Heptanoate Improves Compensatory Mechanism of Glucose Homeostasis in Mitochondrial Long-Chain Fatty Acid Oxidation Defect.

Defects in mitochondrial fatty acid ß-oxidation (FAO) impair metabolic flexibility, which is an essential process for energy homeostasis. Very-long-chain acyl-CoA dehydrogenase (VLCADD; OMIM 609575) deficiency is the most common long-chain mitochondrial FAO disorder presenting with hypoglycemia as a common clinical manifestation. To prevent hypoglycemia, triheptanoin-a triglyceride composed of three heptanoates (C7) esterified with a glycerol backbone-can be used as a dietary treatment, since it is metabolized into precursors for gluconeogenesis. However, studies investigating the effect of triheptanoin on glucose homeostasis are limited. To understand the role of gluconeogenesis in the pathophysiology of long-chain mitochondrial FAO defects, we injected VLCAD-deficient (VLCAD-/-) mice with 13C3-glycerol in the presence and absence of heptanoate (C7). The incorporation of 13C3-glycerol into blood glucose was higher in VLCAD-/- mice than in WT mice, whereas the difference disappeared in the presence of C7. The result correlates with 13C enrichment of liver metabolites in VLCAD-/- mice. In contrast, the C7 bolus significantly decreased the 13C enrichment. These data suggest that the increased contribution of gluconeogenesis to the overall glucose production in VLCAD-/- mice increases the need for gluconeogenesis substrate, thereby avoiding hypoglycemia. Heptanoate is a suitable substrate to induce glucose production in mitochondrial FAO defect.

Maturation of lipid metabolism in the fetal and newborn sheep heart.

At birth, the fetus experiences a dramatic change in environment that is accompanied by a shift in myocardial fuel preference from lactate and glucose in fetal life to fatty acid oxidation after birth. We hypothesized that fatty acid metabolic machinery would mature during fetal life in preparation for this extreme metabolic transformation at birth. We quantified the pre- (94-day and 135-day gestation, term ∼147 days) and postnatal (5 ± 4 days postnatal) gene expression and protein levels for fatty acid transporters and enzymes in hearts from a precocial species, the sheep. Gene expression of fatty acid translocase (CD36), acyl-CoA synthetase long-chain 1 (ACSL1), carnitine palmitoyltransferase 1 (CPT1), hydroxy-acyl dehydrogenase (HADH), acetyl-CoA acetyltransferase (ACAT1), isocitrate dehydrogenase (IDH), and glycerol phosphate acyltransferase (GPAT) progressively increased through the perinatal period, whereas several genes [fatty acid transport protein 6 (FATP6), acyl-CoA synthetase long chain 3 (ACSL3), long-chain acyl-CoA dehydrogenase (LCAD), very long-chain acyl-CoA dehydrogenase (VLCAD), pyruvate dehydrogenase kinase (PDK4), phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT)] were stable in fetal hearts and had high expression after birth. Protein expression of CD36 and ACSL1 progressively increased throughout the perinatal period, whereas protein expression of carnitine palmitoyltransferase 1a (fetal isoform) (CPT1a) decreased and carnitine palmitoyltransferase 1b (adult isoform) (CPT1b) remained constitutively expressed. Using fluorescent-tagged long-chain fatty acids (BODIPY-C12), we demonstrated that fetal (125 ± 1 days gestation) cardiomyocytes produce 59% larger lipid droplets (P < 0.05) compared with newborn (8 ± 1 day) cardiomyocytes. These results provide novel insights into the perinatal maturation of cardiac fatty acid metabolism in a precocial species.NEW & NOTEWORTHY This study characterized the previously unknown expression patterns of genes that regulate the metabolism of free fatty acids in the perinatal sheep myocardium. This study shows that the prenatal myocardium prepares for the dramatic switch from carbohydrate metabolism to near complete reliance on free fatty acids postnatally. Fetal and neonatal cardiomyocytes also demonstrate differing lipid storage mechanisms where fetal cardiomyocytes form larger lipid droplets compared with newborn cardiomyocytes.

The KLF7/PFKL/ACADL axis modulates cardiac metabolic remodelling during cardiac hypertrophy in male mice.

