Search Results (123)

Background/Objectives: We have previously demonstrated that fatty acid oxidation (FAO) enzymes physically and functionally interact with electron transfer chain supercomplexes (ETC-SC) at two contact points. The FAO trifunctional protein (TFP) and electron transfer flavoprotein dehydrogenase (ETFDH) interact with the NADH⁺-binding domain of ETC complex I (com I) and the core 2 subunit of complex III (com III), respectively. In addition, the FAO enzyme very-long-chain acyl-CoA dehydrogenase (VLCAD) interacts with TFP. These interactions define a functional FAO-ETC macromolecular complex (FAO-ETC MEC) in which FAO-generated NADH⁺ and FADH₂ can safely transfer electron equivalents to ETC in order to generate ATP. Methods: In this study, we use multiple mitochondrial functional studies to demonstrate the effect of added VLCAD protein on mutant mitochondria. Results: We demonstrate that heart mitochondria from a VLCAD knockout (KO) mouse exhibit disrupted supercomplexes, with significantly reduced levels of TFPα and TFPβ subunits, electron transfer flavoprotein a-subunit (ETFα), and NDUFV2 subunit of com I in the FAO-ETC MEC. In addition, the activities of individual oxidative phosphorylation (OXPHOS) enzymes are decreased, as is the transfer of reducing equivalents from palmitoyl-CoA to ETC (FAO-ETC flux). However, the total amount of these proteins did not decrease in VLCAD KO animals. These results suggest that loss of VLCAD affects the interactions of FAO and ETC proteins in the FAO-ETC MEC. Reconstitution of VLCAD-deficient heart mitochondria with recombinant VLCAD improved the levels of FAO-ETC MEC proteins and enzyme activities, as well as restoring FAO-ETC flux. It also reduced mitochondrial ROS levels, previously demonstrated to be elevated in VLCAD-deficient mitochondria. In contrast, incubation of VLCAD KO mitochondria with two VLCADs with mutations in the C-terminal domain of the enzyme (A450P and L462P) did not restore FAO-ETC MECs. Conclusions: These results suggest that VLCAD is a necessary component of the FAO-ETC MEC and plays a major role in assembly of the macro-supercomplex. These studies provide evidence that both the level of enzyme and its structural confirmation are necessary to stabilize the FAO-ETC MEC. Full article

Mitochondrial energy metabolism is fundamental to sperm function, and fatty acid β-oxidation is an important pathway for adenosine triphosphate (ATP) production. Riboflavin, a precursor of key flavin cofactors, plays a critical role in regulating β-oxidation and supports multiple physiological processes. This study aimed to determine whether adding riboflavin to semen dilution media could enhance goat sperm motility and to elucidate the underlying metabolic mechanisms. Goat semen was diluted in tris-citrate-glucose (TCG) medium containing 0, 5, 10, 15, and 20 μM riboflavin and incubated at 37 °C, after which sperm motility, acrosome integrity, mitochondrial membrane potential, ATP levels, malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities, and the NADH/NAD⁺ were evaluated. The localization and expression of the β-oxidation enzymes carnitine palmitoyltransferase 1 (CPT1) and extremely long chain acyl-CoA dehydrogenase (ACADVL) were examined, and CPT1 activity was quantified. The results showed that CPT1 and ACADVL were present in goat sperm, and that 10 μM riboflavin significantly increased sperm motility, acrosome integrity, mitochondrial activity, ATP levels, and the activities of MDH, SDH, and CPT1, while also elevating NADH/NAD⁺ levels (p < 0.05). Notably, these enhancements were suppressed by 100 μM etomoxir, a mitochondrial β-oxidation inhibitor, which reduced total motility, ATP Levels, and CPT1 activity after riboflavin supplementation (p < 0.05). These findings indicate that goat sperm at least partly rely on mitochondrial β-oxidation for ATP generation and that riboflavin supplementation enhances mitochondrial metabolism, thereby improving sperm quality. Full article

