Mitochondrial Fatty Acid Transport Research Articles

Inhibition of Crif1 protects fatty acid-induced POMC neuron-like cell-line damage by increasing CPT-1 function.

Hypothalamic proopiomelanocortin (POMC) neurons are sensors of signals that reflect the energy stored in the body. Inducing mild stress in proopiomelanocortin neurons protects them from the damage promoted by the consumption of a high-fat diet, mitigating the development of obesity; however, the cellular mechanisms behind these effects are unknown. Here, we induced mild stress in a proopiomelanocortin neuron cell line by inhibiting Crif1. In proopiomelanocortin neurons exposed to high levels of palmitate, the partial inhibition of Crif1 reverted the defects in mitochondrial respiration and ATP production; this was accompanied by improved mitochondrial fusion/fission cycling. Furthermore, the partial inhibition of Crif1 resulted in increased reactive oxygen species production, increased fatty acid oxidation, and reduced dependency on glucose for mitochondrial respiration. These changes were dependent on the activity of CPT-1. Thus, we identified a CPT-1-dependent metabolic shift toward greater utilization of fatty acids as substrates for respiration as the mechanism behind the protective effect of mild stress against palmitate-induced damage of proopiomelanocortin neurons.NEW & NOTEWORTHY Saturated fats can damage hypothalamic neurons resulting in positive energy balance, and this is mitigated by mild cellular stress; however, the mechanisms behind this protective effect are unknown. Using a proopiomelanocortin cell line, we show that under exposure to a high concentration of palmitate, the partial inhibition of the mitochondrial protein Crif1 results in protection due to a metabolic shift warranted by the increased expression and activity of the mitochondrial fatty acid transporter CPT-1.

Immunometabolic features of natural killer cells are associated with infection outcomes in critical illness.

Immunosuppression increases the risk of nosocomial infection in patients with chronic critical illness. This exploratory study aimed to determine the immunometabolic signature associated with nosocomial infection during chronic critical illness. We prospectively recruited patients who were admitted to the respiratory care center and who had received mechanical ventilator support for more than 10 days in the intensive care unit. The study subjects were followed for the occurrence of nosocomial infection until 6 weeks after admission, hospital discharge, or death. The cytokine levels in the plasma samples were measured. Single-cell immunometabolic regulome profiling by mass cytometry, which analyzed 16 metabolic regulators in 21 immune subsets, was performed to identify immunometabolic features associated with the risk of nosocomial infection. During the study period, 37 patients were enrolled, and 16 patients (43.2%) developed nosocomial infection. Unsupervised immunologic clustering using multidimensional scaling and logistic regression analyses revealed that expression of nuclear respiratory factor 1 (NRF1) and carnitine palmitoyltransferase 1a (CPT1a), key regulators of mitochondrial biogenesis and fatty acid transport, respectively, in natural killer (NK) cells was significantly associated with nosocomial infection. Downregulated NRF1 and upregulated CPT1a were found in all subsets of NK cells from patients who developed a nosocomial infection. The risk of nosocomial infection is significantly correlated with the predictive score developed by selecting NK cell-specific features using an elastic net algorithm. Findings were further examined in an independent cohort of COVID-19-infected patients, and the results confirm that COVID-19-related mortality is significantly associated with mitochondria biogenesis and fatty acid oxidation pathways in NK cells. In conclusion, this study uncovers that NK cell-specific immunometabolic features are significantly associated with the occurrence and fatal outcomes of infection in critically ill population, and provides mechanistic insights into NK cell-specific immunity against microbial invasion in critical illness.