The main hallmark of myocardial substrate metabolism in cardiac hypertrophy or heart failure is a shift from fatty acid oxidation to greater reliance on glycolysis. However, the close correlation between glycolysis and fatty acid oxidation and underlying mechanism by which causes cardiac pathological remodelling remain unclear. We confirm that KLF7 simultaneously targets the rate-limiting enzyme of glycolysis, phosphofructokinase-1, liver, and long-chain acyl-CoA dehydrogenase, a key enzyme for fatty acid oxidation. Cardiac-specific knockout and overexpression KLF7 induce adult concentric hypertrophy and infant eccentric hypertrophy by regulating glycolysis and fatty acid oxidation fluxes in male mice, respectively. Furthermore, cardiac-specific knockdown phosphofructokinase-1, liver or overexpression long-chain acyl-CoA dehydrogenase partially rescues the cardiac hypertrophy in adult male KLF7 deficient mice. Here we show that the KLF7/PFKL/ACADL axis is a critical regulatory mechanism and may provide insight into viable therapeutic concepts aimed at the modulation of cardiac metabolic balance in hypertrophied and failing heart.

Flavonoids from Scutellaria amoena C. H. Wright alleviate mitochondrial dysfunction and regulate oxidative stress via Keap1/Nrf2/HO-1 axis in rats with high-fat diet-induced nonalcoholic steatohepatitis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is among the most common liver diseases in the world. Flavonoids from Scutellaria amoena (SAF) are used in the treatment of hepatopathy in China. However, the effect and mechanism against NASH remain unclear. We investigated the alleviating effect of SAF on NASH via regulating mitochondrial dysfunction and oxidative stress. METHODS: The effects of SAF on NASH were evaluated using in vitro and in vivo methods. L02 cells were induced by fat emulsion to establish an adipocytes model, followed by treatment with SAF for 24 h. NASH rat models were established by the administration of a high-fat diet for 12 weeks and were administered SAF for six weeks. Changes in body weight, organ indexes, lipid levels, inflammatory cytokines, mitochondrial indicators, and fatty acid metabolism were investigated. RESULTS: SAF significantly improved body weight, organ indexes, lipid levels, liver injury, and inflammatory infiltration in NASH rats. SAF notably regulated interleukin-6, tumor necrotic factor-alpha, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), kelch-like ECH-associated protein 1 (Keap1), nuclear factor-erythroid factor 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1). Additionally, SAF improved mitochondrial dysfunction, increased the levels of GSH, SOD, ATP synthase, complex I and II, and decreased the level of MDA in liver mitochondria. SAF regulated the expression of ß-oxidation genes, including peroxisome proliferator-activated receptor -gamma coactivator-1alpha (PGC-1α), carnitine palmitoyltransferase-1 (CPT1) A, CPT1B, medium-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, very long-chain acyl-CoA dehydrogenase, and PPARα. CONCLUSION: SAF can alleviate NASH by regulating mitochondrial function and oxidative stress via the Keap1/Nrf2/HO-1 axis.

Odd- and even-numbered medium-chained fatty acids protect against glutathione depletion in very long-chain acyl-CoA dehydrogenase deficiency.

Recent trials have reported the ability of triheptanoin to improve clinical outcomes for the severe symptoms associated with long-chain fatty acid oxidation disorders, including very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. However, the milder myopathic symptoms are still challenging to treat satisfactorily. Myopathic pathogenesis is multifactorial, but oxidative stress is an important component. We have previously shown that metabolic stress increases the oxidative burden in VLCAD-deficient cell lines and can deplete the antioxidant glutathione (GSH). We investigated whether medium-chain fatty acids provide protection against GSH depletion during metabolic stress in VLCAD-deficient fibroblasts. To investigate the effect of differences in anaplerotic capacity, we included both even-(octanoate) and odd-numbered (heptanoate) medium-chain fatty acids. Overall, we show that modulation of the concentration of medium-chain fatty acids in culture media affects levels of GSH retained during metabolic stress in VLCAD-deficient cell lines but not in controls. Lowered glutamine concentration in the culture media during metabolic stress led to GSH depletion and decreased viability in VLCAD deficient cells, which could be rescued by both heptanoate and octanoate in a dose-dependent manner. Unlike GSH levels, the levels of total thiols increased after metabolic stress exposure, the size of this increase was not affected by differences in cell culture medium concentrations of glutamine, heptanoate or octanoate. Addition of a PPAR agonist further exacerbated stress-related GSH-depletion and viability loss, requiring higher concentrations of fatty acids to restore GSH levels and cell viability. Both odd- and even-numbered medium-chain fatty acids efficiently protect VLCADdeficient cells against metabolic stress-induced antioxidant depletion.

