Mitochondrial Fatty Acid Beta-oxidation Research Articles

#49 Uremic toxins and inflammation: Metabolic pathways affected in stage 5 kidney disease

Abstract Background and Aims Chronic kidney disease (CKD) is a global health threat, affecting around 12% of the population. The uremic phenotype associated with CKD is considered a predictor for the high cardiovascular and overall mortality in these patients and inflammation is regarded as an essential contributor to it. We seek to discover new inflammation-related metabolites in the CKD population through the use of semitargeted metabolomics, in order to gain insight into the pathways involved and identify potential biomarkers. Method The study employed an observational prospective design to investigate metabolite impact on inflammation in 50 non-dialysis-dependent stage 5 CKD patients. Blood samples were collected before the start of the first haemodialysis session. A semitargeted metabolomic approach was followed, utilising liquid chromatography and high-resolution mass spectrometry with an in-house library of polar compounds. Univariate (Mann-Whitney test) and multivariate (logistic regression with LASSO regularization) analyses identified metabolomic variables associated with inflammation. Results Univariate analysis revealed that adenosine-5′-phosphosulfate (APS) (p-value (p) 0.0211), dimethyglycine (p 0.0015), pyruvate (p 0.0066), lactate (p 0.0156) and 2-ketobutyric acid (p 0.0458) were significantly increased with inflammation, with differences over 1,5-fold changes (inflammation = yes vs inflammation = no). Cholic acid (p 0.01655), homogentisic acid (p 0.0007), 2-phenylpropionic acid (p 0.0351) and 3-indoxylsulfate (p 0.0087) show similar patterns but in the opposite direction, as observed in the volcano plot in Fig. 1. Multivariate analysis revealed significant associations between metabolites and inflammation in CKD stage 5 patients. Specifically, odds ratios for N-Butyrylglycine (OR: 1.87), dimethylglycine (OR: 1.81), 2-Oxoisopentanoic acid (OR: 1.70), and pyruvate (OR: 1.71) were related to a higher probability of experiencing inflammation. Conversely, homogentisic acid (OR: 072), 2-Phenylpropionic acid (OR: 0.77), and 2-Methylglutaric acid (OR: 0.78) exhibited an OR<1, suggestive of decreased probability. This is graphically represented in Fig. 2. Conclusion In our semi-targeted metabolomics study on stage 5 CKD patients, we pinpointed various metabolites linked to inflammation. Notably, dimethylglycine, a uremic toxin, may contribute to vascular damage, while pyruvate's abnormal metabolism is a known feature in heart failure and chronic kidney disease. N-butyrilglycine and 2-oxoisopentanoic acid were also associated with inflammation, potentially tied to mitochondrial fatty acid beta oxidation and incomplete breakdown of branched-chain amino acids. APS, impacting bone development and intestinal permeability, showed a positive correlation with inflammation. Conversely, homogentisic acid and phenylpropionic acid were negatively correlated. While homogentisic acid's anti-inflammatory role isn't clear, phenylpropionic acid exhibits protective effects against hepatotoxicity by reducing immune response. 2-methylglutaric acid and cholic acid were inversely correlated with inflammation, suggesting a protective effect related to fatty acid metabolism and bile acid regulation. These identified metabolites enhance our understanding of CKD-associated inflammation and present strong candidates for future biomarker validation with potential for clinical translation.

Effect of Fasting During Time Restricted Feeding on Mitochondrial Beta-Oxidation Enzyme Expression in the Kidney Cortex of Adult Male Salt-Sensitive Dahl Rats