NF1 deficiency drives metabolic reprogramming in ER+ breast cancer

ObjectiveNF1 is a tumor suppressor gene and its protein product, neurofibromin, is a negative regulator of the RAS pathway. NF1 is one of the top driver mutations in sporadic breast cancer such that 27 % of breast cancers exhibit damaging NF1 alterations. NF1 loss-of-function is a frequent event in the genomic evolution of estrogen receptor (ER)+ breast cancer metastasis and endocrine resistance. Individuals with Neurofibromatosis type 1 (NF) – a disorder caused by germline NF1 mutations – have an increased risk of dying from breast cancer [1–4]. NF-related breast cancers are associated with decreased overall survival compared to sporadic breast cancer. Despite numerous studies interrogating the role of RAS mutations in tumor metabolism, no study has comprehensively profiled the NF1-deficient breast cancer metabolome to define patterns of energetic and metabolic reprogramming. The goals of this investigation were (1) to define the role of NF1 deficiency in estrogen receptor-positive (ER+) breast cancer metabolic reprogramming and (2) to identify potential targeted pathway and metabolic inhibitor combination therapies for NF1-deficient ER + breast cancer. MethodsWe employed two ER+ NF1-deficient breast cancer models: (1) an NF1-deficient MCF7 breast cancer cell line to model sporadic breast cancer, and (2) three distinct, Nf1-deficient rat models to model NF-related breast cancer [1]. IncuCyte proliferation analysis was used to measure the effect of NF1 deficiency on cell proliferation and drug response. Protein quantity was assessed by Western Blot analysis. We then used RNAseq to investigate the transcriptional effect of NF1 deficiency on global and metabolism-related transcription. We measured cellular energetics using Agilent Seahorse XF-96 Glyco Stress Test and Mito Stress Test assays. We performed stable isotope labeling and measured [U–13C]-glucose and [U–13C]-glutamine metabolite incorporation and measured total metabolite pools using mass spectrometry. Lastly, we used a Bliss synergy model to investigate NF1-driven changes in targeted and metabolic inhibitor synergy. ResultsOur results revealed that NF1 deficiency enhanced cell proliferation, altered neurofibromin expression, and increased RAS and PI3K/AKT pathway signaling while constraining oxidative ATP production and restricting energetic flexibility. Neurofibromin deficiency also increased glutamine influx into TCA intermediates and dramatically increased lipid pools, especially triglycerides (TG). Lastly, NF1 deficiency alters the synergy between metabolic inhibitors and traditional targeted inhibitors. This includes increased synergy with inhibitors targeting glycolysis, glutamine metabolism, mitochondrial fatty acid transport, and TG synthesis. ConclusionsNF1 deficiency drives metabolic reprogramming in ER+ breast cancer. This reprogramming is characterized by oxidative ATP constraints, glutamine TCA influx, and lipid pool expansion, and these metabolic changes introduce novel metabolic-to-targeted inhibitor synergies.

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.

Triple Therapy with Metformin, Ketogenic Diet, and Metronomic Cyclophosphamide Reduced Tumor Growth in MYCN-Amplified Neuroblastoma Xenografts.

Neuroblastoma (NB) is a childhood cancer in which amplification of the MYCN gene is the most acknowledged marker of poor prognosis. MYCN-amplified NB cells rely on both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Previously, we demonstrated that a ketogenic diet (KD) combined with metronomic cyclophosphamide (CP) delayed tumor growth in MYCN-amplified NB xenografts. The anti-diabetic drug metformin (MET) also targets complex I of the OXPHOS system. Therefore, MET-induced disruptions of mitochondrial respiration may enhance the anti-tumor effect of CP when combined with a KD. In this study, we found that MET decreased cell proliferation and mitochondrial respiration in MYCN-amplified NB cell lines, while the combination of KD, MET, and low-dose CP (triple therapy) also reduced tumor growth and improved survival in vivo in MYCN-amplified NB xenografts. Gene ontology enrichment analysis revealed that this triple therapy had the greatest effect on the transcription of genes involved in fatty acid ß-oxidation, which was supported by the increased protein expression of CPT1A, a key mitochondrial fatty acid transporter. We suspect that alterations to ß-oxidation alongside the inhibition of complex I may hamper mitochondrial energy production, thus explaining these augmented anti-tumor effects, suggesting that the combination of MET and KD is an effective adjuvant therapy to CP in MYCN-amplified NB xenografts.

Altered acylcarnitine metabolism and inflexible mitochondrial fuel utilization characterize the loss of neonatal myocardial regeneration capacity

Myocardial regeneration capacity declines during the first week after birth, and this decline is linked to adaptation to oxidative metabolism. Utilizing this regenerative window, we characterized the metabolic changes in myocardial injury in 1-day-old regeneration-competent and 7-day-old regeneration-compromised mice. The mice were either sham-operated or received left anterior descending coronary artery ligation to induce myocardial infarction (MI) and acute ischemic heart failure. Myocardial samples were collected 21 days after operations for metabolomic, transcriptomic and proteomic analyses. Phenotypic characterizations were carried out using echocardiography, histology and mitochondrial structural and functional assessments. In both groups, MI induced an early decline in cardiac function that persisted in the regeneration-compromised mice over time. By integrating the findings from metabolomic, transcriptomic and proteomic examinations, we linked regeneration failure to the accumulation of long-chain acylcarnitines and insufficient metabolic capacity for fatty acid beta-oxidation. Decreased expression of the redox-sensitive mitochondrial Slc25a20 carnitine-acylcarnitine translocase together with a decreased reduced:oxidized glutathione ratio in the myocardium in the regeneration-compromised mice pointed to a defect in the redox-sensitive acylcarnitine transport to the mitochondrial matrix. Rather than a forced shift from the preferred adult myocardial oxidative fuel source, our results suggest the facilitation of mitochondrial fatty acid transport and improvement of the beta-oxidation pathway as a means to overcome the metabolic barrier for repair and regeneration in adult mammals after MI and heart failure.

Copper deficiency is an independent risk factor for mortality in patients with advanced liver disease.

