Low ACADM expression predicts poor prognosis and suppressive tumor microenvironment in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) represents a highly frequent renal cancer subtype. However, medium-chain acyl-CoA dehydrogenase (ACADM) encodes an important enzyme responsible for fatty acid ß-oxidation (FAO) and its association with prognosis and immunity in cancers has rarely been reported. Therefore, the present work focused on exploring ACADM's expression and role among ccRCC cases. We used multiple public databases and showed the hypo levels of ACADM protein and mRNA within ccRCC. Additionally, we found that ACADM down-regulation showed a remarkable relation to the advanced stage, high histological grade, as well as dismal prognostic outcome. As suggested by Kaplan-Meier curve analysis, cases showing low ACADM levels displayed shorter overall survival (OS) as well as disease-free survival (DFS). Moreover, according to univariate/multivariate Cox regression, ACADM-mRNA independently predicted the prognosis of ccRCC. In addition, this work conducted immunohistochemistry for validating ACADM protein expression and its prognostic role in ccRCC samples. KEGG and GO analyses revealed significantly enriched genes related to ACADM expression during fatty acid metabolism. The low-ACADM group with more regulatory T-cell infiltration showed higher expression of immune negative regulation genes and higher TIDE scores, which might contribute to poor response to immunotherapies. In conclusion, our results confirmed that downregulated ACADM predicted a poor prognosis for ccRCC and a poor response to immunotherapy. Our results provide important data for developing immunotherapy for ccRCC.

Sensory neuropathy as a manifestation of multiple acyl-coenzyme A dehydrogenase deficiency.

Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is a rare metabolic disorder which typically manifests with muscle weakness. However, despite late-onset MADD being treatable, it is often misdiagnosed, due in part to the heterogeneity of presentations. We report a case of late-onset MADD manifesting first as a sensory neuropathy before progressing to myopathic symptoms and acute metabolic decompensation. Early diagnostic workup with acylcarnitine profiling and organic acid analysis was critical in patient outcome; metabolic decompensation and myopathic symptoms were completely reversed with riboflavin supplementation and dietary modification, although sensory neuropathy persisted. Clinical consideration of MADD as part of the differential diagnosis of neuropathy with myopathy is crucial for a timely diagnosis and treatment of MADD.

The presence of white cell Jordan's anomaly in multiple Acyl-CoA dehydrogenase deficiency: A case report and implications for clinical practice.

BACKGROUND: Multiple Acyl-CoA Dehydrogenase Deficiency (MADD), also known as Glutaric Aciduria Type II, is an exceptionally rare autosomal recessive genetic disorder that disrupts the metabolism of fatty acids, amino acids, and choline. It presents with a wide range of clinical manifestations, from severe neonatal-onset forms to milder late-onset cases, with symptoms including metabolic disturbances and muscle weakness. Jordan's anomaly is a distinctive morphological feature found in peripheral blood white cells and is typically associated with Neutral Lipid Storage Disease (NLSD). CASE REPORT: In our case report, the patient initially presented with symptoms of vomiting, abdominal pain, and altered consciousness. The presence of white cell Jordan's anomaly was detected in the blood smear. Subsequent serum tests revealed elevated levels of transaminases, creatine kinase, uric acid, and multiple acylcarnitines, while blood glucose and free carnitine levels were notably reduced. High-throughput sequencing confirmed heterozygous pathogenic variants in the electron-transferring flavoprotein dehydrogenase (ETFDH) gene, leading to the conclusive diagnosis of MADD. Following a three-month treatment regimen involving high-dose vitamin B2, coenzyme Q10, and other supportive interventions, the patient exhibited significant clinical improvement, ultimately resulting in discharge. CONCLUSION: The identification of Jordan's anomaly in a pediatric patient with late-onset MADD sheds light on its broader implications within the realm of lipid storage myopathies. The significance of this finding extends beyond its conventional association with NLSD, challenging the notion of its exclusivity. This novel observation serves as a compelling reminder of the diagnostic significance this morphological abnormality holds, potentially revolutionizing diagnostic practices within the field.

The male-to-female ratio in late-onset multiple acyl-CoA dehydrogenase deficiency: a systematic review and meta-analysis.