The Dahl salt-sensitive (SS) rat is a well-characterized model of SS hypertension and renal disease that exhibits enhanced Na+ reabsorption in the kidney. Prior research has shown that SS rats experience a reduction in fatty acid beta oxidation (FAO) in the kidney cortex when placed on a high salt (HS) diet. Time restricted feeding (TRF), a type of intermittent fasting in which food consumption is restricted to <10 hrs/day, has been shown to activate FAO. Since the proximal tubule is responsible for the highest amount of Na+ reabsorption, we are interested in understanding if intermittent salt intake via TRF can mitigate dysfunctional cortical metabolism and slow progression of renal damage. We hypothesized that fasting during TRF would activate FAO throughout the body, as well as in the kidney cortex. To study this, 10-week-old male SS rats were provided ad libitum access to 4.0% NaCl (high salt; HS) or 0.4% (low salt; LS) diet for one week. Rats were then placed into a Promethion Core system (Sable Systems International) for metabolic phenotyping, where some continued ad libitum feeding, and others began time restricted feeding (TRF) for an additional week (n=7-9/group). During TRF, food was provided for 8 hours during the active cycle (2200hrs-0600hrs). Rats were euthanized in the last two hours of the final fasting period. We found that during the last hour of the fasting period, when compared to ad libitum rats, both the LS TRF (0.824 ± 0.01 vs 0.986 ± 0.008) and HS TRF (0.830 ± 0.01 vs 0.980 ± 0.006) rats had a significant reduction in their respiratory exchange ratio (2-way ANOVA with Tukey multiple comparisons, p<0.05, mean ± SEM). This suggests that the TRF protocol induced a net whole-body shift in fuel utilization from primarily carbohydrates to a mix of fuel sources by the end of the 16-hour fasting period, regardless of diet. To investigate the effect of fasting during TRF on the kidney cortex, we performed western blot analysis of acyl-CoA dehydrogenase long chain (ACADL) and acetyl-CoA acyltransferase 2 (ACAA2), enzymes involved in initial and final steps of mitochondrial beta-oxidation, respectively. We found that HS rats had lower ACADL and ACAA2 expression than the LS rats (2-way ANOVA; main effect of HS, p<0.05), and there was no difference between ad libitum and TRF groups within LS or HS groups (2-way ANOVA; main effect of TRF, p>0.05). This data suggests that, even though whole-body FAO may be stimulated by TRF fasting, HS-induced suppression of mitochondrial FAO proteins ACADL and ACAA2 was not restored by TRF. Future studies will investigate the effects of the shift in fuel utilization on other metabolic pathways in the cortex and proximal tubules. Supported by the Advancing a Healthier Wisconsin Endowment, the Medical College of Wisconsin, and NIH 5T32HL007852-25. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Management and Outcomes of Very Long-Chain Acyl-CoA Dehydrogenase Deficiency (VLCAD Deficiency): A Retrospective Chart Review.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a rare genetic condition affecting the mitochondrial beta-oxidation of long-chain fatty acids. This study reports on the clinical outcomes of patients diagnosed by newborn screening with VLCAD deficiency comparing metabolic parameters, enzyme activities, molecular results, and clinical management. It is a single-center retrospective chart review of VLCAD deficiency patients who met the inclusion criteria between January 2002 and February 2020. The study included 12 patients, 7 of whom had an enzyme activity of more than 10%, and 5 patients had an enzyme activity of less than 10%. The Pearson correlation between enzyme activity and the C14:1 level at newborn screening showed a p-value of 0.0003, and the correlation between enzyme activity and the C14:1 level at diagnosis had a p-value of 0.0295. There was no clear correlation between the number of documented admissions and the enzyme activity level. Patients who had a high C14:1 value at diagnosis were started on a diet with a lower percentage of energy from long-chain triglycerides. The C14:1 result at diagnosis is the value that has been guiding our initial clinical management in asymptomatic diagnosed newborns. However, the newborn screening C14:1 value is the most sensitive predictor of low enzyme activity and may help guide dietary management.

Uremic Toxins and Inflammation: Metabolic Pathways Affected in Non-Dialysis-Dependent Stage 5 Chronic Kidney Disease.

Chronic kidney disease (CKD) affects approximately 12% of the global population, posing a significant health threat. Inflammation plays a crucial role in the uremic phenotype of non-dialysis-dependent (NDD) stage 5 CKD, contributing to elevated cardiovascular and overall mortality in affected individuals. This study aimed to explore novel metabolic pathways in this population using semi-targeted metabolomics, which allowed us to quantify numerous metabolites with known identities before data acquisition through an in-house polar compound library. In a prospective observational design with 50 patients, blood samples collected before the initial hemodialysis session underwent liquid chromatography and high-resolution mass spectrometer analysis. Univariate (Mann-Whitney test) and multivariate (logistic regression with LASSO regularization) methods identified metabolomic variables associated with inflammation. Notably, adenosine-5'-phosphosulfate (APS), dimethylglycine, pyruvate, lactate, and 2-ketobutyric acid exhibited significant differences in the presence of inflammation. Cholic acid, homogentisic acid, and 2-phenylpropionic acid displayed opposing patterns. Multivariate analysis indicated increased inflammation risk with certain metabolites (N-Butyrylglycine, dimethylglycine, 2-Oxoisopentanoic acid, and pyruvate), while others (homogentisic acid, 2-Phenylpropionic acid, and 2-Methylglutaric acid) suggested decreased probability. These findings unveil potential inflammation-associated biomarkers related to defective mitochondrial fatty acid beta oxidation and branched-chain amino acid breakdown in NDD stage 5 CKD, shedding light on cellular energy production and offering insights for further clinical validation.