Copper is an essential trace metal serving as a cofactor in innate immunity, metabolism, and iron transport. We hypothesize that copper deficiency may influence survival in patients with cirrhosis through these pathways. We performed a retrospective cohort study involving 183 consecutive patients with cirrhosis or portal hypertension. Copper from blood and liver tissues was measured using inductively coupled plasma mass spectrometry. Polar metabolites were measured using nuclear magnetic resonance spectroscopy. Copper deficiency was defined by serum or plasma copper below 80µg/dL for women or 70µg/dL for men. The prevalence of copper deficiency was 17% (N=31). Copper deficiency was associated with younger age, race, zinc and selenium deficiency, and higher infection rates (42% vs. 20%, p=0.01). Serum copper correlated positively with albumin, ceruloplasmin, hepatic copper, and negatively with IL-1β. Levels of polar metabolites involved in amino acids catabolism, mitochondrial transport of fatty acids, and gut microbial metabolism differed significantly according to copper deficiency status. During a median follow-up of 396 days, mortality was 22.6% in patients with copper deficiency compared with 10.5% in patients without. Liver transplantation rates were similar (32% vs. 30%). Cause-specific competing risk analysis showed that copper deficiency was associated with a significantly higher risk of death before transplantation after adjusting for age, sex, MELD-Na, and Karnofsky score (HR: 3.40, 95% CI, 1.18-9.82, p=0.023). In advanced cirrhosis, copper deficiency is relatively common and is associated with an increased infection risk, a distinctive metabolic profile, and an increased risk of death before transplantation.

Altered Liver Metabolism, Mitochondrial Function, Oxidative Status, and Inflammatory Response in Intrauterine Growth Restriction Piglets with Different Growth Patterns before Weaning.

Frequent occurrence of intrauterine growth restriction (IUGR) causes huge economic losses in the pig industry. Accelerated catch-up growth (CUG) in the early stage of life could restore multiple adverse outcomes of IUGR offspring; however, there is little knowledge about this beneficial phenomenon. We previously found that nutrient absorption related to intestinal function was globally promoted in CUG-IUGR piglets before weaning, which might be the dominant reason for CUG, but what this alteration could lead to in subsequent liver metabolism is still unknown. Firstly, a Normal, CUG, and non-catch-up growth (NCUG) piglet model before weaning was established by dividing eighty litters of newborn piglets into normal birth weight (NBW) and IUGR groups according to birth weight, and those piglets with IUGR but above-average weanling body weight were considered CUG, and the piglets with IUGR still below average body weight were considered NCUG at weaning day (d 26). Liver samples were collected and then systematically compared in glycolipid metabolism, mitochondrial function, antioxidant status, and inflammatory status among these three different growth models. Enhanced hepatic uptake of fatty acids, diminished de novo synthesis of fatty acids, and increased oxidation of fatty acids were observed in CUG livers compared to Normal and NCUG. In contrast, the NCUG liver showed enhanced glucose uptake and gluconeogenesis compared to Normal and CUG. We also observed deteriorating hepatic vacuolation in NCUG piglets, while increasing hepatic lipid deposition in CUG piglets. Besides, the expression of genes related to mitochondrial energy metabolism and biogenesis was reduced in CUG piglets and the phosphorylation level of AMPK was significantly higher compared to Normal (p < 0.05). Moreover, NCUG liver showed decreased T-AOC (p < 0.01) and GSH-PX (p < 0.05), increased MDA concentrations (p < 0.01), upregulated phosphorylation levels of ERK and NF-κB (p < 0.05), and elevated pro-inflammatory factors IL-1β, IL-6 and TNF-α (p < 0.05) compared to Normal. Furthermore, correlation analysis revealed a significant positive correlation between glucose metabolism and inflammatory factors, while a negative correlation between mitochondrial function-related genes and fatty acid transport. NGUG piglets showed simultaneous enhancement of glucose uptake and gluconeogenesis, as well as reduced antioxidant capacity and increased inflammatory status, whereas CUG comes at the expense of impaired hepatic mitochondrial function and pathological fat accumulation.

Irisin improves adiposity and exercise tolerance in a rat model of postmenopausal obesity through enhancing adipo-myocyte thermogenesis

The prevalence of obesity and its associated metabolic disorders, along with their healthcare costs, is rising exponentially. Irisin, an adipomyokine, may serve as a critical cross-organ messenger, linking skeletal muscle with adipose tissue and the liver to integrate the energy homeostasis under diet-induced obesity. We aimed to explore the putative role of irisin in the protection against obesity in a postmenopausal rat model by modulating energy expenditure (EE). Bilateral ovariectomy (OVX) was performed. After 3 weeks of recovery, the OVX rats were classified according to their dietary protocol into rats maintained on normal diets (ND) (OVX) or high-fat diet (HFD) groups. The HFD-fed animals were equally divided into OVX/HFD, or irisin-treated OVX/HFD groups. Sham rats, maintained on ND, were selected as the control group. We evaluated anthropometric, EE, and molecular biomarkers of browning and thermogenesis in inguinal white adipose tissue and skeletal muscle, and the activity of the proteins related to mitochondrial long chain fatty acid transport, oxidation, and glycolysis. HFD of OVX further deteriorated the disturbed glucose homeostasis, lipid profile, and the reduced irisin, thermogenic parameters in adipose tissue and skeletal muscle, and EE. Irisin treatment improved the lipid profile and insulin resistance. That was associated with reduced hepatic gluconeogenic enzyme activities and restored hepatic glycogen content. Irisin reduced ectopic lipid infiltration. Irisin augmented EE by activating non-shivering thermogenesis in muscle and adipose tissues and decreasing metabolic efficiency. Our experimental evidence suggests irisin’s use as a potential thermogenic agent, therapeutically targeting obesity in postmenopausal patients.Graphical abstractIrisin modulates the non-shivering thermogenesis in skeletal muscle and adipose tissue in postmenopausal model