Cardiologic evaluation of Turkish mitochondrial fatty acid oxidation disorders.

BACKGROUND: Mitochondrial fatty acid oxidation disorders (FAODs) cause impairment in energy metabolism and can lead to a spectrum of cardiac pathologies including cardiomyopathy and arrhythmias. The frequency of underlying cardiac pathologies and the response to recommended treatment in FAODs was investigated. METHODS: Sixty-eight children (35 males, 33 females) with the diagnosis of a FAOD were included in the study. Cardiac function was evaluated with 12-lead standard electrocardiography, echocardiography, and 24 h Holter monitoring. RESULTS: Forty-five patients (66%) were diagnosed after disease symptoms developed and 23 patients (34%) were diagnosed in the pre-symptomatic period. Among symptomatic patients (n: 45), cardiovascular findings were detected in 18 (40%) patients, including cardiomyopathy in 14 (31.1%) and conduction abnormalities in 4 (8.8%) patients. Cardiac symptoms were more frequently detected in primary systemic carnitine deficiency (57.1%). Patients with multiple acyl-CoA dehydrogenase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and mitochondrial trifunctional protein deficiencies also had an increased frequency of cardiac symptoms. Patients with medium-chain acyl-CoA dehydrogenase, very long-chain acyl-CoA dehydrogenase, and carnitine palmitoyltransferase I deficiencies had a lower prevalence of cardiac symptoms both during admission and during clinical follow up. Cardiomyopathy resolved completely in 8/14 (57%) patients and partially in 2/14 (14.3%) patients with treatment. Two patients with cardiomyopathy died in the newborn period; cardiomyopathy persisted in 1 (7.1%) patient with very long-chain acyl-CoA dehydrogenase deficiency. CONCLUSION: Early diagnosis, treatment and follow up made a significant contribution to the improvement of cardiac symptoms of patients with FAODs.

Expert consensus on diagnosis and treatment of very long-chain acyl-CoA dehydrogenase deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a metabolic disease of long chain fatty acid oxidation. The clinical manifestations are heterogeneous, mainly with heart, liver, skeletal muscle and brain damage, and the onset of which can be from newborn to adult. Cardiomyopathy type is more serious with high mortality. The liver failure type and myopathy type would be potentially lethal, but generally the prognosis is relatively good. Recurrent hypoglycemia, energy metabolism disorder, liver dysfunction, cardiomyopathy and serious arrhythmia are the main causes of death. Most patients can be identified through neonatal screening, and the prognosis is usually good in patients with early diagnosis and treatment. The purpose of this consensus is to standardize the diagnosis, treatment and management of VLCAD deficiency, so as to improve the prognosis of patients and reduce death and disability.

Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

VLCAD inhibits the proliferation and invasion of hepatocellular cancer cells through regulating PI3K/AKT axis

PurposeVery-long-chain acyl-CoA dehydrogenase (VLCAD) is an essential mediator in fatty acid metabolism. The progression of human hepatocellular carcinoma (HCC) is closely associated with the disorder of energy supply. Here, we aimed to investigate the role and underlying molecule mechanism of VLCAD in pathological process of HCC.MethodsIn this study, VLCAD was induced silencing and overexpression using small hairpin RNA (shRNA) and lentiviral-mediated vector in HCC cell lines. The proliferation of HCC cells was determined using CCK-8 assay. Transwell assay and lung metastasis were performed to analysis cell metastasis in vitro and in vivo. ECAR and OCR were used to evaluate the activity of glycolysis and mitochondrial oxidative phosphorylation.ResultsOur data indicated that VLCAD was downregulated in human HCC tissues and cells. VLCAD overexpression strongly suppressed the proliferation and metastasis of HCC cells associating with the decrease of ATP accumulation and glycolysis activity. Importantly, the PI3K/AKT inhibitor LY294002 strongly abolished the role of shVLCAD in HCC cells. Our results suggested that VLCAD suppressed the growth and metastasis in HCC cells by inhibiting the activities of glycolysis and mitochondrial oxidative phosphorylation metabolism via PI3K/AKT pathway.ConclusionsTogether, present findings not only demonstrated the protective role of and molecular network of VLCAD in HCC cells but also indicated its and potential use as a target in the therapy of HCC.

