Crystal structure combined with metabolomics and biochemical studies indicates that FAM3A participates in fatty acid beta-oxidation upon binding of acyl-L-carnitine.

As the first member of the family with sequence similarity 3 (FAM3), FAM3A promotes synthesis of ATP in mitochondria of hepatic cells and cells from other organs. Dysregulations of FAM3A are involved in the development of diabetes and nonalcoholic fatty liver disease (NAFLD). So far, the molecule mechanism under the physiological and pathological functions of FAM3A is largely unexplored. Here, we determined the crystal structure of FAM3A at high resolution of 1.38Å, complexed with an unknown-source compound which was characterized through metabolomics and confirmed as methacholine by thermal shift assay and surface plasmon resonance (SPR). Exploration for natural ligands of FAM3A was conducted through the same molecular interaction assays. The observed binding of acyl-L-carnitine molecules indicated FAM3A participating in fatty acid beta-oxidation. Knockdown and rescue assays coupled with fatty acid oxidation determination confirmed the role of FAM3A in beta-oxidation. This investigation reveals the molecular mechanism for the biological function of FAM3A and would provide basis for identifying drug target for treatment of diabetes and NAFLD.

Differential utilization of vitamin B12-dependent and independent pathways for propionate metabolism across human cells.

Propionic acid links the oxidation of branched-chain amino acids and odd-chain fatty acids to the TCA cycle. Gut microbes ferment complex fiber remnants, generating high concentrations of short chain fatty acids, acetate, propionate and butyrate, which are shared with the host as fuel sources. Analysis of vitamin B12-dependent propionate utilization in skin biopsy samples has been used to characterize and diagnose underlying inborn errors of cobalamin (or B12) metabolism. In these cells, the B12-dependent enzyme, methylmalonyl-CoA mutase (MMUT), plays a central role in funneling propionate to the TCA cycle intermediate, succinate. Our understanding of the fate of propionate in other cell types, specifically, the involvement of the ß-oxidation-like and methylcitrate pathways, is limited. In this study, we have used [14C]-propionate tracing in combination with genetic ablation or inhibition of MMUT, to reveal the differential utilization of the B12-dependent and independent pathways for propionate metabolism in fibroblast versus colon cell lines. We demonstrate that itaconate can be used as a tool to investigate MMUT-dependent propionate metabolism in cultured cell lines. While MMUT gates the entry of propionate carbons into the TCA cycle in fibroblasts, colon-derived cell lines exhibit a quantitatively significant or exclusive reliance on the ß-oxidation-like pathway. Lipidomics and metabolomics analyses reveal that propionate elicits pleiotropic changes, including an increase in odd-chain glycerophospholipids, and perturbations in the purine nucleotide cycle and arginine/nitric oxide metabolism. The metabolic rationale and the regulatory mechanisms underlying the differential reliance on propionate utilization pathways at a cellular, and possibly tissue level, warrant further elucidation.

Decidualized Endometrial Stromal Cells Promote Mitochondrial Beta-Oxidation to Produce the Octanoic Acid Required for Implantation.

Decidualization denotes the morphological and biological differentiating process of human endometrial stromal cells (HESCs). Fatty acid pathways are critical for endometrial decidualization. However, the participation of fatty acids as an energy source and their role in endometrial decidualization have received little attention. To identify fatty acids and clarify their role in decidualization, we comprehensively evaluated free fatty acid profiles using liquid chromatography/Fourier transform mass spectrometry (LC/FT-MS). LC/FT-MS analysis detected 26 kinds of fatty acids in the culture medium of decidualized or un-decidualized HESCs. Only the production of octanoic acid, which is an essential energy source for embryonic development, was increased upon decidualization. The expressions of genes related to octanoic acid metabolism including ACADL, ACADM, and ACADS; genes encoding proteins catalyzing the first step of mitochondrial fatty acid beta-oxidation; and ACSL5 and ACSM5; genes encoding fatty acid synthesis proteins were significantly altered upon decidualization. These results suggest that decidualization promotes lipid metabolism, implying that decidualized HESCs require energy metabolism of the mitochondria in embryo implantation.