P612: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)-DERIVED T-CELLS HAVE DISTURBED FATTY ACID METABOLISM, POSSIBLY CONTRIBUTING TO T-CELL DYSFUNCTION

Background: T-cells from CLL patients are dysfunctional and display reduced activation, proliferation and cytotoxicity upon in-vitro activation. Whilst the role of glucose metabolism in activated T cells is extensively studied, the contribution of other energy sources is less clear. Fatty acid (FA) and lipid uptake in T cells involves the scavenger membrane transporter CD36. CD36-mediated lipid transport has been identified to (metabolically) modulate intratumoral T cells (both Treg and cytotoxic CD8+ T cells). However, lipid metabolism and a potential role for CD36 has not been studied in the context of T cell dysfunction in CLL Aims: To examine the role of FA metabolism and the fatty acid transporter CD36 in acquired T-cell dysfunction in CLL. Methods: Untreated CLL patients with a white blood cell count (WBC) greater than 20×109 cells/L and, as control, age-matched healthy donors (HD) were studied. T cells were analyzed by flow cytometry, confocal microscopy or extracellular flux analysis (Seahorse), either directly after thawing or after a 2 day culture with or without αCD3/αCD28 antibodies. Results: Analysis of CD36+ vs CD36- CD4 and CD8 T-cells from HD revealed that CD36+ cells had higher long-chain fatty acid (LCFA) and glucose uptake, and higher expression of the glucose transporter GLUT-1 and the rate-limiting mitochondrial FA transporter CPT1α (Fig. 1A). The cells expressing CD36 were also more likely to increase their CD25 expression and mitochondrial mass upon stimulation, indicating a metabolic and functional advantage potentially conferred by the surface expression of CD36. The absolute amount and percentage of T cells (CD4 and CD8) expressing FA transporter CD36 was lower in CLL T-cells as compared to HD (Fig. 1B). The levels of LCFA uptake and expression of CPT1α were comparable, indicating that the lower amount of CD36 might successfully cater to the FA demands of resting CLL T cells(Fig. 1C). However, while stimulation of HD T-cells markedly upregulated CD36 abundance, LCFA uptake and CPT1α, concomitantly with the activation marker CD25, CLL-derived T-cells failed to mount a similar response (Fig. 1D). This lack of induction of CD36 expression was also observed in CLL-derived T cells that increased CD25, which implies that this response is independent of T-cell activation. The transcription factors peroxisome proliferator-activated receptors (PPAR) α and γ are important regulators of FA metabolism. We observed reduced expression of these transcription factors in resting CLL T cells. However, distinct from CD36, CLL T cells were indistinguishable from HD T-cells in their ability to increase levels of both PPARα and PPARγ upon stimulation, indicating that alternative mechanisms might be implicated in the regulation of CD36 and FA metabolism in CLL T cells. Image:Summary/Conclusion: Collectively, our results show that disturbances in FA metabolism are present on multiple levels in CLL T cells and that lack of LCFA uptake into CLL T-cells could represent a limiting step in FA metabolism. We also highlight CD36+ cells as a metabolically distinct T-cell subtype that is largely absent in CLL T-cells, independently of their activation status. We are currently investigating strategies to increase CD36 expression and FA metabolism in general in CLL T cells, which could ameliorate CLL T cell dysfunction and provide targets to improve autologous T cell therapies.

Dietary L-Carnitine Affects Leukocyte Count and Function in Dairy Cows Around Parturition.

In early lactation, an energy deficit leading to a negative energy balance (NEB) is associated with increased susceptibility to disease and has been shown to be an important factor during transition in dairy cows. L-carnitine as a key factor in the mitochondrial transport of fatty acids and subsequently for β-oxidation and energy release is known to modulate mitochondrial biogenesis and thus influence metabolism and immune system. In the current study, we characterized hematological changes around parturition and investigated the potential effects of dietary L-carnitine supplementation on immune cell functions. For this approach, dairy cows were assigned either to a control (CON, n = 30) or an L-carnitine group [CAR, n = 29, 25 g rumen-protected L-carnitine per cow and day (d)]. Blood samples were taken from d 42 ante partum (ap) until d 110 post-partum (pp), with special focus and frequent sampling from 0.5 to72 h post-calving to clarify the impact of L-carnitine supplementation on leukocyte count, formation of reactive oxygen species (ROS) in polymorphonuclear cells (PMN) and peripheral mononuclear cells (PBMC) and their phagocytosis activity. Blood cortisol concentration and the capacity of PBMC proliferation was also investigated. All populations of leukocytes were changed during the peripartal period, especially granulocytes showed a characteristic increase up to 4 h pp. L-carnitine supplementation resulted in increased levels of eosinophils which was particularly pronounced one day before to 4 h pp, indicating a possible enhanced support for tissue repair and recovery. Non-supplemented cows showed a higher phagocytic activity in PBMC as well as a higher phagocytic capacity of PMN during the most demanding period around parturition, which may relate to a decrease in plasma levels of non-esterified fatty acids reported previously. L-carnitine, on the other hand, led to an increased efficiency to form ROS in stimulated PMN. Finally, a short period around calving proved to be a sensitive period in which L-carnitine administration was effective.