Hypertrophic cardiomyopathy and lactic acidosis in a child with acyl-CoA dehydrogenase 9 deficiency. Review of the literature and clinical observation

Introduction. Acyl-CoA dehydrogenase 9 deficiency (mitochondrial complex I deficiency) is an autosomal recessive disease from the heterogeneous group of disorders of mitochondrial β-oxidation of fatty acids caused by mutations in the ACAD9 gene. The disease is characterized by a wide range of clinical manifestations, the most common of which are metabolic acidosis, hypertrophic cardiomyopathy, muscle hypotonicity, and impaired motor skills. The article presents the first Russian clinical observation of a rare variant of hypertrophic cardiomyopathy with early debut in a patient with mitochondrial complex I deficiency caused by homozygous mutation c.659C>T (p.A220V) in the ACAD9 gene and emphasizes the importance of early diagnosis of the disease and complex drug therapy to prevent the development of severe complications.
 Objective: to describe the clinical course and management of a patient with the pathogenic c.659C>T (p.A220V) variant of the ACAD9 gene.
 Detailed analysis of anamnesis data, results of clinical, laboratory, instrumental diagnostic methods, and molecular genetic research performed using high-throughput sequencing and direct Sanger sequencing technology. 
 The article presents a literature review and detailed data on clinical observation of a child with homozygous c.659C>T (p.A220V) mutation in the ACAD9 gene diagnosed on the basis of the cardiology department of the National Medical Research Center for Children’s Health. Early disease markers and possibilities of complex drug therapy to prevent the development of severe complications are described.
 Conclusion. Disruption of mitochondrial beta-oxidation of fatty acids is a heterogeneous group of inherited diseases due to abnormal mitochondrial beta-oxidation and transport of carnitine and fatty acids in mitochondria. A feature of these diseases is the multisystem nature of the lesion and its progressive course. In some cases, the initial clinical manifestations may be various disorders of the cardiovascular system (cardiomyopathy, heart rhythm disturbances), which may cause death in neonatal period and early childhood. Early molecular genetic research provides accurate diagnosis and, accordingly, timely prescription of complex therapy.

Abstract 18959: Glycosphingolipid-Mediated Cardiomyocyte and Immune Cell Dysfunction in Hypoplastic Left Heart Syndrome

Introduction: Hypoplastic Left Heart Syndrome (HLHS) is the leading indication for heart transplant and the most common cause of cardiovascular death in infants. However, the mechanisms associated with heart failure progression in HLHS are poorly understood. Glycosphingolipids (GSLs) have been implicated in a variety of biological and disease processes, however, the role of GSLs in the context of HLHS pathophysiology have not been explored. Here we test the hypothesis that aberrant GSLs drive maladaptive cardiac and immune cell responses and predispose HLHS patients to pathologic remodeling and eventual cardiac dysfunction. Methods: GSL content was measured by mass-spectrometry. To test the direct effect of the specific GSL species, lactosylceramide (LacCer), cardiac and peripheral immune cells (PBMCs) were treated with liposomes containing 3uM LacCer (C18:0 and C24:0). Mitochondrial respiration was assessed using the Seahorse Bioanalyzer (Agilent). Expression of metabolic proteins were assessed by western blot. Statistical analysis between 2 groups was performed using an unpaired T-test. Results: LacCer is significantly increased in heart tissue and in isolated PBMCs from patients with HLHS (A), and isolated mitochondria are highly enriched with LacCer. Moreover, exogenous LacCer is sufficient to impair cardiomyocyte mitochondrial fatty acid beta-oxidation (B), induce ROS generation, and decrease the expression of metabolic proteins Additionally, exogenous LacCer is sufficient to promote inflammation and decrease mitochondrial bioenergetics in PBMCs (C). Conclusions: These data highlight a novel role for LacCer in modulating cardiomyocyte and immune cell phenotypes and suggest aberrant GSLs contribute to the HLHS pathophysiology. Elucidation of the molecular mechanisms involved in impaired immune cell and cardiomyocyte responses in HLHS is crucial and these data suggest a unique avenue for therapeutic intervention in this population.