ACAD10 and ACAD11 allow entry of 4-hydroxy fatty acids into ß-oxidation.

Hydroxylated fatty acids are important intermediates in lipid metabolism and signaling. Surprisingly, the metabolism of 4-hydroxy fatty acids remains largely unexplored. We found that both ACAD10 and ACAD11 unite two enzymatic activities to introduce these metabolites into mitochondrial and peroxisomal ß-oxidation, respectively. First, they phosphorylate 4-hydroxyacyl-CoAs via a kinase domain, followed by an elimination of the phosphate to form enoyl-CoAs catalyzed by an acyl-CoA dehydrogenase (ACAD) domain. Studies in knockout cell lines revealed that ACAD10 preferentially metabolizes shorter chain 4-hydroxy fatty acids than ACAD11 (i.e. 6 carbons versus 10 carbons). Yet, recombinant proteins showed comparable activity on the corresponding 4-hydroxyacyl-CoAs. This suggests that the localization of ACAD10 and ACAD11 to mitochondria and peroxisomes, respectively, might influence their physiological substrate spectrum. Interestingly, we observed that ACAD10 is cleaved internally during its maturation generating a C-terminal part consisting of the ACAD domain, and an N-terminal part comprising the kinase domain and a haloacid dehalogenase (HAD) domain. HAD domains often exhibit phosphatase activity, but negligible activity was observed in the case of ACAD10. Yet, inactivation of a presumptive key residue in this domain significantly increased the kinase activity, suggesting that this domain might have acquired a regulatory function to prevent accumulation of the phospho-hydroxyacyl-CoA intermediate. Taken together, our work reveals that 4-hydroxy fatty acids enter mitochondrial and peroxisomal fatty acid ß-oxidation via two enzymes with an overlapping substrate repertoire.

Metabolomic Disparities in Intraocular Fluid Across Varied Stages of Cataract Progression: Implications for the Analysis of Cataract Development.

Introduction: The lens's metabolic demands are met through a continuous circulation of aqueous humor, encompassing a spectrum of components such as organic and inorganic ions, carbohydrates, glutathione, urea, amino acids, proteins, oxygen, carbon dioxide, and water. Metabolomics is a pivotal tool, offering an initial insight into the complexities of integrated metabolism. In this investigative study, we systematically scrutinize the composition of intraocular fluid in individuals afflicted with cataracts. Methods: The investigation involved a comprehensive analysis of aqueous humor samples from a cohort comprising 192 patients. These individuals were stratified by utilizing the SPONCS classification system, delineating distinct groups characterized by the hardness of cataracts. The analytical approach employed targeted quantitative metabolite analysis using HILIC-based liquid chromatography coupled with high-resolution mass spectrometric detection. The metabolomics data analysis was performed with MetaboAnalyst 5.0. Results: The results of the enrichment analysis have facilitated the inference that the discerned disparities among groups arise from disruptions in taurine and hypotaurine metabolism, variations in tryptophan metabolism, and modifications in mitochondrial beta-oxidation of short-chain saturated fatty acids and pyrimidine metabolism. Conclusion: A decline in taurine concentration precipitates diminished glutathione activity, prompting an elevated requirement for NAD+ and instigating tryptophan metabolism along the kynurenine pathway. Activation of this pathway is additionally prompted by interferon-gamma and UV radiation, leading to the induction of IDO. Concurrently, heightened mitochondrial beta-oxidation signifies a distinctive scenario in translocating fatty acids into the mitochondria, enhancing energy production.

