, proteomics and imaging analysis.Trial registration ClinicalTrials.gov NCT04044131 Registered 17 July 2019, https://clinicaltrials.gov/ct2/show/NCT04044131

Abstract 5603: Heterodimeric IL-15 (hetIL-15) immunotherapy reverses CD8+T cell metabolic dysfunction in murine breast tumors

Abstract Introduction: Metabolic fitness and T cell survival are crucial in anti-tumor responses because nutrients are often scarce and other regulatory molecules may be unfavorable in the tumor microenvironment leading to T cell dysfunction, stress, and apoptosis. Tumor-infiltrating cytotoxic CD8+T cells often acquire an altered state of differentiation referred to as “exhaustion” and, as a result, they fail to control tumor outgrowth. IL-15 cytokine stimulates the generation, the proliferation and the cytotoxic function of tumor specific CD8+ T cells and NK cells. The objective of this study was to assess the effects of hetIL-15 immunotherapy after locoregional administration in triple negative breast cancer tumors (TNBC) and to evaluate the metabolic profile of the tumor infiltrating T cells. Study design and methods: We studied the therapeutic efficacy of hetIL-15 immunotherapy in murine EO771 orthotopic breast cancer model. We monitored the effect of the treatment on tumor size and immune infiltration using flow cytometry and immunohistochemistry. We also evaluated hetIL-15 effects on the cell metabolism and the mitochondrial function of the tumor-infiltrating immune cells, using flow cytometry, Seahorse flux analysis, Mitotracker, 2-NBDG and/or Bodipy staining. Results: hetIL-15 peritumoral administration, as monotherapy resulted in complete regression in 40% of the treated animals and increased survival. We demonstrated that tumor infiltrating cytotoxic CD8+T and NK cells were increased in hetIL-15 treated tumors and showed enhanced activation and proliferation, resulting in enhanced intratumoral immunological killing. Metabolic flux analysis of the tumor-infiltrating CD8+T cells from treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity, which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. Consistent with the above finding, tumor infiltrated CD8+T cells from hetIL-15 treated mice showed increased mitochondrial potential and fatty acid uptake, as evidenced by increased Mitotracker and Bodipy staining, respectively. In addition, tumor infiltrating CD8+T cells from hetIL-15 treated mice presented elevated extracellular acidification rate (ECAR) and showed a pronounced shift in the OCR to ECAR ratio in comparison to control, confirming their increased proliferating status. Conclusions: Our results indicate that hetIL-15 not only increases the infiltration and the cytotoxic properties of the CD8+T cells inside the tumors, but also enhances the metabolic reprogramming of T cells to achieve superior antitumor efficacy. We suggest that metabolic reprogramming of tumor-specific CD8+T cells might represent a strategy to promote survival in the metabolically hostile TME as part of an approach to enhance the clinical efficacy of immunotherapy. Citation Format: Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Breana Myers, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 (hetIL-15) immunotherapy reverses CD8+T cell metabolic dysfunction in murine breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5603.

Abstract 2465: Optical imaging captures chemo-induced metabolic reprogramming during disease regression