Reprogramming of lipid metabolism in hepatocellular carcinoma resulting in downregulation of phosphatidylcholines used as potential markers for diagnosis and prediction

ABSTRACT Background Aberrant methylation and metabolic perturbations may deepen our understanding of hepatocarcinogenesis and help identify novel biomarkers for diagnosing hepatocellular carcinoma (HCC). We aimed to develop an HCC model based on a multi-omics. Research design and methods Four hundred patient samples (200 with HCC and 200 with hepatitis B virus-related liver disease (HBVLD)) were subjected to liquid chromatography-mass spectrometry and multiplex bisulfite sequencing. Integrative analysis of clinical data, CpG data, and metabolome for the 20 complete imputation datasets within a for-loopwas used to identify biomarker. Results Totally, 1,140 metabolites were annotated, of which 125 were differentially expressed. Lipid metabolism reprogramming in HCC, resulting in phosphatidylcholines (PC) significantly downregulated, partly due to the altered mitochondrial beta-oxidation of fatty acids with diverse chain lengths. Age, sex, serum-fetoprotein levels, cg05166871,cg14171514, cg18772205, PC (O-16:0/20:3(8Z, 11Z, 14Z)), and PC (16:1(9Z)/P-18:0) were used to develop the HCC model. The model presented a good diagnostic and an acceptable predictive performance. The cumulative incidence of HCC in low- and high-risk groups of HBVLD patients were 1.19% and 21.40%, respectively (p = 0.0039). Conclusions PCs serve as potential plasma biomarkers and help identify patients with HBVLD at risk of HCC who should be screened for early diagnosis and intervention.

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle.

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 105 TCID50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

Abstract 4852: Metabolomic rewiring in endocrine therapy resistant estrogen receptor positive breast cancer

Abstract Breast cancer (BC) is one of the most commonly diagnosed cancers worldwide and the most common in women in the United States. The majority of the BC are estrogen receptor alpha positive (ER+) and are likely to respond to endocrine therapy. Tamoxifen and fulvestrant are the most widely used hormonal treatments for ER+ BC. However, nearly half of patients receiving endocrine therapy suffer from the risk of recurrence due to either intrinsic or acquired resistance. Thus, it is vital to understand the mechanisms of resistance to endocrine therapy in ER+ BC. Metabolic reprogramming is one of the significant hallmarks of cancer and it is now known that many tumors utilize mitochondrial oxidative phosphorylation (OXPHOS) for their energy needs. Our lab previously reported that metastatic triple-negative breast cancer (TNBC), which does not express ER, has a high energy dependency on mitochondrial fatty acid beta-oxidation (FAO). In this project, we developed a gene signature by integrating the transcriptomic, metabolomic, and lipidomic data from FAO rate-limiting gene (CPT1) modulated TNBC cells. Our signature represented the FAO-regulated gene set relevant to metabolic or lipidomic changes. We discovered a significant reliance on this TNBC signature in endocrine-resistant ER+ BC. Clinical data suggests that our gene signature predicts the survival of endocrine-treated ER+ BC patients. Though increased fatty acid consumption is reported in tamoxifen-resistant cells, the role of FAO in endocrine treatment resistance is largely unclear. We then analyzed the metabolic reprogramming in ER+ BC cell lines after tamoxifen or fulvestrant therapy. Molecular, genetic, and metabolic analyses suggested that endocrine therapy-induce AMPK-FAO-OXPHOS signal activation in ER+ BC cells. Furthermore, the knockdown of CPT1, the rate-limiting enzyme of FAO, or treatment with FAO inhibitors significantly enhanced the response to endocrine therapies. We have also reported that FAO induces the autophosphorylation of the c-Src proto-oncogene in TNBC tumors. Thus, we analyzed this phenomenon in the endocrine-resistant ER+ BC cells. As expected, endocrine therapy-induced FAO activated the Src pathway in ER+ BC also. Moreover, in vitro, and in vivo studies confirmed that endocrine-resistant ER+ BC cells have increased sensitivity to FAO, OXPHOS, and Src inhibitors. Finally, analysis of clinical data suggests that low expression of FAO rate-limiting genes in the ER+ primary tumors have better recurrence-free and distant metastasis-free survival after endocrine therapy. Overall, our findings suggest that the gene signature generated from FAO-modulated TNBC cells predicts response to endocrine therapy in ER+ BC. Moreover, metabolic reprogramming in endocrine-resistant ER+ tumors induce FAO, OXPHOS, and Src pathways, providing potential targets to overcome endocrine-resistance ER+ BC patients. Citation Format: Songyeon Ahn, Junhyoung Park, Sandra L. Grimm, Badrajee WB Piyarathna, Nagireddy Putluri, Gokul Das, Cristian Coarfa, Benny A. Kaipparettu. Metabolomic rewiring in endocrine therapy resistant estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4852.