BACKGROUND: Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common lipid storage myopathy. There are sex differences in fat metabolism and it is not known whether late-onset MADD affects men and women equally. METHODS: In this systematic review and meta-analysis, the PubMed, Embase, Web of Science, CNKI, CBM, and Wanfang databases were searched until 01/08/2023. Studies reporting sex distribution in patients with late-onset MADD were included. Two authors independently screened studies for eligibility, extracted data, and assessed risk of bias. Pre-specified outcomes of interest were the male-to-female ratio (MFR) of patients with late-onset MADD, the differences of clinical characteristics between the sexes, and factors influencing the MFR. RESULTS: Of 3379 identified studies, 34 met inclusion criteria, yielding a total of 609 late-onset MADD patients. The overall pooled percentage of males was 58% (95% CI, 54-63%) with low heterogeneity across studies (I2 = 2.99%; P = 0.42). The mean onset ages, diagnostic delay, serum creatine kinase (CK), and allelic frequencies of 3 hotspot variants in ETFDH gene were similar between male and female patients (P > 0.05). Meta-regressions revealed that ethnic group was associated with the MFR in late-onset MADD, and subgroup meta-analyses demonstrated that East-Asian patients had a higher percentage of male, lower CK, and higher proportion of hotspot variants in ETFDH gene than non-East-Asian patients (P < 0.05). CONCLUSIONS: Male patients with late-onset MADD were more common than female patients. Ethnicity was proved to be a factor influencing the MFR in late-onset MADD. These findings suggest that male sex may be a risk factor for the disease.

Assessing the quality and value of metabolic chart data for capturing core outcomes for pediatric medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

BACKGROUND: Generating rigorous evidence to inform care for rare diseases requires reliable, sustainable, and longitudinal measurement of priority outcomes. Having developed a core outcome set for pediatric medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, we aimed to assess the feasibility of prospective measurement of these core outcomes during routine metabolic clinic visits. METHODS: We used existing cohort data abstracted from charts of 124 children diagnosed with MCAD deficiency who participated in a Canadian study which collected data from birth to a maximum of 11 years of age to investigate the frequency of clinic visits and quality of metabolic chart data for selected outcomes. We recorded all opportunities to collect outcomes from the medical chart as a function of visit rate to the metabolic clinic, by treatment centre and by child age. We applied a data quality framework to evaluate data based on completeness, conformance, and plausibility for four core MCAD outcomes: emergency department use, fasting time, metabolic decompensation, and death. RESULTS: The frequency of metabolic clinic visits decreased with increasing age, from a rate of 2.8 visits per child per year (95% confidence interval, 2.3-3.3) among infants 2 to 6 months, to 1.0 visit per child per year (95% confidence interval, 0.9-1.2) among those ≥ 5 years of age. Rates of emergency department visits followed anticipated trends by child age. Supplemental findings suggested that some emergency visits occur outside of the metabolic care treatment centre but are not captured. Recommended fasting times were updated relatively infrequently in patients' metabolic charts. Episodes of metabolic decompensation were identifiable but required an operational definition based on acute manifestations most commonly recorded in the metabolic chart. Deaths occurred rarely in these patients and quality of mortality data was not evaluated. CONCLUSIONS: Opportunities to record core outcomes at the metabolic clinic occur at least annually for children with MCAD deficiency. Methods to comprehensively capture emergency care received at outside institutions are needed. To reduce substantial heterogeneous recording of core outcome across treatment centres, improved documentation standards are required for recording of recommended fasting times and a consensus definition for metabolic decompensations needs to be developed and implemented.

A gene therapy targeting medium-chain acyl-CoA dehydrogenase (MCAD) did not protect against diabetes-induced cardiac pathology.

Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. KEY MESSAGES: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications.

Structural insights into the pathogenicity of point mutations in human acyl-CoA dehydrogenase homotetramers.