Adiponectin overexpression in C2C12 myocytes increases lipid oxidation and myofiber transition.

Metabolic syndrome and obesity have detrimental effects on the metabolic function of the skeletal muscle. Mounting evidence indicates that patients with those conditions may present an increased ratio of glycolytic to oxidative fibers associated with a decrease in oxidative capacity. In this regard, adiponectin, a hormone mainly secreted by adipocytes that regulates glucose and lipid metabolism, has emerged as a myokine that could play an important role in this process. We aimed to investigate whether adiponectin overexpression in skeletal muscle might be a local protective mechanism, favoring fatty acid utilization. To that end, we generated an in vitro model of myocytes with upregulated endogenous adiponectin using a lentiviral carrier. We demonstrated that the adiponectin-transduced myocytes were able to produce and secrete fully functional adiponectin complexes. Adiponectin overexpression remarkably upregulated the mRNA level of myogenic regulatory factors as well as genes implicated in lipolysis (HSL, ATGL) and cellular and mitochondrial fatty acid transport (LPL, CD36, CPT1B). This was accompanied by increased isoproterenol-induced lipolysis and β-oxidation and reduced lipogenesis, whereas insulin-stimulated glucose uptake was unaltered in transduced myocytes. Lastly, the relative expression of the more glycolytic myofibers (MyHC IIb) compared to the more oxidative ones (MyHC I) was notably reduced. Our results showed that the released adiponectin acted in an autocrine/paracrine manner, increasing lipid oxidation in myocytes and leading to a transition of myofibers from the glycolytic to the oxidative type. In conclusion, muscle adiponectin overexpression might be a way to relieve muscle diseases caused by oxidative muscle fiber deficiency.

Resting skeletal muscle PNPLA2 (ATGL) and CPT1B are associated with peak fat oxidation rates in men and women but do not explain observed sex differences

What is the central question of this study? What is the relationship between proteins in skeletal muscle and adipose tissue determined at rest and at peak rates of fat oxidation in men and women? What is the main finding and its importance? The resting contents of proteins in skeletal muscle involved in triglyceride hydrolysis and mitochondrial lipid transport were more strongly associated with peak fat oxidation rates than proteins related to lipid transport or hydrolysis in adipose tissue. Although females displayed higher relative rates of fat oxidation than males, this was not explained by the proteins measured in this study, suggesting that other factors determine sex differences in fat metabolism. We explored key proteins involved in fat metabolism that might be associated with peak fat oxidation (PFO) and account for sexual dimorphism in fuel metabolism during exercise. Thirty-six healthy adults [15 women; 40±11years of age; peak oxygen consumption 42.5±9.5ml(kg body mass)-1 min-1 ; mean±SD] completed two exercise tests to determine PFO via indirect calorimetry. Resting adipose tissue and/or skeletal muscle biopsies were obtained to determine the adipose tissue protein content of PLIN1, ABHD5 (CGI-58), LIPE (HSL), PNPLA2 (ATGL), ACSL1, CPT1B and oestrogen receptorα (ERα) and the skeletal muscle protein content of FABP 3 (FABPpm), PNPLA2 (ATGL), ACSL1, CTP1B and ESR1 (ERα). Moderate strength correlations were found between PFO [in milligrams per kilogram of fat-free mass (FFM) per minute] and the protein content of PNPLA2 (ATGL) [rs =0.41 (0.03-0.68), P<0.05] and CPT1B [rs =0.45 (0.09-0.71), P<0.05] in skeletal muscle. No other statistically significant bivariate correlations were found consistently. Females had a greater relative PFO than males [7.1±1.9 vs. 4.5±1.3 and 7.3±1.7 vs. 4.8±1.2mg(kg FFM)-1 min-1 in the adipose tissue (n=14) and skeletal muscle (n=12) subgroups, respectively (P<0.05)]. No statistically significant sex differences were found in the content of these proteins. The regulation of PFO might involve processes relating to intramyocellular triglyceride hydrolysis and mitochondrial fatty acid transport, and adipose tissue is likely to play a more minor role than muscle. Sex differences in fat metabolism are likely to be attributable to factors other than the resting content of proteins in skeletal muscle and adipose tissue relating to triglyceride hydrolysis and fatty acid transport.