Mitochondrial Fatty Acid Beta-Oxidation Disorders in Children: Literature Review

Congenital mitochondrial fatty acid beta-oxidation disorders are a heterogeneous group of metabolic disorders characterized by impaired fatty acid metabolism in mitochondria. It results in central nervous system, skeletal muscle, cardiovascular system, and liver damage, as well as the development of nonketotic hypoglycemia. The age of disease manifestation and its severity range from severe (neonatal) to milder myopathic (adult) forms. The extension of the mass screening program in Russian Federation allows to detect these diseases during the first weeks of life. The availability of effective therapy for mitochondrial fatty acid beta-oxidation disorders, especially during early diagnosis, enables timely stabilization of the patient's condition and prevention of severe complications. Awareness of pediatricians, neonatologists, neurologists, and cardiologists about such diseases is the urgent task of modern pediatrics.

Abstract 15870: Integrated Transcriptomic and Proteomics Analyses Identify Lipid Metabolism as a Key Regulator of RV Size and Function in a Porcine Pulmonary Artery Banded Model

Introduction: Right ventricular (RV) dysfunction is the leading cause of death in pulmonary arterial hypertension (PAH), but the molecular determinants of RV dysfunction are not well defined. In addition, most molecular evaluations of RV dysfunction use small animal models, and data from large animal models is sparse. Methods: Control and pulmonary artery banded (PAB) male piglets were subjected to cardiac MRI to quantify RV ejection fraction (EF), mass, and end systolic volume (ESV). Total RNA was isolated from RV free wall specimens and subjected to Next Gene RNAsequencing. RV mitochondrial enrichments were analyzed with quantitative mass spectrometry to define RV proteome changes. Bioinformatics analyses integrated transcriptomics, proteomics, and MRI data to identified pathways associated with RV dysfunction, hypertrophy, and volume. Results: Both transcriptomic and proteomic profiling revealed significantly different molecular signatures when comparing control to PAB piglets. Both analyses identified metabolism of lipids, lysosome, and mitochondrial fatty acid beta-oxidation pathways were associated with RVEF, while dilated cardiomyopathy, and arrhythmogenic RV cardiomyopathy pathways correlated with RV mass, and metabolism, metabolic pathways, citric acid cycle, and respiratory electron transport pathways were associated with RVESV. Fatty acid metabolism was identified on our analyses, and ferroptosis, a nonapoptotic form of cell death initiated by lipid peroxidation, was associated with RVESV and RV mass. Finally, in addition to coding genes we identified 72 lncRNAs that were strongly (r>|0.85|) associated with RVEF. Conclusions: A combined proteomics and transcriptomics analysis identified multiple lncRNAs and lipid metabolism pathways associated with pathological RV remodeling and RV function. Future studies are needed to define how modulation of these pathways could impact modulate RV size and function in large animal models.

Proteomic analysis for the effects of non-saponin fraction with rich polysaccharide from Korean Red Ginseng on Alzheimer's disease in a mouse model

BackgroundThe most common type of dementia, Alzheimer's disease (AD), is marked by the formation of extracellular amyloid beta (Aβ) plaques. The impairments of axons and synapses appear in the process of Aβ plaques formation, and this damage could cause neurodegeneration. We previously reported that non-saponin fraction with rich polysaccharide (NFP) from Korean Red Ginseng (KRG) showed neuroprotective effects in AD. However, precise molecular mechanism of the therapeutic effects of NFP from KRG in AD still remains elusive. MethodsTo investigate the therapeutic mechanisms of NFP from KRG on AD, we conducted proteomic analysis for frontal cortex from vehicle-treated wild-type, vehicle-treated 5XFAD mice, and NFP-treated 5XFAD mice by using nano-LC-ESI-MS/MS. Metabolic network analysis was additionally performed as the effects of NFP appeared to be associated with metabolism according to the proteome analysis. ResultsStarting from 5,470 proteins, 2,636 proteins were selected for hierarchical clustering analysis, and finally 111 proteins were further selected for protein-protein interaction network analysis. A series of these analyses revealed that proteins associated with synapse and mitochondria might be linked to the therapeutic mechanism of NFP. Subsequent metabolic network analysis via genome-scale metabolic models that represent the three mouse groups showed that there were significant changes in metabolic fluxes of mitochondrial carnitine shuttle pathway and mitochondrial beta-oxidation of polyunsaturated fatty acids. ConclusionOur results suggested that the therapeutic effects of NFP on AD were associated with synaptic- and mitochondrial-related pathways, and they provided targets for further rigorous studies on precise understanding of the molecular mechanism of NFP.

Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts.

Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblasts-the effector cells of muscle growth and regeneration-are affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts. In Dmdmdx myoblasts lacking full-length dystrophin, the expression of 170 genes was significantly altered. Myod1 and key genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology analysis indicated enrichment in genes involved in muscle development and function. Functionally, we found increased myoblast proliferation, reduced chemotaxis and accelerated differentiation, which are all essential for myoregeneration. The defects were caused by the loss of expression of full-length dystrophin, as similar and not exacerbated alterations were observed in dystrophin-null Dmdmdx-βgeo myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and a dystrophic mouse muscle cell line, confirming the cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts showed alterations in the rate of glycolysis/gluconeogenesis, leukotriene metabolism, and mitochondrial beta-oxidation of various fatty acids. These results reveal the disease continuum: DMD defects in satellite cells, the myoblast dysfunction affecting muscle regeneration, which is insufficient to counteract muscle loss due to myofiber instability. Contrary to the established belief, our data demonstrate that DMD abnormalities occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease.

Exploratory lipidome and metabolome profiling contributes to understanding differences in high and normal ultimate pH beef

The aim of this work was to compare the lipidome and metabolome profiling in the Longissimus thoracis muscle early and late postmortem from high and normal ultimate pH (pHu) beef. Lipid profiling discriminated between high and normal pHu beef based on fatty acid metabolism and mitochondrial beta-oxidation of long chain saturated fatty acids at 30 min postmortem, and phospholipid biosynthesis at 44 h postmortem. Metabolite profiling also discriminated between high and normal pHu beef, mainly through glutathione, purine, arginine and proline, and glycine, serine and threonine metabolisms at 30 min postmortem, and glycolysis, TCA cycle, glutathione, tyrosine, and pyruvate metabolisms at 44 h postmortem. Lipid and metabolite profiles showed reduced glycolysis and increased use of alternative energy metabolic processes that were central to differentiating high and normal pHu beef. Phospholipid biosynthesis modification suggested high pHu beef experienced greater oxidative stress.

The Regulators of Peroxisomal Acyl-Carnitine Shuttle CROT and CRAT Promote Metastasis in Melanoma

Circulating tumor cells (CTCs) are the key link between a primary tumor and distant metastases, but once in the bloodstream loss of adhesion induces cell death. To identify mechanisms relevant for melanoma CTC survival we performed RNAseq and discovered that detached melanoma cells and isolated melanoma CTCs rewire lipid metabolism by up-regulating fatty acid transport and fatty acid beta-oxidation (FAO) related genes. In melanoma patients high expression of fatty acid transporters and FAO enzymes significantly correlates with reduced progression free and overall survival. Amongst the highest expressed regulators in melanoma CTCs were the carnitine-transferases CROT and CRAT, which control the shuttle of peroxisome derived medium-chain fatty acids (MCFAs) towards mitochondria to fuel mitochondrial FAO. Knockdown of CROT or CRAT and short-term treatment with peroxisomal or mitochondrial FAO inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. CROT and CRAT depletion could be rescued by MCFA supplementation, indicating that the peroxisomal supply of fatty acids is crucial for the survival of non-adherent melanoma cells. Our study identifies targeting the fatty acid based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the anti-metastatic activity of the FDA-approved drug ranolazine carries translational potential.

Ct-OATP1B3 promotes high-grade serous ovarian cancer metastasis by regulation of fatty acid beta-oxidation and oxidative phosphorylation

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy mainly due to its extensive metastasis. Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3), a newly discovered splice variant of solute carrier organic anion transporter family member 1B3 (SLCO1B3), has been reported to be overexpressed in several types of cancer. However, the biological function of Ct-OATP1B3 remains largely unknown. Here, we reveal that Ct-OATP1B3 is overexpressed in HGSOC and promotes the metastasis of HGSOC in vivo and in vitro. Mechanically, Ct-OATP1B3 directly interacts with insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), an RNA-binding protein, which results in enhancement of the mRNA stability and expression of carnitine palmitoyltransferase 1A (CPT1A) and NADH:Ubiquinone Oxidoreductase Subunit A2 (NDUFA2), leading to increased mitochondrial fatty acid beta-oxidation (FAO) and oxidative phosphorylation (OXPHOS) activities. The increased FAO and OXPHOS activities further facilitate adenosine triphosphate (ATP) production and cellular lamellipodia formation, which is the initial step in the processes of tumor cell migration and invasion. Taken together, our study provides an insight into the function and underlying mechanism of Ct-OATP1B3 in HGSOC metastasis, and highlights Ct-OATP1B3 as a novel prognostic marker as well as therapeutic target in HGSOC.