Acyl-CoA dehydrogenase deficiency (ACAD) is an inherited and potentially fatal disorder with variable clinical symptoms. The relationship between pathogenicity and deleterious point mutations is investigated here in ACAD structures of short (SCAD) and medium-chain (MCAD) types. Structures and dynamic features of native and mutant forms of enzymes models were compared. A total of 2.88 µs molecular dynamics simulations were performed at four different temperatures. Total energy, RMSD, protein ligand interactions and affinity, RMSF measures, secondary structure changes, and important interactions were studied. Mutations in the three main domains of ACADs are pathogenic, while those located at linker turns are not. Mutations affect mostly tetramer formations, secondary structures, and many contacts and interactions. In R206H (MCAD mutant) which is experimentally known to cause a huge turnover decrease, the lack of a single H-bond between substrate and FAD was observed. Secondary structures showed temperature-dependent changes, and SCAD activity was found to be highly correlated to the enzyme helix 3-10 content. Finally, RMSF patterns pointed to one important loop that maintains the substrate close to the active site and is a cause of substrate wobbling upon mutation. Despite similar structure, function, and cellular location, SCAD and MCAD may have different optimum temperatures that are related to the structure taken at that specific temperature. In conclusion, new insight has been provided on the effect of various SCAD and MCAD pathogenic mutations on the structure and dynamical features of the enzymes.

Analysis of genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine. / ä¸é °åŸºè‚‰ç¢±ä»£è°¢å¼‚å¸¸æ–°ç”Ÿå„¿çš„åŸºå› åž‹å’Œç”ŸåŒ–è¡¨åž‹åˆ†æž.

OBJECTIVES: To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4). METHODS: One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children's Hospital, Zhejiang University School of Medicine from January 2018 to June 2023 were included. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. Next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. Variant information was obtained by bioinformatic analysis and the pathogenicity were classified according to the American College of Medical Genetics and Genomics criteria. The Wilcoxon rank sum test was used to analyze the differences in C4 levels among neonates with different variation types. RESULTS: In total, 32 variants in ACAD8 gene were detected, of which 7 variants were reported for the first time; while 41 variants of ACADS gene were detected, of which 17 variants have not been previously reported. There were 39 cases with ACAD8 biallelic variations and 3 cases with ACAD8 monoallelic variations; 34 cases with ACADS biallelic variations and 36 cases with ACADS monoallelic variations. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variations. Inter group comparison showed that the multiples of C4 reference range in initial screening and re-examination of the ACAD8 biallelic variations and ACADS biallelic variations groups were significantly higher than those of the ACADS monoallelic variations group (all P<0.01), while the multiples in the ACAD8 biallelic variations group were significantly higher than those in the ACADS biallelic variations group (all P<0.01). The multiples of C4 reference range in the initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variations, while only 25% (9/36) in neonates carrying ACADS monoallelic variations. CONCLUSIONS: ACAD8 and/or ACADS gene variants are the main genetic causes for elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variations are significantly higher than those of neonates with monoallelic variations. The cut-off value for C4 level could be modestly elevated, which could reduce the false positive rate in tandem mass spectrometry neonatal screening.

Personalised modelling of clinical heterogeneity between medium-chain acyl-CoA dehydrogenase patients.

BACKGROUND: Monogenetic inborn errors of metabolism cause a wide phenotypic heterogeneity that may even differ between family members carrying the same genetic variant. Computational modelling of metabolic networks may identify putative sources of this inter-patient heterogeneity. Here, we mainly focus on medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common inborn error of the mitochondrial fatty acid oxidation (mFAO). It is an enigma why some MCADD patients-if untreated-are at risk to develop severe metabolic decompensations, whereas others remain asymptomatic throughout life. We hypothesised that an ability to maintain an increased free mitochondrial CoA (CoASH) and pathway flux might distinguish asymptomatic from symptomatic patients. RESULTS: We built and experimentally validated, for the first time, a kinetic model of the human liver mFAO. Metabolites were partitioned according to their water solubility between the bulk aqueous matrix and the inner membrane. Enzymes are also either membrane-bound or in the matrix. This metabolite partitioning is a novel model attribute and improved predictions. MCADD substantially reduced pathway flux and CoASH, the latter due to the sequestration of CoA as medium-chain acyl-CoA esters. Analysis of urine from MCADD patients obtained during a metabolic decompensation showed an accumulation of medium- and short-chain acylcarnitines, just like the acyl-CoA pool in the MCADD model. The model suggested some rescues that increased flux and CoASH, notably increasing short-chain acyl-CoA dehydrogenase (SCAD) levels. Proteome analysis of MCADD patient-derived fibroblasts indeed revealed elevated levels of SCAD in a patient with a clinically asymptomatic state. This is a rescue for MCADD that has not been explored before. Personalised models based on these proteomics data confirmed an increased pathway flux and CoASH in the model of an asymptomatic patient compared to those of symptomatic MCADD patients. CONCLUSIONS: We present a detailed, validated kinetic model of mFAO in human liver, with solubility-dependent metabolite partitioning. Personalised modelling of individual patients provides a novel explanation for phenotypic heterogeneity among MCADD patients. Further development of personalised metabolic models is a promising direction to improve individualised risk assessment, management and monitoring for inborn errors of metabolism.