Nickel is a pervasive heavy metal with the potential for multi-route exposure, raising significant concerns regarding systemic toxicity. Although Ni²⁺ has been implicated in nickel sulfate NiSO₄-induced neurotoxicity, its underlying mechanisms remain incompletely elucidated. The present study investigates the role of NiSO₄-induced ferroptosis as a potential contributor to neurotoxicity. C57BL/6 mice were administered NiSO₄ daily via oral gavage at doses of 50, 100, and 200 mg/kg over 28 days. Neurobehavioral assessments, histopathological examination, transmission electron microscopy, and molecular profiling were conducted to evaluate brain injury and ferroptotic activity. Gut microbiota composition and intestinal barrier integrity were systematically evaluated. In vitro, HT22 cells were subjected to NiSO₄ treatment, followed by integrative transcriptomic analysis complemented by pharmacological and genetic manipulation to delineate the contributions of ferroptosis and autophagy. The results demonstrated that NiSO₄ exposure inhibited body weight gain, elicited depression-like behaviors, and initiated ferroptosis, evidenced by ultrastructural mitochondrial damage and dysregulated expression of glutathione peroxidase 4/acyl-CoA synthetase long chain family member 4 (GPX4/ACSL4). Furthermore, NiSO₄ caused gut microbiota dysbiosis and compromised the intestinal barrier, which was correlated with the induction of ferroptosis in neuronal cells of the brain. In HT22 cells, NiSO₄ elicited dose-dependent cytotoxicity and lactate dehydrogenase (LDH) release. KEGG pathway enrichment analysis further revealed that NiSO₄ treatment significantly upregulated pathways associated with ferroptosis, autophagy, and lysosomal function. Moreover, both ferrostatin-1 and rapamycin attenuated NiSO₄-induced cytotoxicity and ferroptosis, indicating that autophagy serves a protective function against ferroptotic cell death. Additionally, overexpression of Transcription Factor EB (TFEB) attenuated NiSO₄-induced ferroptosis by downregulating ACSL4, and upregulating GPX4, implicating the autophagy–lysosome pathway in the protective regulation of this cell death process. In summary, our findings indicated that NiSO₄-induced neurotoxicity was strongly associated with gut microbiota dysbiosis and coincided with ferroptosis in the brain, while stimulation of the autophagy–lysosome pathway conferred neuroprotective effects via modulating TFEB-dependent anti-ferroptotic mechanisms. These findings offer novel insights for risk assessment and therapeutic strategies of nickel-related neurotoxicity. Full article

This study evaluates the incidence of metabolic disorders detected from January 2005 to December 2024 and their clinical outcomes. Data were retrospectively collected from the Louisiana Newborn Screening database. Clinical outcomes were obtained through review of corresponding medical records. In addition, an electronic questionnaire assessing educational attainment and neurodevelopmental disorders was sent to the patients’ families. Of 1,230,356 infants screened, 478 were diagnosed with metabolic disorders, corresponding to an incidence of 1 in 2574 live births. The three most commonly identified conditions were biotinidase deficiency, phenylketonuria (PKU), and medium-chain acyl-CoA dehydrogenase deficiency (MCADD). During the study period, at least 11 patients died. The program demonstrated a false-positive rate of 0.93%. Twelve patients (7%) were symptomatic before or at the time of NBS result notification. Recurrent metabolic decompensations occurred in 3 of 4 maple syrup urine disease (MSUD) cases, 7 of 7 methylmalonic acidemia (MMA) cases, 1 of 4 propionic acidemia (PA) cases and 1 of 7 urea cycle defect cases. Regarding long-term outcomes, 45.7% of survey respondents reported adverse neurodevelopmental outcomes of varying severity. Early detection and timely intervention have contributed to normal or near-normal outcomes in many cases. However, the morbidity and mortality observed in some patients despite early diagnosis highlights the severity and complexity of certain metabolic conditions. Additionally, the relatively high false positive rate underscores the need for ongoing efforts to improve the specificity of screening protocols to reduce unnecessary follow-ups and mitigate potential stress for families. Full article

Introduction. Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is an autosomal recessive metabolic disorder resulting from mutations in the genes that encode the electron transfer flavoprotein (ETF) or its associated dehydrogenase (ETFDH), resulting in defects in mitochondrial fatty acid oxidation and a broad range of clinical presentations, most notably in the form of muscle weakness; exercise intolerance; and, in some cases, life-threatening metabolic crises. Late-onset MADD represents the most common form of lipid storage myopathy, but its diagnosis can be elusive due to its varied and often nonspecific clinical symptoms and may resemble other neuromuscular conditions, like inflammatory myopathies or other peripheral neuropathies, complicating the diagnostic process and delaying appropriate treatment. Aims. This case series aims to provide additional insights into the clinical presentation of MADD, highlighting diagnostic challenges in differentiating metabolic myopathies and emphasizing the role of muscle biopsy in diagnosing these conditions. Results. We describe five clinical cases of patients who were diagnosed with MADD, their clinical manifestations, and the diagnostic processes undertaken to arrive at this diagnosis. Notably, three patients were initially misdiagnosed with inflammatory myopathy, and one was misdiagnosed with Guillain–Barré syndrome. The correct diagnosis was established following a muscle biopsy, which revealed characteristic findings consistent with lipid storage myopathy and prompted subsequent biochemical analyses and genetic testing that confirmed the diagnosis of MADD. Conclusions. MADD is an underdiagnosed condition, and the description of new patients with various clinical presentations could support the development of clinical tools to promptly recognize this disease and allow physicians to deliver the most appropriate and effective therapy protocol, with Riboflavin and Carnitine supplementations, avoiding inappropriate treatments. The muscle biopsy was essential for a correct diagnostic assessment. Full article