Sex Differences in Insulin Sensitivity are Related to Muscle Tissue Acylcarnitine But Not Subcellular Lipid Distribution.

Sex differences in insulin sensitivity are present throughout the life-span, with men having a higher prevalence of insulin resistance and diabetes compared with women. Differences in lean mass, fat mass, and fat distribution-particularly ectopic fat-have all been postulated to contribute to the sexual dimorphism in diabetes risk. Emerging data suggest ectopic lipid composition and subcellular localization are most relevant; however, it is not known whether they explain sex differences in obesity-induced insulin resistance. To address this gap, this study evaluated insulin sensitivity and subcellular localization of intramuscular triacylglycerol, diacylglycerol, and sphingolipids as well as muscle acylcarnitines and serum lipidomics in people with obesity. Insulin sensitivity was significantly lower in men (P < 0.05); however, no sex differences were found in localization of intramuscular triacylglycerol, diacylglycerol, or sphingolipids in skeletal muscle. In contrast, men had higher total muscle acylcarnitine (P < 0.05) and long-chain muscle acylcarnitine (P < 0.05), which were related to lower insulin sensitivity (r = -0.42, P < 0.05). Men also displayed higher serum ceramide (P = 0.05) and lysophosphatidylcholine (P < 0.01). These data reveal novel sex-specific associations between lipid species involved in the coupling of mitochondrial fatty acid transport, β-oxidation, and tricarboxylic acid cycle flux that may provide therapeutic targets to improve insulin sensitivity.

Abstract PS17-28: Abrupt involution of lactating mammary gland induces metabolic reprogramming conducive to pro-tumorigenic changes

Abstract Background: Epidemiological studies indicate that prolonged breast feeding reduces the risk of triple negative breast cancer (TNBC), which carries the worst prognosis. Prolonged breastfeeding allows gradual involution (GI) of the breast while lack of or short-term breast feeding leads to abrupt involution (AI). We developed a novel murine model mimicking AI and GI of breast, and found that GI offers better protection to mammary glands from tissue remodeling associated injuries. Our data showed that AI leads to the development of pro-tumorigenic microenvironment and ductal hyperplasia1. Tissue remodeling involves orchestrated cell death and repopulation, and is closely associated with metabolic reprogramming from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis. Such metabolic alterations can contribute to cellular changes aiding malignant transformation2. We used our murine model to evaluate whether AI affects cellular metabolism differentially when compared to GI. Methods: Wild-type mice of FVB background were used in all our experiments. Twelve to fourteen week old uniparous mice were allowed to nurse (6 pups/dam) for 7 days postpartum. All pups were removed on day7 postpartum from the dams in AI cohort and three each on day28 and day31 from dams in GI cohort. Whole mammary glands and sorted luminal progenitor (LP) cells harvested on postpartum day28 were subjected to Affymetrix microarray analysis. Gene Set Enrichment Analysis (GSEA) was used to compute pathway enrichments in AI vs. GI glands. Differentially expressed genes were validated using qRT-PCR. Mammary glands harvested on day28 postpartum were subjected to mass spectrometry based untargeted metabolic profiling using Agilent QTOF. Raw data were analyzed using XCMS to assess key metabolic networks altered in AI vs GI glands. Targeted analysis for lactate, pyruvate, succinate and palmitic acid were performed using C13 labelled internal standards to compare OXPHOS vs glycolysis reliance in the AI and GI glands. Results: We observed enrichment of mitochondrial OXPHOS pathway, fatty acid metabolism and Myc target genes in both whole mammary gland and LP cells of AI vs. GI mice. Adipogenesis and hypoxia related genes were enriched in AI-glands. We observed significant upregulation of genes involved in glucose transport and fatty acid synthesis in AI glands, namely, Glut-5, Cidea, Acss2, Acsm3, Acly, Atp6v0d2, Acot11, and Elvol3. Several factors indicating a higher reliance on OXPHOS vs glycolysis, such as, Ppar-γ, Pgc1α, Cpt-2, Srebp1c and Chrebp were upregulated in AI glands. Upregulation of Cpt-2 and Srebp1c in the AI glands indicate higher flux through fatty acid oxidation and reliance on cholesterol synthesis. Metabolomic profiling revealed significant alteration in L-carnitine, GMP and XMP in AI glands which reflect mitochondrial fatty acid transport and nucleotide biosynthesis via guanine-guanosine salvage pathway. Pyruvate and lactate associated with glycolysis were increased in GI vs. AI glands. Conclusion: We show for the first time that in the abruptly involuting (AI) mammary glands following short-term breast feeding, there is a significant shift in the metabolic pathways towards mitochondrial OXPHOS and fatty acid oxidation compared to GI glands. Studies are underway to determine the effect of this metabolic shift on cellular transformation and tumorigenesis and the potential to target these pathways to reverse the detrimental effects of AI. 1. Basree MM, Shinde N, Koivisto C, et al. Breast Cancer Res. 2019; 21(1):80. 2. Ward PS, Thompson CB. Cancer Cell. 2012; 21(3):297. Citation Format: Neelam Shinde, Kirti Kaul, Allen Zhang, Saba Mehra, Resham Mawalkar, Hee Kyung Kim, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Abrupt involution of lactating mammary gland induces metabolic reprogramming conducive to pro-tumorigenic changes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-28.