Heptanoic and medium branched-chain fatty acids as anaplerotic treatment for medium chain acyl-CoA dehydrogenase deficiency.

Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.

Decreased Fatty Acid Oxidation Gene Expression in Pre-Eclampsia According to the Onset and Presence of Intrauterine Growth Restriction.

Mitochondrial fatty acid oxidation (FAO) is lower in placentas with pre-eclampsia. The aim of our study was to compare the placental mRNA expression of FAO enzymes in healthy pregnancies vs. different subgroups of pre-eclampsia according to the severity, time of onset, and the presence of intrauterine growth restriction (IUGR). By using real-time qPCR, we measured the mRNA levels of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), medium-chain acyl-CoA dehydrogenase (MCAD), and carnitine palmitoyltransferases 1A and 2 (CPT1A, CPT2) on the maternal side (anchoring villi in the basal decidua) and on the fetal side (chorionic plate) of the placenta (n = 56). When compared to the controls, LCHAD, MCAD, and CPT2 mRNA had decreased in all pre-eclampsia subgroups globally and on the fetal side. On the maternal side, LCHAD mRNA was also lower in all pre-eclampsia subgroups; however, MCAD and CPT2 mRNA were only reduced in severe and early-onset disease, as well as CPT2 in IUGR (p < 0.05). There were no differences in CPT1A mRNA expression. We conclude that the FAO enzymes mRNA in the placenta was lower in pre-eclampsia, with higher reductions observed in severe, early-onset, and IUGR cases and more striking reductions on the fetal side.

Paeonol represses A549 cell glycolytic reprogramming and proliferation by decreasing m6A modification of Acyl-CoA dehydrogenase.

Aberrant glycolytic reprogramming is involved in lung cancer progression by promoting the proliferation of non-small cell lung cancer cells. Paeonol, as a traditional Chinese medicine, plays a critical role in multiple cancer cell proliferation and inflammation. Acyl-CoA dehydrogenase (ACADM) is involved in the development of metabolic diseases. N6-methyladenosine (m6A) modification is important for the regulation of messenger RNA stability, splicing, and translation. Here, we investigated whether paeonol regulates the proliferation and glycolytic reprogramming via ACADM with m6A modification in A549 cells (human non-small cell lung cancer cells). Cell counting kit 8, 5-Bromo-2-deoxyuridine, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, flow cytometry analysis, western blotting and seahorse XFe24 extracellular flux analyzer assays showed that paeonol had a significant inhibitory effect against A549 cell proliferation and glycolysis. Mechanistically, ACADM was a functional target of paeonol. We also showed that the m6A reader YTH domain containing 1 plays an important role in m6A-modified ACADM expression, which is negatively regulated by paeonol, and is involved in A549 cell proliferation and glycolytic reprogramming. These results indicated the central function of paeonol in regulating A549 cell glycolytic reprogramming and proliferation via m6A modification of ACADM.

Exosomal ACADM sensitizes gemcitabine-resistance through modulating fatty acid metabolism and ferroptosis in pancreatic cancer.

This study aimed to evaluate the potential of exosomes from cancer cells to predict chemoresistance in pancreatic cancer (PC) and explore the molecular mechanisms through RNA-sequencing and mass spectrometry. We sought to understand the connection between the exosomal Medium-chain acyl-CoA dehydrogenase (ACADM) level and the reaction to gemcitabine in vivo and in patients with PC. We employed loss-of-function, gain-of-function, metabolome mass spectrometry, and xenograft models to investigate the effect of exosomal ACADM in chemoresistance in PC. Our results showed that the molecules involved in lipid metabolism in exosomes vary between PC cells with different gemcitabine sensitivity. Exosomal ACADM (Exo-ACADM) was strongly correlated with gemcitabine sensitivity in vivo, which can be used as a predictor for postoperative gemcitabine chemosensitivity in pancreatic patients. Moreover, ACADM was found to regulate the gemcitabine response by affecting ferroptosis through Glutathione peroxidase 4 (GPX4) and mevalonate pathways. It was also observed that ACADM increased the consumption of unsaturated fatty acids and decreased intracellular lipid peroxides and reactive oxygen species (ROS) levels. In conclusion, this research suggests that Exo-ACADM may be a viable biomarker for predicting the responsiveness of patients to chemotherapy.