CircRNA and miRNA expression profiles during remote ischemic postconditioning attenuate brain ischemia/reperfusion injury.

Remote ischemic postconditioning (RIPostC) is a protective procedure for brain damage caused by ischemia/reperfusion (IR), yet the mechanism of this treatment remains to be elucidated. Circular RNAs (circRNAs) are endogenous non-coding RNAs that have recently been recognized to play vital roles in ischemic brain injury. The aim of this study was to explore the role of circRNAs in the protective mechanism of RIPostC and to analyze the circRNA-microRNA (miRNA) regulation network in RIPostC. Nine rats were assigned randomly into three groups (three rats per group): sham, IR, and RIPostC. Their brain tissues were extracted for next-generation RNA sequencing and bioinformatics analysis was performed for two comparisons: sham vs. IR and IR vs. RIPostC. The expression patterns of selected circRNAs and miRNAs were validated by quantitative real-time PCR (qPCR). We detected 82 upregulated and 51 downregulated circRNAs and 137 upregulated and 127 downregulated miRNAs in the IR group compared with the sham group, and 41 upregulated and 100 downregulated circRNAs and 45 upregulated and 64 downregulated miRNAs in the RIPostC group compared with the IR group. The proposed competitive endogenous RNA (ceRNA) network, which included 24 circRNAs, 20 miRNAs, and 145 mRNAs, indicated that the dysregulated circRNAs played important roles in brain IR injury. On the basis of the expression patterns of selected circRNAs, miRNAs, and mRNAs obtained by qPCR, we proposed a circRNA_0002286-miR-124-3p-VLCAD pathway. In PC12 cell, the expression level of miR-124-3p was significantly upregulated when the expression of circRNA_0002286 was repressed and the expression level of VLCAD (very-long chain acyl-CoA dehydrogenase) was significantly downregulated, which suggested that circRNA_0002286 may act as a miRNA sponge for miR-124-3p to regulate the expression of VLCAD. We found that upregulation of circRNA_0002286 attenuated IR injury and was associated with downregulation of miR-124-3p and upregulation of VLCAD. This is the first time that circRNAs have been shown to be closely related to brain IR injury and RIPostC and suggests that targeting the circRNA_0002286-miR-124-3p-VLCAD pathway might attenuate brain IR injury.

A mitochondrial long-chain fatty acid oxidation defect leads to transfer RNA uncharging and activation of the integrated stress response in the mouse heart.

AIMS: Cardiomyopathy and arrhythmias can be severe presentations in patients with inherited defects of mitochondrial long-chain fatty acid ß-oxidation (FAO). The pathophysiological mechanisms that underlie these cardiac abnormalities remain largely unknown. We investigated the molecular adaptations to a FAO deficiency in the heart using the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse model. METHODS AND RESULTS: We observed enrichment of amino acid metabolic pathways and of ATF4 target genes among the upregulated genes in the LCAD KO heart transcriptome. We also found a prominent activation of the eIF2α/ATF4 axis at the protein level that was independent of the feeding status, in addition to a reduction of cardiac protein synthesis during a short period of food withdrawal. These findings are consistent with an activation of the integrated stress response (ISR) in the LCAD KO mouse heart. Notably, charging of several transfer RNAs (tRNAs), such as tRNAGln was decreased in LCAD KO hearts, reflecting a reduced availability of cardiac amino acids, in particular, glutamine. We replicated the activation of the ISR in the hearts of mice with muscle-specific deletion of carnitine palmitoyltransferase 2. CONCLUSIONS: Our results show that perturbations in amino acid metabolism caused by long-chain FAO deficiency impact cardiac metabolic signalling, in particular the ISR. These results may serve as a foundation for investigating the role of the ISR in the cardiac pathology associated with long-chain FAO defects.Translational Perspective: The heart relies mainly on mitochondrial fatty acid ß-oxidation (FAO) for its high energy requirements. The heart disease observed in patients with a genetic defect in this pathway highlights the importance of FAO for cardiac health. We show that the consequences of a FAO defect extend beyond cardiac energy homeostasis and include amino acid metabolism and associated signalling pathways such as the integrated stress response.