Abstract Less than 20% of Triple Negative Breast Cancer (TNBC) patients experience long-term responses to mainstay chemotherapy, as tumors develop chemo-resistance. Prediction of chemo-resistance will guide treatment planning, thus improving response while reducing toxicity and cost associated with ineffective therapies. Spatial and temporal metabolic reprogramming holds promise as a biomarker of therapy response as resistant tumor subpopulations utilize alternate metabolic pathways to escape therapy, enter dormancy and recur. Currently, there are no tools to temporally evaluate heterogenous changes along distinct metabolic axes in vivo at a spatial resolution capable of resolving vulnerabilities of residual tumor subpopulations. We developed an optical-imaging based platform to correlate long-term treatment outcomes to early chemo-induced metabolic changes along three relevant axes of TNBC chemoresistance (Glycolysis, Oxidative Phosphorylation and Fatty Acid Oxidation) at high spatial resolutions. Here, we validated our system’s ability to capture metabolic signatures of disease regression that accompany chemo-resistance by assessing metabolic reprogramming and tumor kinetics in MDA-MB-231 TNBC xenografts. Xenografts were established by orthotopic cell injection and mice were treated with Paclitaxel (PTX), a commonly used chemotherapeutic drug in TNBC treatment, under a conventional maximum dose density regimen. Mice were monitored for drug response via caliper measurements. Xenografts were partially sensitive to PTX: tumor kinetics showed an initial response to PTX, a dormancy period, and a resurgence in tumor volume at ~60 days post drug withdrawal (n=3). A separate cohort of mice were implanted with window chambers on the primary tumor and imaged every two days with previously validated fluorophores 2-NBDG, TMRE and Bodipy to directly report on glucose uptake, mitochondrial membrane potential or fatty acid uptake, respectively. Wide field fluorescence imaging showed a significant increase in TMRE as early as two days after the 3rd PTX dose (n=5, p<0.05) and a significant decrease in 2-NBDG as early as two days after 5th PTX dose (n=5, p<0.05). No significant changes in Bodipy were seen. Decreased 2-NBDG and increased TMRE remained up to 40 days after drug withdrawal. Consistent with the literature, our results point towards a metabolic switch from glycolysis to mitochondrial respiration and suggests decreased use of glucose as a metabolic substrate. For this cell line, no altered uptake of fatty acids was detected, possibly suggesting amino acid catabolism as a fuel for mitochondrial respiration. This study confirms our platform’s ability to capture the resulting metabolic changes following chemotherapy and during disease regression. We aim to use this system to visualize and exploit in vivo metabolic vulnerabilities of disease regression that correlate to local and distal recurrences. Citation Format: Enakshi Devi Sunassee, Elizabeth Maydew, Sultan Erturk, Megan Madonna, Brian Crouch, Nirmala Ramanujam. Optical imaging captures chemo-induced metabolic reprogramming during disease regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2465.

The Role of Annexin A6 in Triple‐negative breast cancer metabolism and disease progression

The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying therapeutic resistance in solid tumors, including the hard‐to‐treat triple‐negative breast cancer (TNBC) subtype. Here, we report the synergism between Annexin A6 (AnxA6) and Lapatinib‐resistance (Lap‐R) in the context of metabolic reprogramming. Metabolic profiling of TNBC cell lines show extensive diversity amongst the proliferative basal‐like (BSL) vs the highly motile mesenchymal‐like (MSL) molecular subtypes. In this study, we demonstrate that down regulation of AnxA6 in AnxA6‐, expressing and Lap‐R cell lines attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, diminishing the overall energy phenotype of the cell. Additionally, AnxA6‐depletion altered lipid metabolism by decreasing lipid droplet accumulation, but enhancing the mitochondrial uptake and degradation of free fatty acids for ATP production via fatty acid oxidation (FAO). Our NMR‐based metabolomics revealed that AnxA6 depleted and Lap‐R TNBC cells rely on active amino acid metabolites and gluconeogenic precursors including alanine, glycine, and lactic acid to sustain their high proliferative rate. Collectively, these results demonstrate that the expression of AnxA6 underlies TNBC metabolic dependency and hence provide novel insights into the failure of EGFR‐targeted therapies as therapeutic options for TNBC patients.

Abstract PO-148: The role of annexin A6 in triple-negative breast cancer metabolism and disease progression

Abstract Triple-negative breast cancer (TNBC), which accounts for up to 17% of all breast cancer cases in the Unites States, disproportionately affects premenopausal African American and Hispanic women. Here, we report the synergistic function of the tumor suppressor annexin-A6 (AnxA6) and Lapatinib-resistance (Lap-R) in the context of cellular bioenergetics. Metabolic profiling of TNBC cell lines show extensive diversity amongst basal-like (BSL) vs mesenchymal-like (MSL) molecular subtypes. Down regulation of AnxA6 in AnxA6- expressing and Lap-R cell lines attenuated mitochondrial respiration, glycolytic function, and cellular ATP production capacity, decreasing the overall metabolic plasticity of the cell. Additionally, AnxA6-depletion altered lipid metabolism by enhancing the mitochondrial uptake of cytosolic fatty acids for β-oxidation. NMR-based metabolomics revealed that AnxA6 depleted and/or Lap-R TNBC cells have a greater dependency on gluconeogenic precursors including glycine, alanine, lactic acid, and oxaloacetic acid as survival mechanisms. Taken together, this study proposes that altered expression of AnxA6 is accompanied by significant bioenergetic adaptations and hence provide novel insights into the failure of EGFR-targeted therapies as therapeutic options and disease progression in patients with triple-negative breast cancer. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. The role of annexin A6 in triple-negative breast cancer metabolism and disease progression [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-148.