[Analysis of clinical characteristics and ACADM gene variants in four children with Medium chain acyl-CoA dehydrogenase deficiency].

OBJECTIVE: To explore the clinical and genetic characteristics of four patients with medium-chain acyl-CoA dehydrogenase deficiency (MCADD). METHODS: Four children who had presented at the Children's Hospital Affiliated to Zhengzhou University between August 2019 and August 2021 were selected as the study subjects. Clinical data of the children were collected. The children were subjected to whole exome sequencing (WES). RESULTS: All of the four children were diagnosed with MCADD. Blood amino acid and ester acyl carnitine spectrum test showed that the concentration of octanoyl carnitine (C8) was significantly increased. The main clinical manifestations included poor mental response (3 cases), intermittent diarrhea with abdominal pain (1 case), vomiting (1 case), increased transaminase (3 cases), and metabolic acidosis (2 cases). Five variants were identified by genetic testing, among which c.341A>G (p.Y114C) was unreported previously. Three were missense variants, one was frameshift variant and one was splicing variant. CONCLUSION: The clinical heterogeneity of MCADD is obvious, and the severity of the disease may vary. WES can assist with the diagnosis. Delineation of the clinical symptoms and genetic characteristics of the disease can facilitate early diagnosis and treatment of the disease.

Acyl-CoA dehydrogenase substrate promiscuity: Challenges and opportunities for development of substrate reduction therapy in disorders of valine and isoleucine metabolism.

Toxicity of accumulating substrates is a significant problem in several disorders of valine and isoleucine degradation notably short-chain enoyl-CoA hydratase (ECHS1 or crotonase) deficiency, 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, propionic acidemia (PA), and methylmalonic aciduria (MMA). Isobutyryl-CoA dehydrogenase (ACAD8) and short/branched-chain acyl-CoA dehydrogenase (SBCAD, ACADSB) function in the valine and isoleucine degradation pathways, respectively. Deficiencies of these acyl-CoA dehydrogenase (ACAD) enzymes are considered biochemical abnormalities with limited or no clinical consequences. We investigated whether substrate reduction therapy through inhibition of ACAD8 and SBCAD can limit the accumulation of toxic metabolic intermediates in disorders of valine and isoleucine metabolism. Using analysis of acylcarnitine isomers, we show that 2-methylenecyclopropaneacetic acid (MCPA) inhibited SBCAD, isovaleryl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase, but not ACAD8. MCPA treatment of wild-type and PA HEK-293 cells caused a pronounced decrease in C3-carnitine. Furthermore, deletion of ACADSB in HEK-293 cells led to an equally strong decrease in C3-carnitine when compared to wild-type cells. Deletion of ECHS1 in HEK-293 cells caused a defect in lipoylation of the E2 component of the pyruvate dehydrogenase complex, which was not rescued by ACAD8 deletion. MCPA was able to rescue lipoylation in ECHS1 KO cells, but only in cells with prior ACAD8 deletion. SBCAD was not the sole ACAD responsible for this compensation, which indicates substantial promiscuity of ACADs in HEK-293 cells for the isobutyryl-CoA substrate. Substrate promiscuity appeared less prominent for 2-methylbutyryl-CoA at least in HEK-293 cells. We suggest that pharmacological inhibition of SBCAD to treat PA should be investigated further.

Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid ß-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.

Bulk and single-cell transcriptome profiling reveal extracellular matrix mechanical regulation of lipid metabolism reprograming through YAP/TEAD4/ACADL axis in hepatocellular carcinoma.