Multi-layered metabolic effects of trehalose on the liver proteome in apoE-knockout mice model of liver steatosis.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease has been well documented as a key independent risk factor for the development of atherosclerosis. A growing body of evidence suggests that due to its numerous favorable molecular effects, trehalose may exert beneficial effects in counteracting liver steatosis. In our previous study, we described the antiatherosclerotic and antisteatotic properties of trehalose, which we attributed to the induction of autophagy. Considering the pleiotropic activities of trehalose, our present study aimed to extend our preliminary results with the comprehensive examination of proteome-wide changes in the livers of high-fat-fed apoE-/- mice. METHODS: Thus, we applied modern, next-generation proteomic methodology to comprehensively analyze the effects of trehalose on the alterations of liver proteins in apoE-/- mice. RESULTS: Our proteomic analysis showed that the administration of trehalose elicited profound changes in the liver proteome of apoE-/- mice. The collected data allowed the identification and quantitation of 3 681 protein groups of which 129 were significantly regulated in the livers of trehalose-treated apoE-/- mice. CONCLUSIONS: The presented results are the first to highlight the effects of disaccharide on the induction of proteins mainly related to the metabolism and elimination of lipids, especially by peroxisomal ß-oxidation. Our study provides evidence for the pleiotropic activity of trehalose, extending our initial observations of its potential mechanisms responsible for mitigating of liver steatosis, which paves the way for new pharmacological strategies in fatty liver disease.

Sodium-glucose cotransporter-2 inhibitor (SGLT2i) plus glucagon-like peptide type 1 receptor combination is more effective than SGLT2i plus dipeptidyl peptidase-4 inhibitor combination in treating obese mice metabolic dysfunction-associated steatotic liver disease (MASLD).

BACKGROUND: Monotherapy to treat obesity-associated liver insult is limited. OBJECTIVES: In diet-induced obese mice showing metabolic dysfunction-associated steatotic liver disease (MASLD), we aimed to compare the combinations of sodium-glucose cotransporter-2 inhibitor (SGLT2i, empagliflozin, E), dipeptidyl peptidase-4 inhibitor (DPP4i, linagliptin, L), and glucagon-like peptide type 1 receptor agonist (GLP1RA, dulaglutide, D). METHODS: Male 3-month-old C57BL/6J mice were fed for 12 weeks in a control (C, n = 10) or high-fat (HF, n = 30) diet. Then, mice were followed for three additional weeks: C, HF, HF E + L, and HF E + D (n = 10/group). RESULTS: HF versus C showed higher hepatic triacylglycerol (TAG, +82%), steatosis (+850%), glucose intolerance (+71%), insulin (+98%), and insulin resistance (+68%). Compared to the HF group, HF E + L showed lower glucose intolerance (-60%), insulin (-61%), insulin resistance (-46%), TAG (-61%), and steatosis (-58%), and HF E + D showed lower glucose intolerance (-71%), insulin (-58%), insulin resistance (-62%), TAG (-61%), and steatosis (-82%). The principal component analysis (PCA) placed the HF group and the HF E + D group on opposite sides, while the HF E + L group was placed between C and HF E + D. CONCLUSION: PCA separated the groups considering the metabolism-related genes (glucose and lipid), mitochondrial biogenesis, and steatosis. The two pharmacological combinations showed beneficial effects in treating obesity and MASLD. However, the combination of SGLT2i and GLP1RA showed more potent beneficial effects on MASLD than SGLT2i and DPP4i and, therefore, should be the recommended combination.

Mitochondrial fatty acid beta-oxidation: a possible therapeutic target for skeletal muscle lipotoxicity in peripheral artery disease myopathy.

Peripheral artery disease (PAD) is an atherosclerotic disease impacting over 200 million individuals and the prevalence increases with age. PAD occurs when plaque builds up within the peripheral arteries, leading to reduced blood flow and oxygen supply to the outer extremities. Individuals who experience PAD suffer from ischemia, which is typically accompanied by significant damage to skeletal muscles. Additionally, this tissue damage affects mitochondria, causing them to become dysregulated and dysfunctional, resulting in decreased metabolic rates. As there is no known cure for PAD, researchers are exploring potential therapeutic targets by examining coexisting cardiovascular conditions and metabolic risk factors, such as the aging process. Among these comorbidities, type-two diabetes mellitus and obesity are particularly common in PAD cases. These conditions, along with aging itself, are associated with an elevated accumulation of ectopic lipids within skeletal muscles, similar to what is observed in PAD. Researchers have attempted to reduce excess lipid accumulation by increasing the rate of fatty acid beta oxidation. Manipulating acetyl coenzyme A carboxylase 2, a key regulatory protein of fatty acid beta oxidation, has been the primary focus of such research. When acetyl coenzyme A carboxylase 2 is inhibited, it interrupts the conversion of acetyl-CoA into malonyl-CoA, resulting in an increase in the rate of fatty acid beta oxidation. By utilizing samples from PAD patients and applying the pharmacological strategies developed for acetyl coenzyme A carboxylase 2 in diabetes and obesity to PAD, a potential new therapeutic avenue may emerge, offering hope for improved quality of life for individuals suffering from PAD.