IL-13 Alleviates Cardiomyocyte Apoptosis by Improving Fatty Acid Oxidation in Mitochondria.

Sepsis-induced cardiac injury (SIC) is one of the most common complications in the intensive care unit (ICU) with high morbidity and mortality. Mitochondrial dysfunction is one of the main reasons for SIC, and Interleukin-13 (IL-13) is a master regulator of mitochondria biogenesis. The aim of the present study was to investigate the role of IL-13 in SIC and explore the underlying mechanism. It was found that reactive oxygen species (ROS) production and apoptosis were significantly increased in lipopolysaccharide (LPS)-stimulated primary cardiomyocytes, which was accompanied with obvious mitochondria dysfunction. The results of RNA-sequencing (RNA-seq), mitochondrial membrane potential, fatty acid uptake and oxidation rate suggested that treatment with IL-13 could restore the function and morphology of mitochondria, indicating that it played an important role in protecting septic cardiomyocytes. These findings demonstrated that IL-13 alleviated sepsis-induced cardiac inflammation and apoptosis by improving mitochondrial fatty acid uptake and oxidation, suggesting that IL-13 may prove to be a potential promising target for SIC treatment.

Cyclic AMP-binding protein Epac1 acts as a metabolic sensor to promote cardiomyocyte lipotoxicity

Cyclic adenosine monophosphate (cAMP) is a master regulator of mitochondrial metabolism but its precise mechanism of action yet remains unclear. Here, we found that a dietary saturated fatty acid (FA), palmitate increased intracellular cAMP synthesis through the palmitoylation of soluble adenylyl cyclase in cardiomyocytes. cAMP further induced exchange protein directly activated by cyclic AMP 1 (Epac1) activation, which was upregulated in the myocardium of obese patients. Epac1 enhanced the activity of a key enzyme regulating mitochondrial FA uptake, carnitine palmitoyltransferase 1. Consistently, pharmacological or genetic Epac1 inhibition prevented lipid overload, increased FA oxidation (FAO), and protected against mitochondrial dysfunction in cardiomyocytes. In addition, analysis of Epac1 phosphoproteome led us to identify two key mitochondrial enzymes of the the β-oxidation cycle as targets of Epac1, the long-chain FA acyl-CoA dehydrogenase (ACADL) and the 3-ketoacyl-CoA thiolase (3-KAT). Epac1 formed molecular complexes with the Ca2+/calmodulin-dependent protein kinase II (CaMKII), which phosphorylated ACADL and 3-KAT at specific amino acid residues to decrease lipid oxidation. The Epac1-CaMKII axis also interacted with the α subunit of ATP synthase, thereby further impairing mitochondrial energetics. Altogether, these findings indicate that Epac1 disrupts the balance between mitochondrial FA uptake and oxidation leading to lipid accumulation and mitochondrial dysfunction, and ultimately cardiomyocyte death.

Increasing fatty acid oxidation elicits a sex-dependent response in failing mouse hearts

BackgroundReduced fatty acid oxidation (FAO) is a hallmark of metabolic remodeling in heart failure. Enhancing mitochondrial long-chain fatty acid uptake by Acetyl-CoA carboxylase 2 (ACC2) deletion increases FAO and prevents cardiac dysfunction during chronic stresses, but therapeutic efficacy of this approach has not been determined. MethodsMale and female ACC2 f/f-MCM (ACC2KO) and their respective littermate controls were subjected to chronic pressure overload by TAC surgery. Tamoxifen injection 3 weeks after TAC induced ACC2 deletion and increased FAO in ACC2KO mice with pathological hypertrophy. ResultsACC2 deletion in mice with pre-existing cardiac pathology promoted FAO in female and male hearts, but improved cardiac function only in female mice. In males, pressure overload caused a downregulation in the mitochondrial oxidative function. Stimulating FAO by ACC2 deletion caused unproductive acyl-carnitine accumulation, which failed to improve cardiac energetics. In contrast, mitochondrial oxidative capacity was sustained in female pressure overloaded hearts and ACC2 deletion improved myocardial energetics. Mechanistically, we revealed a sex-dependent regulation of PPARα signaling pathway in heart failure, which accounted for the differential response to ACC2 deletion. ConclusionMetabolic remodeling in the failing heart is sex-dependent which could determine the response to metabolic intervention. The findings suggest that both mitochondrial oxidative capacity and substrate preference should be considered for metabolic therapy of heart failure.