Emerging studies have revealed matrix stiffness promotes hepatocellular carcinoma (HCC) development. We studied metabolic dysregulation in HCC using the TCGA-LIHC database (n=374) and GEO datasets (GSE14520). HCC samples were classified into three heterogeneous metabolic pathway subtypes with different metabolic profiles: Cluster 1, an ECM-producing subtype with upregulated glycan metabolism; Cluster 2, a hybrid subtype with partial pathway dysregulation. Cluster 3, a lipogenic subtype with upregulated lipid metabolism; These three subtypes have different prognosis, clinical features and genomic alterations. We identified key enzymes that respond to matrix stiffness and regulate lipid metabolism through bioinformatic analysis. We found long-chain acyl-CoA dehydrogenase (ACADL) is a mechanoreactive enzyme that reprograms HCC cell lipid metabolism in response to extracellular matrix stiffness. ACADL is also regarded as tumor suppressor in HCC. We found that increased extracellular matrix stiffness led to activation of Yes-associated protein (YAP) and the YAP/TEA Domain transcription factor 4 (TEAD4) transcriptional complex was able to directly repress ACADL at the transcriptional level. The ACADL-dependent mechanoresponsive pathway is a potential therapeutic target for HCC treatment.

Functional and structural impact of 10 ACADM missense mutations on human medium chain acyl-Coa dehydrogenase.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is associated with ACADM gene mutations, leading to an impaired function and/or structure of MCAD. Importantly, after import into the mitochondria, MCAD must incorporate a molecule of flavin adenine dinucleotide (FAD) per subunit and assemble into tetramers. However, the effect of MCAD amino acid substitutions on FAD incorporation has not been investigated. Herein, the commonest MCAD variant (p.K304E) and 11 additional rare variants (p.Y48C, p.R55G, p.A88P, p.Y133C, p.A140T, p.D143V, p.G224R, p.L238F, p.V264I, p.Y372N, and p.G377V) were functionally and structurally characterized. Half of the studied variants presented a FAD content <65 % compared to the wild-type. Most of them were recovered as tetramers, except the p.Y372N (mainly as dimers). No correlation was found between the levels of tetramers and FAD content. However, a correlation between FAD content and the cofactor's affinity, proteolytic stability, thermostability, and thermal inactivation was established. We showed that the studied amino acid changes in MCAD may alter the substrate chain-length dependence and the interaction with electron-transferring-flavoprotein (ETF) necessary for a proper functioning electron transfer thus adding additional layers of complexity to the pathological effect of ACADM missense mutations. Although the majority of the variant MCADs presented an impaired capacity to retain FAD during their synthesis, some of them were structurally rescued by cofactor supplementation, suggesting that in the mitochondrial environment the levels and activity of those variants may be dependent of FAD's availability thus contributing for the heterogeneity of the MCADD phenotype found in patients presenting the same genotype.

Fenofibrate Attenuates Renal Tubular Cell Apoptosis by Up-Regulating MCAD in Diabetic Kidney Disease.

Background: Diabetic kidney disease (DKD) is a major diabetic microvascular complication. Fatty acid-induced lipotoxicity and apoptosis were associated with the exacerbation of DKD. However, the association of lipotoxicity with renal tubular apoptosis and the effects of fenofibrate on DKD are not fully understood. Methods: Eight-week-old db/db mice were given fenofibrate or saline by gavage for 8 weeks. Human kidney proximal tubular epithelial (HK2) cells stimulated with palmitic acid (PA) and high glucose (HG) were used as a model of lipid metabolism disorders. Apoptosis was assessed with or without fenofibrate. The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and AMPK inhibitor Compound C were used to determine the involvement of AMPK and Medium-chain acyl-CoA dehydrogenase (MCAD) in the regulation of lipid accumulation by fenofibrate. MCAD silencing was achieved by small interfering RNA (siRNA) transfection. Results: Fenofibrate reduced triglyceride (TG) content and lipid accumulation in DKD. Importantly, renal function and tubular cell apoptosis were significantly improved by fenofibrate. Fenofibrate reduced apoptosis, accompanied by increased activation of the AMPK/FOXA2/MCAD pathway. MCAD silencing resulted in apoptosis and lipid accumulation despite fenofibrate treatment. Conclusion: Fenofibrate improves lipid accumulation and apoptosis through the AMPK/FOXA2/MCAD pathway. MCAD may be a potential therapeutic target of DKD, and the use of fenofibrate as a treatment for DKD warrants further study.