VLCAD inhibits the proliferation and invasion of hepatocellular cancer cells through regulating PI3K/AKT axis.

PURPOSE: Very-long-chain acyl-CoA dehydrogenase (VLCAD) is an essential mediator in fatty acid metabolism. The progression of human hepatocellular carcinoma (HCC) is closely associated with the disorder of energy supply. Here, we aimed to investigate the role and underlying molecule mechanism of VLCAD in pathological process of HCC. METHODS: In this study, VLCAD was induced silencing and overexpression using small hairpin RNA (shRNA) and lentiviral-mediated vector in HCC cell lines. The proliferation of HCC cells was determined using CCK-8 assay. Transwell assay and lung metastasis were performed to analysis cell metastasis in vitro and in vivo. ECAR and OCR were used to evaluate the activity of glycolysis and mitochondrial oxidative phosphorylation. RESULTS: Our data indicated that VLCAD was downregulated in human HCC tissues and cells. VLCAD overexpression strongly suppressed the proliferation and metastasis of HCC cells associating with the decrease of ATP accumulation and glycolysis activity. Importantly, the PI3K/AKT inhibitor LY294002 strongly abolished the role of shVLCAD in HCC cells. Our results suggested that VLCAD suppressed the growth and metastasis in HCC cells by inhibiting the activities of glycolysis and mitochondrial oxidative phosphorylation metabolism via PI3K/AKT pathway. CONCLUSIONS: Together, present findings not only demonstrated the protective role of and molecular network of VLCAD in HCC cells but also indicated its and potential use as a target in the therapy of HCC.

Integrative multi-omic analysis of radiation-induced skin injury reveals the alteration of fatty acid metabolism in early response of ionizing radiation.

BACKGROUND: Radiation-induced skin injury is a serious concern during radiotherapy and accidental exposure to radiation. OBJECTIVE: This study aims to investigate the molecular events in early response to ionizing radiation of skin tissues and underlying mechanism. METHODS: Mice and rats were irradiated with an electron beam. Skin tissues were used for liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, mRNA-Seq and single-cell RNA sequencing (scRNA-Seq). Human keratinocytes (HaCaT) and skin fibroblasts (WS1) were used for functional studies. RESULTS: The integrated analysis of metabolomics and transcriptomics showed that 6 key fatty acid-associated metabolites, 9 key fatty acid-associated genes and multiple fatty acid-associated pathways were most obviously enriched and increased in the irradiated skins. Among them, acyl-CoA dehydrogenase very long chain (ACADVL) was investigated in greater detail due to its most obvious expression difference and significance in fatty acid metabolism. ScRNA-Seq of rat skin from irradiated individuals revealed that ACADVL was expressed in all subpopulations of skin tissues, with variations at different timepoints after radiation. Immunohistochemistry confirmed an increased ACADVL expression in the epidermis from human sample and various animal models, including monkeys, rats and mice. The knockdown of ACADVL increased the radiosensitivity of human keratinocytes and human skin fibroblasts. Silencing of ACADVL facilitated the expression of apoptosis and pyroptosis-related proteins following ionizing radiation. CONCLUSION: This study illustrated that cutaneous fatty acid metabolism was altered in the early response of ionizing radiation, and fatty acid metabolism-associated ACADVL is involved in radiation-induced cell death.

Lipidomic and Proteomic Alterations Induced by Even and Odd Medium-Chain Fatty Acids on Fibroblasts of Long-Chain Fatty Acid Oxidation Disorders.