Plasma acylcarnitines and risk of lower-extremity functional impairment in older adults: a nested case\u2013control study

Elevated concentrations of acylcarnitines have been associated with higher risk of obesity, type 2 diabetes and cardiovascular disease. The aim of the present study was to assess the association between L-carnitine and acylcarnitine profiles, and 2-year risk of incident lower-extremity functional impairment (LEFI). This case–control study is nested in the Seniors-ENRICA cohort of community-dwelling older adults, which included 43 incident cases of LEFI and 86 age- and sex- matched controls. LEFI was assessed with the Short Physical Performance Battery. Plasma L-carnitine and 28 acylcarnitine species were measured. After adjusting for potential confounders, medium-chain acylcarnitines levels were associated with 2-year incidence of LEFI [odds ratio per 1-SD increase: 1.69; 95% confidence interval: 1.08, 2.64; p = 0.02]. Similar results were observed for long-chain acylcarnitines [odds ratio per 1-SD increase: 1.70; 95% confidence interval: 1.03, 2.80; p = 0.04]. Stratified analyses showed a stronger association between medium- and long-chain acylcarnitines and incidence of LEFI among those with body mass index and energy intake below the median value. In conclusion, higher plasma concentrations of medium- and long-chain acylcarnitines were associated with higher risk of LEFI. Given the role of these molecules on mitochondrial transport of fatty acids, our results suggest that bioenergetics dysbalance contributes to LEFI.

Crucial Role of Mammalian Glutaredoxin 3 in Cardiac Energy Metabolism in Diet-induced Obese Mice Revealed by Transcriptome Analysis.

Obesity is often associated with metabolic dysregulation and oxidative stress with the latter serving as a possible unifying link between obesity and cardiovascular complications. Glutaredoxins (Grxs) comprise one of the major antioxidant systems in the heart. Although Grx3 has been shown to act as an endogenous negative regulator of cardiac hypertrophy and heart failure, its metabolic impact on cardiac function in diet-induced obese (DIO) mice remains largely unknown. In the present study, analysis of Grx3 expression indicated that Grx3 protein levels, but not mRNA levels, were significantly increased in the hearts of DIO mice. Cardiac-specific Grx3 deletion (Grx3 CKO) mice were viable and grew indistinguishably from their littermates after being fed a high fat diet (HFD) for one month, starting at 2 months of age. After being fed with a HFD for 8 months (starting at 2 months of age); however, Grx3 CKO DIO mice displayed left ventricular systolic dysfunction with a significant decrease in ejection fraction and fractional shortening that was associated with heart failure. ROS production was significantly increased in Grx3 CKO DIO cardiomyocytes compared to control cells. Gene expression analysis revealed a significant decline in the level of transcripts corresponding to genes associated with processes such as fatty acid uptake, mitochondrial fatty acid transport and oxidation, and citrate cycle in Grx3 CKO DIO mice compared to DIO controls. In contrast, an increase in the level of transcripts corresponding to genes associated with glucose uptake and utilization were found in Grx3 CKO DIO mice compared to DIO controls. Taken together, these findings indicate that Grx3 may play a critical role in redox balance and as a metabolic switch in cardiomyocytes contributing to the development and progression of heart failure.

In Silico Identification of Novel Interactions for FABP5 (Fatty Acid-Binding Protein 5) with Nutraceuticals: Possible Repurposing Approach.

Fatty Acid Binding-Protein 5 (FABP5) is a cytoplasmic protein, which binds long-chain fatty acids and other hydrophobic ligands. This protein is implicated in several physiological processes including mitochondrial β-oxidation and transport of fatty acids, membrane phospholipid synthesis, lipid metabolism, inflammation and pain. In the present study, we used molecular docking tools to determine the possible interaction of FABP5 with six selected compounds retrieved form Drugbank. Our results showed that FABP5 binding pocket included 31 polar and non-polar amino acids, and these residues may be related to phosphorylation, acetylation, ubiquitylation, and mono-methylation. Docking results showed that the most energetically favorable compounds are NADH (-9.12kcal/mol), 5'-O-({[(Phosphonatooxy)phosphinato]oxy}phosphinato)adenosine (-8.62kcal/mol), lutein (-8.25kcal/mol), (2S)-2-[(4-{[(2-Amino-4-oxo-1,4,5,6,7,8-hexahydro-6-pteridinyl)methyl]amino}benzoyl)amino]pentanedioate (-7.17kcal/mol), Pteroyl-L-glutamate (-6.86kcal/mol) and (1S,3R,5E,7Z)-9,10-Secocholesta-5,7,10-triene-1,3,25-triol (-6.79kcal/mol). Common interacting residues of FABP5 with nutraceuticals included SER16, LYS24, LYS34, LYS40 and LYS17. Further, we used the SwissADME server to determine the physicochemical and pharmacokinetic characteristics and to predict the ADME parameters of the selected nutraceuticals after molecular analysis by docking with the FABP5 protein. Amongst all compounds, pteroyl-L-glutamate is the only one meeting the Lipinski's rule of five criteria, demonstrating its potential pharmacological use. Finally, our results also suggest the importance of FABP5 in mediating the anti-inflammatory activity of the nutraceutical compounds.