Neonatal screening programs for inborn errors of metabolism are essential for early diagnosis and intervention. However, false-positive results can cause unnecessary psychological stress for caregivers. This study investigated the emotional impact on a small number of caregivers in Oita Prefecture in Japan, whose infants received false-positive screening results for very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Particular attention was given to caregivers’ concerns regarding episodes of transient fasting suggestive of nutritional deficiency, as well as their perspectives on appropriate feeding practices for newborns. Nineteen infants in Oita Prefecture were identified as having elevated acylcarnitines, which were later confirmed as false positives. Of these cases, 11 mothers consented to participate in a survey and long-term growth evaluation using health check records. Thirty children with normal screening results were included as controls. While no differences in physical growth were found between groups by 3.5 years of age, some mothers of false-positive infants reported persistent anxiety. Their concerns included regret for inadequate breastfeeding and latent adverse effects on long-term growth or development. Conversely, caregivers’ anxiety diminished over time as they directly observed their infants’ normal growth and development. No regret was expressed regarding breastfeeding, and concerns about VLDCAD were not observed. Caregivers’ responses may help reduce their psychological burden. Full article

Background and Aims: Little is known about metabolic dysfunction-associated steatotic liver disease (MASLD) as a risk factor for hepatocellular carcinoma (HCC) in non-cirrhotic (HCC-NC) patients. In-house developed mouse models with defective lipid-metabolizing enzyme long-chain 3-hydroxy acyl-CoA dehydrogenase (LCHAD), coded by hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA) gene, result in MASLD (steatosis) without cirrhosis leading to HCC-NC. The aims of the current investigations are to assess molecular markers and the associated molecular events that may lead to HCC-NC. Methods: cDNA array study of HCC patients was conducted to assess the expression of HADHA transcripts. Differentially expressed proteins identified between wild-type (WT) and heterozygous mice with no cancer (HT) from a previous study were subjected to Ingenuity Pathway Analysis (IPA). Western blotting was performed to assess the expression of proteins. Results: IPA of the differentially expressed proteins between WT and HT mice results in two biological networks (network 1 and network 2), which pointed to an important role of p53 in HCC-NC. Validation of the levels of MDM2 and p53 also highlights the role of MDM2-p53 axis in HCC-NC. All the focus molecules in network 1 and network 2 are either presented as tumor suppressor/promoter of carcinogenesis or serum markers for early HCC diagnosis. The hepatotoxicity report from IPA further identified four functional groups including liver steatosis, glutathione depletion, hepatocellular carcinoma, and liver hyperplasia/hyperproliferation. Conclusions: This study suggests that impaired fatty oxidation may play a role in the development of HCC associated with steatosis but without cirrhosis (HCC-NC). Defective LCHAD is a novel etiology for HCC. Full article

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessive disorder of fatty acid oxidation that can have life-threatening consequences if not promptly treated. Early diagnosis by means of newborn screening (NBS) has the potential to reduce morbidity and mortality. This study investigates the incidence and molecular characteristics of MCADD in Italy over a five-year period within the framework of the expanded NBS program. Between January 2019 and December 2023, a total of 1,976,473 newborns were screened. Ninety unrelated neonates were diagnosed with MCADD, providing an estimated incidence of 1/21,960 live births (95% CI: 1:17,780–1:27,200), comparable to rates reported in other Mediterranean populations. Molecular analysis identified c.985A>G (p.Lys329Glu) as the most frequent pathogenic ACADM gene variant, observed in 56 patients (63%), including eighteen patients (20%) who were homozygous and thirty-eight (43%) who were compound heterozygotes for this variant. To our knowledge, this study represents the first comprehensive investigation to document the high prevalence of MCADD among the Italian population. Full article

Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD) is a metabolic disorder caused by mutations in the ACADM gene, leading to impaired fatty acid oxidation. The present study aims to analyze the prevalence and genetic mutation characteristics of MCADD among newborns in Hefei, China, providing insights for the diagnosis, treatment, and prevention of MCADD. A retrospective analysis was conducted on data from newborns diagnosed with MCADD at the Hefei Newborn Disease Screening Center between January 2016 and December 2024. Screening was performed using tandem mass spectrometry (MS/MS), complemented by next-generation sequencing (NGS) for genetic testing. Out of 880,224 screened newborns, 16 cases of MCADD were diagnosed, resulting in a prevalence of 1 in 55,014. A total of 31 mutation sites in the ACADM gene were identified, with 18 different mutation types. The hotspot mutations were c.449-452del (p.T150Rfs*4) and c.1085G>A (p.G362E), each with a mutation frequency of 16.13% (5 out of 31). Additionally, three novel mutations were identified: c.468+5G>A, c.854C>G, and c.428_431delinsTCTTCTTTTGTT. Following diagnosis, patients received health education, dietary guidance, and symptomatic treatment, all resulting in favorable prognoses without any acute metabolic decompensation events. The prevalence of MCADD is lower in Asia compared to Europe and America. The hotspot mutations for MCADD in Hefei are c.449-452del and c.1085G>A. Diagnosis should integrate results from both octanoylcarnitine (C8) levels and genetic testing. Early screening, diagnosis, treatment, and scientific prevention strategies are essential for reducing adverse outcomes in children with MCADD. Full article

Acyl-CoA dehydrogenase 9 deficiency is considered as a rare neuromuscular syndrome with an autosomal recessive transmission. The ACAD9 protein presents two essential functions, i.e., the limiting step enzyme of the fatty acid β-oxidation pathway and one of the complex’s compounds involved in the respiratory chain complex I assembly. Thus, loss-of-function mutations are known to convey mitochondrial cytopathologies. A patient with a mild and late-onset phenotype, suffering from exercise intolerance and hypertrophic cardiomyopathy, was diagnosed as a compound heterozygote of the ACAD9 gene. The first c.1240C> T p.Arg414Cys variant has been previously reported and is known to be responsible for ACAD9 deficiency. However, the second c.1636G> A p.Val546Met variant has never been described. The goal was to investigate the eventual pathogenicity of this new genetic variant. For this purpose, molecular cloning was generated to express the ACAD9 gene with the V546M variant in a cell line (ACAD9mut) and compared to cells expressing the wild-type ACAD9. Then, the mitochondrial respiration, ATP production, the mitochondrial network, and the oxidative phosphorylation’s composition were investigated to reveal the effects of the V546M variant. While avoiding to affect the amount of the respiratory chain’s complexes, the new ACAD9 variant was entirely responsible for reducing over 50% of the mitochondrial complex I activity. Full article

Multiple acyl-CoA dehydrogenase deficiency (MADD), also known as glutaric acidemia/glutaric aciduria type II (GA II), is an inborn error of fatty acid, amino acid, and choline metabolism. The chronic management of MADD involves both dietary fat and protein restriction to reduce the substrates of the dehydrogenases affected, the avoidance of prolonged fasting as in any fat metabolism disorder, and monitoring for potential complications. Due to its rarity, there is little published experience on the management of MADD in pregnancy. Herein, we report the successful management of a pregnancy in a patient with late-onset or type III MADD, with considerations for preconception, antepartum, intrapartum, and postpartum care. Full article

Background/Objectives: Hexaraphane, also known as 6-methylsulfinylhexyl isothiocyanate, derived from wasabi (Eutrema japonicum), increases heme oxygenase-1 (HO-1) and aldehyde dehydrogenase 2 (ALDH2) mRNA expression by activating nuclear factor erythroid 2-related factor 2 (Nrf2) in both HepG2 cells and the mouse liver. Given the presence of a peroxisome proliferator-activated receptor (PPAR) response element (PPRE) in the HO-1 and ALDH2 promoters, the present study aimed to determine the effects of hexaraphane on PPARα-associated genes, age-related weight gain, plasma triglyceride levels, and hepatic senescence. Methods: HepG2 cells were treated with hexaraphane to evaluate PPARα target gene expression and PPRE transcriptional activity. Male C57BL/6J young control, aged control, and aged mice administered with hexaraphane for 16 weeks were assessed for food and water intake, body and tissue weights, plasma parameters, and hepatic PPARα-related gene expression. Results: Hexaraphane increased HO-1 mRNA expression levels in HepG2 cells, which was inhibited by GW6471, a PPARα antagonist. It elevated PPRE transcriptional activity and increased carnitine palmitoyltransferase 1A (CPT1A) mRNA expression levels, indicating PPARα activation. In aged mice, hexaraphane intake reduced body weight gain by decreasing the adipose tissue weight. Increased CPT1A expression levels and a tendency toward increased acyl-CoA oxidase 1 (ACOX1) expression levels in the liver of aged mice administered hexaraphane were associated with reduced plasma triglyceride levels and body weight gain. Increased hepatic Sirt1 expression levels in aged mice administered hexaraphane was associated with lower plasma triglyceride levels. Increased hepatic PPARα mRNA expression levels in aged mice administered hexaraphane suggest a positive feedback loop between PPARα and Sirt1. The expression levels of hepatic p21 mRNA, a senescence marker regulated by Sirt1, were upregulated in aged mice but suppressed by hexaraphane intake. Conclusions: Hexaraphane may prevent age-related body weight gain, elevated plasma triglyceride levels, and hepatic senescence by activating PPARα, potentially contributing to longevity. Full article