Phosphoglycerate Mutase 5 Expression Modulates Hepatocellular Steatosis

Phosphoglycerate Mutase 5 (PGAM5) is a serine/threonine phosphatase that plays a role in oxidant injury sensing, mitochondrial biogenesis, mitophagy, and multiple cell death pathways.Pgam5 null mice are protected from high‐fat diet induced obesity. This metabolic phenotype was attributed to upregulation of adaptive thermogenesis in brown adipose tissue, but hepatocyte specific lipid metabolism has not been evaluated. We hypothesize that hepatic PGAM5 expression level modulates hepatic steatosis. Using primary cultures of Pgam5 knockout hepatocytes, we have shown that palmitate induced steatosis is attenuated and that treatment with the long‐chain fatty acid impairs oxygen consumption rate specifically reducing the maximal respiratory capacity. Further, over‐expression of PGAM5 in human hepatoma cells exacerbates palmitate induced steatosis. Cumulatively, these results suggest that PGAM5 regulates hepatic lipogenesis and mitochondrial long‐chain fatty acid beta‐oxidation.

Fatty acid oxidation protects cancer cells from apoptosis by increasing mitochondrial membrane lipids.

Overcoming resistance to chemotherapies remains a major unmet need for cancers, such as triple-negative breast cancer (TNBC). Therefore, mechanistic studies to provide insight for drug development are urgently needed to overcome TNBC therapy resistance. Recently, an important role of fatty acid β-oxidation (FAO) in chemoresistance has been shown. But how FAO might mitigate tumor cell apoptosis by chemotherapy is unclear. Here, we show that elevated FAO activates STAT3 by acetylation via elevated acetyl-coenzyme A (CoA). Acetylated STAT3 upregulates expression of long-chain acyl-CoA synthetase 4 (ACSL4), resulting in increased phospholipid synthesis. Elevating phospholipids in mitochondrial membranes leads to heightened mitochondrial integrity, which in turn overcomes chemotherapy-induced tumor cell apoptosis. Conversely, in both cultured tumor cells and xenograft tumors, enhanced cancer cell apoptosis by inhibiting ASCL4 or specifically targeting acetylated-STAT3 is associated with a reduction in phospholipids within mitochondrial membranes. This study demonstrates a critical mechanism underlying tumor cell chemoresistance.

Bone Marrow Adipocytes Induce Metabolic Reprogramming of Multiple Myeloma Cells

Obesity-induced increases in bone marrow adipocyte (BMAd) numbers and volume are associated with an increased risk of multiple myeloma (MM). We analyzed gene expression from previously published public datasets and found that 11 out of 47 genes associated with fatty acid (FA) metabolism showed increasing trend from MGUS (monoclonal gammopathy of undetermined significance), MM, to plasma cell leukemia. These genes included ACC1, the first and rate-limiting step of de novo fatty acid biosynthesis and ECHS1, the second step of the mitochondrial fatty acid beta-oxidation pathway. We have previously shown that adipocytes support the growth of MM cells. However, the molecular mechanisms of interaction between MM cells and BMAd remain largely unknown. Here, we hypothesize that BMAd support MM cells through metabolic reprogramming.Here we sought to identify the molecular pathways involved in MM/ BMAd interaction. BM aspirates of MGUS, smoldering MM (SMM), and newly diagnosed MM (NDMM) patients were used to isolate fat-enriched BM fraction and BM stromal cells (BMSCs). Murine BMSC cell-line OP9, murine MM cell-line 5TGM1, and human MM cell lines MM.1S and OPM2 were obtained from ATCC or provided by collaborators and cultured as their respective standard procedures. In-vitro adipogenesis was induced in OP9 cells or BMSCs by supplementing media with dexamethasone, indomethacin, insulin, and IBMX. MM cells were co-cultured directly with pre- or mature adipocytes. Cell proliferation was assessed using CyQUANT NF Cell Proliferation Assay. Lipolysis was assessed by High Sensitivity Lipolysis Assay Kit (Sigma). Lipid uptake in MM cells was assessed by flow-cytometry analysis of the incorporation of fluorescent 12- or 16-carbon long-chain fatty acids BODIPY-FL-C12 and BODIPY-FL-C16, respectively, or LipidTox labelled FA from co-cultured adipocytes. In-vivo effects of excess FA on MM cell growth were assessed using a plasmacytoma model in CB17 SCID mice.In-vitro co-culture revealed that BMSC-derived adipocytes (Ad) from MGUS, SMM and NDMM donors increased the proliferation of MM.1S MM cells significantly. Similarly, mature murine OP9 Ad cells also increased the proliferation of 5TGM1 murine MM cells. Interestingly, co-cultures showed dramatic decrease in LipidTox-stained lipid-droplet size distribution, suggesting increased lipolysis in Ad. In the process of lipolysis, various lipase enzymes hydrolyze stored triglycerides into free fatty acids (FFA) and glycerol. Co-culture of 5TGM1 or OPM2 cells with OP9 mature Ad increased glycerol secretion in the conditioned media by more than 3-fold indicating that MM induce lipolysis in Ad. Consequently, BMAd from MGUS, SMM, and NDMM patients showed increased expression of genes responsible for lipolysis (NR1H3) and increased FA desaturation (SCD1, FASD2). Although MM cells lacked intracellular lipid storage, OPM2 and 5TGM1 MM cells rapidly took up BODIPY-C12 and -C16 FAs. The FA secreted from Ad were directly taken up by MM cells as shown by transfer of LipidTox-labeled lipids from OP9 Ad to unstained 5TGM1 or OPM2 MM cells as assessed by flow cytometry. The addition of Acipomox, a small-molecule inhibitor of lipolysis, decreased LipidTox signal in MM cells compared to untreated OP9 cells. FA are primarily transported into cells through FATP (1-6) or CD36 receptors. Bioinformatic analysis of public database showed that FATP1 and FATP4 were highly expressed in 21 human MM cell lines. Indeed, MM cells from NDMM patients expressed high levels of FATP1 and FATP4. The uptake of BODIPY-C12 and -C16 by 5TGM1 or OPM2 MM cells was significantly reduced in the presence of Lipofermata, a pharmacological small-molecule inhibitor of FATP. Lipidomic analysis of BM aspirates from MM patients showed altered expression of various FA, including arachidonic acid (AA). Low doses of AA (0.125 - 2 µM) increased the proliferation and viability of MM cells whereas high doses (25- 100µM) dramatically decreased it, indicating a bimodal cellular effect of AA. Peritumoral AA treatment in a plasmacytoma model in CB17 SCID mice using MM.1S cells dramatically decreased tumor volume along with the markers of proliferation.In summary, we show that MM cells induce lipolysis in BMAd and that the released FFA are then taken up by MM cells through FATPs. Inhibition of either BMAd lipolysis or FFA transporter into MM cells could be a potential novel strategy to prevent MM progression. DisclosuresFulzele: Constellation Pharma: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other.