Mitochondrial Bioenergetics at the Onset of Drug Resistance in Hematological Malignancies: An Overview.

The combined derangements in mitochondria network, function and dynamics can affect metabolism and ATP production, redox homeostasis and apoptosis triggering, contributing to cancer development in many different complex ways. In hematological malignancies, there is a strong relationship between cellular metabolism, mitochondrial bioenergetics, interconnections with supportive microenvironment and drug resistance. Lymphoma and chronic lymphocytic leukemia cells, e.g., adapt to intrinsic oxidative stress by increasing mitochondrial biogenesis. In other hematological disorders such as myeloma, on the contrary, bioenergetics changes, associated to increased mitochondrial fitness, derive from the adaptive response to drug-induced stress. In the bone marrow niche, a reverse Warburg effect has been recently described, consisting in metabolic changes occurring in stromal cells in the attempt to metabolically support adjacent cancer cells. Moreover, a physiological dynamic, based on mitochondria transfer, between tumor cells and their supporting stromal microenvironment has been described to sustain oxidative stress associated to proteostasis maintenance in multiple myeloma and leukemia. Increased mitochondrial biogenesis of tumor cells associated to acquisition of new mitochondria transferred by mesenchymal stromal cells results in augmented ATP production through increased oxidative phosphorylation (OX-PHOS), higher drug resistance, and resurgence after treatment. Accordingly, targeting mitochondrial biogenesis, electron transfer, mitochondrial DNA replication, or mitochondrial fatty acid transport increases therapy efficacy. In this review, we summarize selected examples of the mitochondrial derangements in hematological malignancies, which provide metabolic adaptation and apoptosis resistance, also supported by the crosstalk with tumor microenvironment. This field promises a rational design to improve target-therapy including the metabolic phenotype.

Abstract 283: Western Diet Induces Diastolic Dysfunction Linked to Impaired Cardiac Lipid Storage Dynamics That Predate Systolic Dysfunction

Western diet (WD), containing high fat and high refined sugar, is associated with increased risk of cardiovascular disease. However, mechanisms linking WD to cardiomyopathy are far from understood. We hypothesize that WD induces cardiac dysfunction by dysregulation of cardiac lipid dynamics. Adult C57BL/6J mice were fed with either standard chow (CON, fat 4.5 %, sucrose 0%) or western diet (WD, fat 45%, sucrose 21%). At 20 weeks, WD mice exhibited higher body mass, reduced glucose tolerance, and increased isovolumetric relaxation time (IVRT), with no systolic dysfunction vs. CON. However, at 24 weeks, WD caused both systolic and diastolic dysfunction with decreases in left ventricular ejection fraction (66±2% CON vs. 54±2 WD, P &lt;0.05) and fractional shortening (35±2% CON vs. 27±1 WD), with elevated IVRT and E/E’ ratio vs CON. Real time, dynamic-mode 13 C NMR and endpoint LC/MS of isolated perfused hearts supplied 13 C palmitate and 13 C oleate, with unlabeled glucose and lactate, revealed elevated TG content (28% increase vs. CON) with reduced TG turnover in WD hearts (35% decrease vs. CON), concurrent with early diastolic dysfunction at 20 weeks and preceding systolic dysfunction at 24 weeks. WD induced lower content of the TG lipase, ATGL (48% vs. CON), with no change in the TG synthase, DGAT1, suggesting elevated cardiac TG and lower TG turnover are due to reduced TG hydrolysis and not TAG synthesis. Impaired lipid storage dynamics can cause increased generation of potentially lipotoxic acyl intermediates, leading to cardiac dysfunction. The mitochondrial long chain fatty acid transporter, CPT1b was reduced (50%), with no change in the lesser, co-expressed CPT1a isoform in WD hearts. Despite reduced CPT1b in WD hearts, contributions from LCFAs to acetyl CoA production in the citric acid cycle were unchanged, suggesting that the WD provided sufficient LCFAs for mitochondrial oxidation and that the reduced CPT1b was not limiting. In conclusion, western diet expands the TG pool but impairs fat storage dynamics via low ATGL, leading to early diastolic dysfunction and eventual systolic dysfunction. Nutrient overload from WD leads to impaired cardiac lipid dynamics with early diastolic dysfunction that is distinguished from HFpEF due to later systolic dysfunction.