Metabolic Remodeling in the Pressure-Loaded Right Ventricle: Shifts in Glucose and Fatty Acid Metabolism-A Systematic Review and Meta-Analysis.

BackgroundRight ventricular (RV) failure because of chronic pressure load is an important determinant of outcome in pulmonary hypertension. Progression towards RV failure is characterized by diastolic dysfunction, fibrosis and metabolic dysregulation. Metabolic modulation has been suggested as therapeutic option, yet, metabolic dysregulation may have various faces in different experimental models and disease severity. In this systematic review and meta‐analysis, we aimed to identify metabolic changes in the pressure loaded RV and formulate recommendations required to optimize translation between animal models and human disease.Methods and ResultsMedline and EMBASE were searched to identify original studies describing cardiac metabolic variables in the pressure loaded RV. We identified mostly rat‐models, inducing pressure load by hypoxia, Sugen‐hypoxia, monocrotaline (MCT), pulmonary artery banding (PAB) or strain (fawn hooded rats, FHR), and human studies. Meta‐analysis revealed increased Hedges’ g (effect size) of the gene expression of GLUT1 and HK1 and glycolytic flux. The expression of MCAD was uniformly decreased. Mitochondrial respiratory capacity and fatty acid uptake varied considerably between studies, yet there was a model effect in carbohydrate respiratory capacity in MCT‐rats.ConclusionsThis systematic review and meta‐analysis on metabolic remodeling in the pressure‐loaded RV showed a consistent increase in glucose uptake and glycolysis, strongly suggest a downregulation of beta‐oxidation, and showed divergent and model‐specific changes regarding fatty acid uptake and oxidative metabolism. To translate metabolic results from animal models to human disease, more extensive characterization, including function, and uniformity in methodology and studied variables, will be required.

Lipidomics Reveals Triacylglycerol Accumulation Due to Impaired Fatty Acid Flux in Opa1-Disrupted Fibroblasts.

OPA1 is a dynamin GTPase implicated in mitochondrial membrane fusion. Despite its involvement in lipid remodeling, the function of OPA1 has never been analyzed by whole-cell lipidomics. We used a nontargeted, reversed-phase lipidomics approach, validated for cell cultures, to investigate OPA1-inactivated mouse embryonic fibroblasts ( Opa1 -/- MEFs). This led to the identification of a wide range of 14 different lipid subclasses comprising 212 accurately detected lipids. Multivariate and univariate statistical analyses were then carried out to assess the differences between the Opa1 -/- and Opa1 +/+ genotypes. Of the 212 lipids identified, 69 were found to discriminate between Opa1 -/- MEFs and Opa1 +/+ MEFs. Among these lipids, 34 were triglycerides, all of which were at higher levels in Opa1 -/- MEFs with fold changes ranging from 3.60 to 17.93. Cell imaging with labeled fatty acids revealed a sharp alteration of the fatty acid flux with a reduced mitochondrial uptake. The other 35 discriminating lipids included phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamine, and sphingomyelins, mainly involved in membrane remodeling, and ceramides, gangliosides, and phosphatidylinositols, mainly involved in apoptotic cell signaling. Our results show that the inactivation of OPA1 severely affects the mitochondrial uptake of fatty acids and lipids through membrane remodeling and apoptotic cell signaling.