To explore the differences in protein quality among classic medicinal entomopathogenic fungi and to evaluate their metabolic adaptability, we analyzed the amino acid composition and proteomic characteristics of Cordyceps sinensis (CS), Cordyceps militaris (CM), and Cordyceps cicadae (CC). Quantitative analysis showed CM contained the highest crude protein and lysine, methionine, threonine, and valine. CS adapted to high-altitude hypoxia and exhibited lower protein but elevated leucine, isoleucine, and histidine contents, which may contribute to membrane stabilization and oxidative stress resistance. CC displayed higher non-essential amino acids such as arginine, proline, and tyrosine, reflecting active nitrogen metabolism. Four-dimensional data-independent acquisition (4D-DIA) proteomics identified 495 differentially expressed proteins (DEPs). Compared with CS, CM and CC displayed upregulated glutamate oxaloacetate transaminases 2 (GOT2), glutamate dehydrogenase (GDH), and argininosuccinate synthase 1 (ASS1) coordinately regulate nitrogen flux through the alanine-aspartate-glutamate metabolic network and urea cycle, supporting metabolic intermediate replenishment for energy metabolism. The upregulation of branched-chain keto acid dehydrogenase E1 subunit alpha (BCKDHA) and acyl-CoA dehydrogenase short/branched chain (ACADSB) in CM and CC facilitated the integration of branched-chain amino acid catabolism with the TCA cycle, explaining species-specific differences in protein content. This study presents the first application of 4D-DIA proteomics to compare CS, CM, and CC, providing insights into quality divergence mechanisms in medicinal fungi. Full article

Background: Very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is caused by biallelic pathogenic variants in ACADVL (acyl-CoA dehydrogenase very-long-chain), leading to impaired fatty acid oxidation and the accumulation of long-chain acylcarnitine. We report a single case of a two-year-old girl, whose neonatal metabolic screening revealed an acylcarnitine profile suggestive of VLCADD, with residual enzymatic activity of 19.8%. Methods: We performed ACADVL whole-gene sequencing. We then carried out an in silico analysis of the potential effects of the variants with dedicated tools, assessing splicing, RNA structure, RNA binding factors, and protein structure. We also conducted gene expression analysis. Results: Genetic testing identified her as compound heterozygous for the pathogenic ACADVL variant (NM_000018.3):c.848T>C, inherited from her mother, and for the two paternal variants, c.-64T>C in the basal promoter and c.957G>A, a synonymous substitution in exon 10. Gene expression analysis revealed reduced ACADVL mRNA levels in the proband’s blood cells but without abnormal isoform production. A decreased expression of the paternal allele carrying the 957A was also observed. Despite this significant reduction in mRNA levels, the underlying mechanism remains unclear. Conclusions: Although currently healthy, due to the VLCAD residual activity within the range associated with the mild form of the disease, the child might be at potential risk for metabolic decompensation or late-onset VLCADD. Our results indicated an allelic imbalance in mRNA expression and c.957G>A is identified as a hypomorphic allele. This suggests that deep ACADVL sequencing is a valuable tool for correlating genetic variants with enzymatic activity levels. Full article

The detection of elevated long-chain acylcarnitine levels, particularly C14:1 and the C14:1/C2 ratio, during neonatal screening may indicate very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), although similar findings can result from postnatal starvation. We investigated the relationship between false-positive results, postnatal weight loss, and subsequent growth. Additionally, we explored potential diagnostic markers of postnatal starvation. The following neonates from Oita Prefecture (April 2014–March 2024) were included in this study: patients identified as false-positive for VLCADD (n = 19), patients with VLCADD (n = 3), and children negative in mass screening who completed their 3-year-old health check-up (n = 30). The false-positive group exhibited significant weight loss at blood sampling for neonatal screening. An acylcarnitine analysis showed significant increases in various short- to long-chain fatty acids in the false-positive group, likely owing to enhanced fatty acid catabolism via β-oxidation. Elevation of a broad range of fatty acids and reduced amino acid levels seemed to be associated with significant weight loss at blood sampling. Full article