Medium-chain fatty acids (mc-FAs) are currently applied in the treatment of long-chain fatty acid oxidation disorders (lc-FAOD) characterized by impaired ß-oxidation. Here, we performed lipidomic and proteomic analysis in fibroblasts from patients with very long-chain acyl-CoA dehydrogenase (VLCADD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies after incubation with heptanoate (C7) and octanoate (C8). Defects of ß-oxidation induced striking proteomic alterations, whereas the effect of treatment with mc-FAs was minor. However, mc-FAs induced a remodeling of complex lipids. Especially C7 appeared to act protectively by restoring sphingolipid biosynthesis flux and improving the observed dysregulation of protein homeostasis in LCHADD under control conditions.

Impact of Adenosine Analogue, Adenosine-5'-N-Ethyluronamide (NECA), on Insulin Signaling in Skeletal Muscle Cells.

MATERIALS AND METHODS: Rat L6 skeletal muscle cells were cultured in 25 cm2 flasks. These differentiated cells were treated, and then, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (probe-based) was used to measure the relative mRNA expression level for metabolic, inflammatory, and nuclear receptor genes including peroxisome proliferator-activated receptor gamma (PGC-1α), carnitine palmitoyl transferase 1 beta (CPT1B), long-chain acyl-CoA de hydrogenase (LCAD), acetyl-CoA carboxylase beta (ACCß), pyruvate dehydrogenase kinase 4 (PDK4), hexokinase II (HKII), phosphofructokinase (PFK), interleukin-6 (IL-6), and nuclear receptor subfamily 4, group A (NR4A) at different treatment conditions. RESULTS: Adenosine-5'-N-ethyluronamide (NECA), a stable adenosine analogue, significantly stimulate inflammatory mediator (IL-6) (p < 0.001) and nuclear receptors (NR4A) (p < 0.05) and significantly modulate metabolic (PFK, LCAD, PGC-1α, and CPT1B) gene expressions in skeletal muscle cells (p < 0.05, p < 0.05, p < 0.001, and p < 0.01, respectively). This present study shows that there is a noteworthy crosstalk between NECA and insulin at various metabolic levels including glycolysis (HKII), fatty acid oxidation (ACCß), and insulin sensitivity (PDK4). CONCLUSIONS: A novel crosstalk between adenosine analogue and insulin has been demonstrated for the first time; evidence has been gathered in vitro for the effects of NECA and insulin treatment on intracellular signaling pathways, in particular glycolysis and insulin sensitivity in skeletal muscle cells.

Very long chain fatty acid metabolism is required in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells have an atypical metabolic phenotype characterized by increased mitochondrial mass, as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. To exploit this altered metabolism, we assessed publicly available databases to identify FAO enzyme overexpression. Very long chain acyl-CoA dehydrogenase (VLCAD; ACADVL) was found to be overexpressed and critical to leukemia cell mitochondrial metabolism. Genetic attenuation or pharmacological inhibition of VLCAD hindered mitochondrial respiration and FAO contribution to the tricarboxylic acid cycle, resulting in decreased viability, proliferation, clonogenic growth, and AML cell engraftment. Suppression of FAO at VLCAD triggered an increase in pyruvate dehydrogenase activity that was insufficient to increase glycolysis but resulted in adenosine triphosphate depletion and AML cell death, with no effect on normal hematopoietic cells. Together, these results demonstrate the importance of VLCAD in AML cell biology and highlight a novel metabolic vulnerability for this devastating disease.

Mitochondrial morphology, bioenergetics and proteomic responses in fatty acid oxidation disorders.

Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondrial deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

Downregulation of fatty acid oxidation by involvement of HIF-1α and PPARγ in human gastric adenocarcinoma and related clinical significance.