Evaluation of Biosafety, Antiobesity, and Endothelial Cells Proliferation Potential of Basil Seed Extract Loaded Organic Solid Lipid Nanoparticle.

The present study aimed to synthesize solid lipid nanoparticles to enhance liposome-assisted intracellular uptake of basil seed active components in adipocytes and vascular smooth muscle cells to attain increased bioavailability. To obtain solid lipid nanoparticle (SLNp), the water phase containing basil seed extract (BSE) was encapsulated with lipid matrix containing chia seed phospholipids using homogenization and cold ultra-sonication method. The physicochemical characterization of BSE loaded solid lipid nanoparticles (BSE-SLNp) has been analyzed using Zetasizer, FT-IR, and TEM. The BSE-SLNp showed an average diameter of 20–110 nm on the day of preparation and it remains the same after 60 days of storage. The cytotoxicity assay confirmed that the BSE-SLNp did not produce toxicity in hMSCs, preadipocytes, or human umbilical vein endothelial cells (HUVECs) until the tested higher dose up to 64 μg/ml. During effective dose determination, 4 μg/ml of BSE-SLNp confirmed non-toxic and enhanced metabolic function in hMSCs, preadipocytes, and HUVECs. Biosafety assay confirmed normal nuclear morphology in PI staining and high mitochondrial membrane potential in JC-1 assay within 48 h in hMSCs. The maturing adipocyte treated with 4 μg/ml of BSE-SLNp significantly increased the mitochondrial efficiency and fatty acid beta-oxidation (PPARγC1α, UCP-1, and PRDM-16) related gene expression levels. Oxidative stress induced HUVECs treated with 4 μg/ml of BSE-SLNp potentially enhanced antioxidant capacity, cell growth, and microtubule development within 48 h H2O2 induced oxidative stressed HUVECs have shown 39.8% viable cells, but treatment with BSE-SLNp has shown 99% of viable cells within 48 h confirmed by Annexin-V assay. In addition, mitochondrial membrane potential (Δψm) increased to 89.4% confirmed by JC-1 assay. The observed DNA integrity, cell viability was confirmed by increased antioxidant and tumor suppressor-related gene expression levels. VEGF expression has been significantly increased and pro-inflammation-related mRNA levels were decreased in BSE-SLNp treated cells. In conclusion, enhanced adipocyte fatty acid oxidation is directly associated with decreased adipocytokine secretion which arrests obesity-associated comorbidities. In addition, suppressing vascular cell oxidative stress and metabolic inflammation supports vascular cell proliferation and arrests ageing-related vascular diseases.