Ketone flux through BDH1 supports metabolic remodeling of skeletal and cardiac muscles in response to intermittent time-restricted feeding.

Time-restricted feeding (TRF) has gained attention as a dietary regimen that promotes metabolic health. This study questioned if the health benefits of an intermittent TRF (iTRF) schedule require ketone flux specifically in skeletal and cardiac muscles. Notably, we found that the ketolytic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1) is uniquely enriched in isolated mitochondria derived from heart and red/oxidative skeletal muscles, which also have high capacity for fatty acid oxidation (FAO). Using mice with BDH1 deficiency in striated muscles, we discover that this enzyme optimizes FAO efficiency and exercise tolerance during acute fasting. Additionally, iTRF leads to robust molecular remodeling of muscle tissues, and muscle BDH1 flux does indeed play an essential role in conferring the full adaptive benefits of this regimen, including increased lean mass, mitochondrial hormesis, and metabolic rerouting of pyruvate. In sum, ketone flux enhances mitochondrial bioenergetics and supports iTRF-induced remodeling of skeletal muscle and heart.

miR-30d-5p inhibits proliferation, invasion and migration of breast cancer cells by targeting SERPINE1 and promoting fatty acid ß-oxidation.

Breast cancer (BC) is among the top three most prevalent cancers across the world, especially in women, and its pathogenesis is still unknown. Fatty acid ß-oxidation is highly associated with breast cancer. Serpin family E member 1 (SERPINE1)-induced down-regulation of fatty acid ß-oxidation can facilitate BC cell proliferation, invasion, and metastasis. In this paper, the difference of miR-30d-5p expressions in both cancerous tissues and para-carcinoma tissues was first detected. Next, the expressions of SERPINE1, long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) in the aforementioned tissues were analyzed. Finally, miR-30d-5p mimics were supplemented to breast cancer cells to observe the miR-30d-5p effect upon breast cancer cells. Via immunofluorescence assay and Western blotting, it was found that cancerous tissues had lower expressions of miR-30d-5p, MCAD and LCAD and a higher expression of SERPINE1 than para-carcinoma tissues. The miR-30d-5p mimic group had a decreased SERPINE1 expression and increased MCAD and LCAD expressions compared with the NC group, thus inhibiting BC cell proliferation, invasion, and metastasis. To sum up, miR-30d-5p blocks the cell proliferation, invasion and metastasis by targeting SERPINE1 and promoting fatty acid ß-oxidation. Preclinical studies are further required to establish a fatty acid ß-oxidation-targeting therapy for breast cancer.

An acyl-CoA thioesterase is essential for the biosynthesis of a key dauer pheromone in C. elegans.

Methyl ketone (MK)-ascarosides represent essential components of several pheromones in Caenorhabditis elegans, including the dauer pheromone, which triggers the stress-resistant dauer larval stage, and the male-attracting sex pheromone. Here, we identify an acyl-CoA thioesterase, ACOT-15, that is required for the biosynthesis of MK-ascarosides. We propose a model in which ACOT-15 hydrolyzes the ß-keto acyl-CoA side chain of an ascaroside intermediate during ß-oxidation, leading to decarboxylation and formation of the MK. Using comparative metabolomics, we identify additional ACOT-15-dependent metabolites, including an unusual piperidyl-modified ascaroside, reminiscent of the alkaloid pelletierine. The ß-keto acid generated by ACOT-15 likely couples to 1-piperideine to produce the piperidyl ascaroside, which is much less dauer-inducing than the dauer pheromone, asc-C6-MK (ascr#2, 1). The bacterial food provided influences production of the piperidyl ascaroside by the worm. Our work shows how the biosynthesis of MK- and piperidyl ascarosides intersect and how bacterial food may impact chemical signaling in the worm.

Long-term prognosis of fatty-acid oxidation disorders in adults: Optimism despite the limited effective therapies available.