Mitochondrial changes synergize with long chain fatty acid derivatives to support Th17 inflammation in diabetes

Abstract Obesity-associated Type 2 diabetes (T2D) is driven by chronic inflammation. A combinatorial Th17 cytokine profile characterizes and mathematically predicts T2D in people, but the mechanisms that generate the Th17 profile are not clear. We tested the possibility that anaerobic glycolysis, which fuels inflammatory cytokine production from multiple immune cell types, drives the T2D-associated Th17 profile. We showed that activated PBMCs and purified CD4+T cells from T2D subjects prefer anaerobic glucose metabolism to produce ATP regardless of fuel availability. Unexpectedly, glucose starvation did not abrogate the T2D-predictive Th17 profile. Gene expression array suggested that mitochondrial fatty acid uptake catalyzing protein CPT1a differentiated PBMCs from T2D and ND subjects. CPT1a inhibition by etomoxir strongly down-regulated the Th17 profile, surprisingly independent of CPT1a-mediated fatty acid oxidation (OXPHOS). These data suggest that OXPHOS-independent mitochondrial changes that are also glucose-independent support the Th17 profile in T2D. Analyses from T2D vs ND PBMCs showed a lower CACT:CPT1a protein ratio in T2D, indicating defects in lipid uptake and thus defects in lipid flux. We knocked down CACT protein and overloaded PBMCs from lean subjects with fatty acylcarnitines to mimic lipid uptake defects in T2D. 16C-fatty acylcarnitine, but not 6C- or 10C-derivatives, increased frequency of CD4+IL-17+T cells, and phenocopied the Th17 profile only in CACT knockdown cells. 16C-fatty acylcarnitine alone had no effect. We conclude that excessive long chain fatty acylcarnitine combines with dysfunctional mitochondria to support a T2D-associated Th17 profile largely independent of glycolysis.

Exercise Training Induces Depot-Specific Adaptations to White and Brown Adipose Tissue

SummaryExercise affects whole-body metabolism through adaptations to various tissues, including adipose tissue (AT). Recent studies investigated exercise-induced adaptations to AT, focusing on inguinal white adipose tissue (WAT), perigonadal WAT, and interscapular brown adipose tissue (iBAT). Although these AT depots play important roles in metabolism, they account for only ∼50% of the AT mass in a mouse. Here, we investigated the effects of 3 weeks of exercise training on all 14 AT depots. Exercise induced depot-specific effects in genes involved in mitochondrial activity, glucose metabolism, and fatty acid uptake and oxidation in each adipose tissue (AT) depot. These data demonstrate that exercise training results in unique responses in each AT depot; identifying the depot-specific adaptations to AT in response to exercise is essential to determine how AT contributes to the overall beneficial effect of exercise.

The saturation degree of fatty acids and their derived acylcarnitines determines the direct effect of metabolically active thyroid hormones on insulin sensitivity in skeletal muscle cells.

Using differentiated rat L6 cells, we studied the direct effect of 3,5,3'-triiodo-l-thyronine (T3) and 3,5-diiodo-l-thyronine (T2) on the response to insulin in presence of fatty acids with a varying degree of saturation. We found that T3 and T2 both invert the response to insulin by modulating Akt Ser473 phosphorylation in the presence of palmitate and oleate. Both hormones prevented palmitate-induced insulin resistance, whereas increased insulin sensitivity in the presence of oleate was reduced, with normalization to (or, in the case of T3, even below) control levels. Both hormones effectively reduced intracellular acylcarnitine concentrations. Interestingly, insulin sensitization was lowered by incubation of the myotubes with relevant concentrations of palmitoylcarnitines (C16) and increased by oleylcarnitines and linoleylcarnitines (C18:1 and C18:2, respectively). The efficiency of mitochondrial respiration decreased in the order palmitate-oleate-linoleate; in the presence of palmitate, only T3 increased ATP synthesis-independent cellular respiration and mitochondrial respiratory complex activities. Both hormones modulated gene expression and enzyme activities related to insulin sensitivity, glucose metabolism, and lipid handling. Although T2 and T3 differentially regulated the expression of relevant genes involved in glucose metabolism, they equally stimulated related metabolic activities. T2 and T3 differentially modulated mitochondrial fatty acid uptake and oxidation in the presence of each fatty acid. The results show that T2 and T3 both invert the fatty acid-induced response to insulin but through different mechanisms, and that the outcome depends on the degree of saturation of the fatty acids and their derived acylcarnitines.-Giacco, A., delli Paoli, G., Senese, R., Cioffi, F., Silvestri, E., Moreno, M., Ruoppolo, M., Caterino, M., Costanzo, M., Lombardi, A., Goglia, F., Lanni, A., de Lange, P. The saturation degree of fatty acids and their derived acylcarnitines determines the direct effect of metabolically active thyroid hormones on insulin sensitivity in skeletal muscle cells.