Lipid metabolism rewiring in gastric adenocarcinoma (GA) pathogenesis is still not clearly elucidated. This study aimed to describe the role of lipid catabolism in GA patient outcomes and possible therapeutic targets by analyzing the effect of hypoxia-inducible factor-1α (HIF-1α) on fatty acid oxidation (FAO). AGS cell line was cultured in normoxic and hypoxic conditions, and FAO-related genes were analyzed by real-time-PCR and Western-blot. The study group comprised 108 newly diagnosed GA patients and 152 control cases. Serum concentrations of medium and long-chain acyl-CoA dehydrogenases (MCAD and LCAD) proteins were measured using ELISA, and local expression of HIF-1α, carnitine palmitoyl transferase 1 (CPT1A) and peroxisome proliferator-activated receptor γ (PPARγ) was evaluated by immunohistochemistry. In addition, gene expression of PPARγ, CPT1A, LCAD, and MCAD was assessed by real-time-PCR. In vitro findings indicate HIF-1α upregulation and FAO-related genes and proteins reduction in the hypoxic culture of AGS cells. GA patients had significantly lower circulating levels of LCAD compared to controls. Higher protein expression of HIF-1α and downregulated CPT1A and PPARγ were observed in GA tissues versus controls. Gene expression of CPT1A, PPARγ, LCAD, and MCAD were repressed in GA tissues compared to controls. Moreover, reduced expression of CPT1A, PPARγ, and MCAD were correlated with HIF-1α upregulation in GA. Poor patient outcome was associated with lower PPARγ and LCAD expression in GA. HIF-1α upregulation in human GA patients and AGS cells was paralleled by downregulation of lipid catabolism genes potentially via reduced PPARγ-mediated FAO. This metabolic adaptation to hypoxic condition may play a role in GA pathogenesis and might have clinical and therapeutic value in GA patients.

The Mitochondrial Antioxidant Sirtuin3 Cooperates with Lipid Metabolism to Safeguard Neurogenesis in Aging and Depression.

Neural stem cells (NSCs), crucial for memory in the adult brain, are also pivotal to buffer depressive behavior. However, the mechanisms underlying the boost in NSC activity throughout life are still largely undiscovered. Here, we aimed to explore the role of deacetylase Sirtuin 3 (SIRT3), a central player in mitochondrial metabolism and oxidative protection, in the fate of NSC under aging and depression-like contexts. We showed that chronic treatment with tert-butyl hydroperoxide induces NSC aging, markedly reducing SIRT3 protein. SIRT3 overexpression, in turn, restored mitochondrial oxidative stress and the differentiation potential of aged NSCs. Notably, SIRT3 was also shown to physically interact with the long chain acyl-CoA dehydrogenase (LCAD) in NSCs and to require its activation to prevent age-impaired neurogenesis. Finally, the SIRT3 regulatory network was investigated in vivo using the unpredictable chronic mild stress (uCMS) paradigm to mimic depressive-like behavior in mice. Interestingly, uCMS mice presented lower levels of neurogenesis and LCAD expression in the same neurogenic niches, being significantly rescued by physical exercise, a well-known upregulator of SIRT3 and lipid metabolism. Our results suggest that targeting NSC metabolism, namely through SIRT3, might be a suitable promising strategy to delay NSC aging and confer stress resilience.

Nutrition management guideline for very-long chain acyl-CoA dehydrogenase deficiency (VLCAD): An evidence- and consensus-based approach.

The nutrition management guideline for very-long chain acyl-CoA dehydrogenase deficiency (VLCAD) is the fourth in a series of web-based guidelines focusing on the diet treatment for inherited metabolic disorders and follows previous publication of guidelines for maple syrup urine disease (2014), phenylketonuria (2016) and propionic acidemia (2019). The purpose of this guideline is to establish harmonization in the treatment and monitoring of individuals with VLCAD of all ages in order to improve clinical outcomes. Six research questions were identified to support guideline development on: nutrition recommendations for the healthy individual, illness management, supplementation, monitoring, physical activity and management during pregnancy. This report describes the methodology used in its development including review, critical appraisal and abstraction of peer-reviewed studies and unpublished practice literature; expert input through two Delphi surveys and a nominal group process; and external review from metabolic physicians and dietitians. It includes the summary statements of the nutrition management recommendations for each research question, followed by a standardized rating based on the strength of the evidence. Online, open access of the full published guideline allows utilization by health care providers, researchers and collaborators who advise, advocate and care for individuals with VLCAD and their families and can be accessed from the Genetic Metabolic Dietitians International (https://GMDI.org) and Southeast Regional Genetics Network (https://southeastgenetics.org/ngp) websites.