INTRODUCTION: Fatty-acid oxidation disorders (FAODs) are recessive genetic diseases. MATERIALS AND METHODS: We report here clinical and paraclinical data from a retrospective study of 44 adults with muscular FAODs from six French reference centers for neuromuscular or metabolic diseases. RESULTS: The study cohort consisted of 44 adult patients: 14 with carnitine palmitoyl transferase 2 deficiency (32%), nine with multiple acyl-CoA deficiency (20%), 13 with very long-chain acyl-CoA dehydrogenase deficiency (30%), three with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (7%), and five with short-chain acyl-CoA dehydrogenase deficiency (11%). Disease onset occurred during childhood in the majority of patients (59%), with a mean age at onset of 15 years (range = 0.5-35) and a mean of 12.6 years (range = 0-58) from disease onset to diagnosis. The principal symptoms were acute muscle manifestations (rhabdomyolysis, exercise intolerance, myalgia), sometimes associated with permanent muscle weakness. Episodes of rhabdomyolysis were frequent (84%), with a mean creatinine kinase level of 68,958 U/L (range = 660-300,000). General metabolic complications were observed in 58% of patients, respiratory manifestations in 18% of cases, and cardiological manifestations in 9% of cases. Fasting acylcarnitine profile was used to orient genetic explorations in 65% of cases. After a mean follow-up of 10 years, 33% of patients were asymptomatic and 56% continued to display symptoms after exercise. The frequency of rhabdomyolysis decreased after diagnosis in 64% of cases. CONCLUSION: A standardized register would complete this cohort description of muscular forms of FAODs with exhaustive data, making it possible to assess the efficacy of therapeutic protocols in real-life conditions and during the long-term follow-up of patients.

Association between F2-Isoprostane Metabolites and Weight Change in Older Women: A Longitudinal Analysis.

INTRODUCTION: Theoretically, some metabolic traits may predispose older individuals to weight loss during aging, leading to increased all-cause mortality and many serious health issues. Biomarkers to robustly predict progressive weight loss during aging are, however, lacking. We prospectively assessed if urinary levels of F2-isoprostanes and their peroxisomal ß-oxidation metabolite, 2,3-dinor-5,6-dihydro-15-F2t-isoprostane (F2-IsoP-M), were associated with subsequent weight loss in middle-aged and older women. METHODS: Included in the analysis were 2,066 women aged 40-70 years, a subset of a prospective cohort study. F2-isoprostanes (F2-IsoPs) and its ß-oxidation metabolite, F2-IsoP-M, were measured in urine using gas chromatography-mass spectrometry. Measurements of anthropometry and exposures to major determinants of body weight were performed at baseline and repeated thrice over 15-year follow-up. The longitudinal associations of F2-IsoP-M and the F2-IsoP-M to its parent compound, F2-IsoP, ratio (MPR) with repeatedly measured weight changes were examined using linear mixed-effect models. RESULTS: After adjusting for time-varying covariates: energy intake, physical activity, and comorbidity index, among others, levels of F2-IsoP-M and the MPR were both inversely associated with percentage of weight change. Weight in the highest quartile of these two biomarkers was 1.33% (95% CI = -2.41, -0.24) and 1.09% (95% CI = -2.16, -0.02) lower than those in the lowest quartile group, with p for trend of 0.01 and 0.03, respectively. The inverse association was consistently seen across follow-up periods, although appearing stronger with prolonged follow-up. There was no association between the parent compound, F2-IsoPs, and weight change. CONCLUSION: This study demonstrates the first piece of evidence to associate F2-IsoP metabolism, peroxisomal ß-oxidation, with weight loss in older women. Further investigations into the role of lipid peroxidation and peroxisomal ß-oxidation in weight change among older individuals are warranted.

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.

Fatty acids are crucial to fuel NK cells upon acute retrovirus infection.

Natural killer (NK) cells are cytotoxic innate immune cells, able to recognize and eliminate virus-infected as well as cancer cells. Metabolic reprogramming is crucial for their activity as they have enhanced energy and nutritional demands for their functions during an infection. Fatty acids (FAs) represent an important source of cellular energy and are essential for proliferation of immune cells. However, the precise role of FAs for NK cells activity in retrovirus infection was unknown. Here we show that activated NK cells increase the expression of the FA uptake receptor CD36 and subsequently the uptake of FAs upon acute virus infection. We found an enhanced flexibility of NK cells to utilize FAs as source of energy compare to naïve NK cells. NK cells that were able to generate energy from FAs showed an augmented target cell killing and increased expression of cytotoxic parameters. However, NK cells that were unable to generate energy from FAs exhibited a severely decreased migratory capacity. Our results demonstrate that NK cells require FAs in order to fight acute virus infection. Susceptibility to severe virus infections as it is shown for people with malnutrition may be augmented by defects in the FA processing machinery, which might be a target to therapeutically boost NK cell functions in the future.

Lipin-1 restrains macrophage lipid synthesis to promote inflammation resolution.

Macrophages are critical to maintaining and restoring tissue homeostasis during inflammation. The lipid metabolic state of macrophages influences their function, but a deeper understanding of how lipid metabolism is regulated in pro-resolving macrophage responses is needed. Lipin-1 is a phosphatidic acid phosphatase with a transcriptional coregulatory activity (TC) that regulates lipid metabolism. We previously demonstrated that lipin-1 supports pro-resolving macrophage responses, and here, myeloid-associated lipin-1 is required for inflammation resolution, yet how lipin-1-regulated cellular mechanisms promote macrophage pro-resolution responses is unknown. We demonstrated that the loss of lipin-1 in macrophages led to increased free fatty acid, neutral lipid, and ceramide content and increased phosphorylation of acetyl-CoA carboxylase. The inhibition of the first step of lipid synthesis and transport of citrate from the mitochondria in macrophages reduced lipid content and restored efferocytosis and inflammation resolution in lipin-1mKO macrophages and mice. Our findings suggest macrophage-associated lipin-1 restrains lipid synthesis, promoting pro-resolving macrophage function in response to pro-resolving stimuli.

The Physiological and Pathological Role of Acyl-CoA Oxidation.

Fatty acid metabolism, including ß-oxidation (ßOX), plays an important role in human physiology and pathology. ßOX is an essential process in the energy metabolism of most human cells. Moreover, ßOX is also the source of acetyl-CoA, the substrate for (a) ketone bodies synthesis, (b) cholesterol synthesis, (c) phase II detoxication, (d) protein acetylation, and (d) the synthesis of many other compounds, including N-acetylglutamate-an important regulator of urea synthesis. This review describes the current knowledge on the importance of the mitochondrial and peroxisomal ßOX in various organs, including the liver, heart, kidney, lung, gastrointestinal tract, peripheral white blood cells, and other cells. In addition, the diseases associated with a disturbance of fatty acid oxidation (FAO) in the liver, heart, kidney, lung, alimentary tract, and other organs or cells are presented. Special attention was paid to abnormalities of FAO in cancer cells and the diseases caused by mutations in gene-encoding enzymes involved in FAO. Finally, issues related to α- and ω- fatty acid oxidation are discussed.

Case report: Episodic psychosis caused by a novel SCP2 splicing mutation.

SCPx deficiency is a rare disorder of peroxisomal beta-oxidation dysfunction, and it has only been documented in two patients thus far. In the previously reported patients, both patients were primarily presented with slowly progressive dystonia or ataxia, and they both displayed symmetrical lesions in the thalamus and brainstem on magnetic resonance imaging. This study presents the third patient exhibiting a similar neuroimaging abnormality but a notably different clinical phenotype characterized by episodic psychosis. Through whole-exome sequencing, we identified a homozygous splicing mutation in SCP2 (c.674 + 1G > C), and further RNA sequencing revealed exon 8 skipping in the mature transcripts of SCP2. This study significantly expands our understanding of the genotypic and phenotypic spectrum associated with SCP2-related metabolic encephalopathy.

CLPX regulates mitochondrial fatty acid ß-oxidation in liver cells.

Mitochondrial fatty acid oxidation (ß-oxidation) is an essential metabolic process for energy production in eukaryotic cells, but the regulatory mechanisms of this pathway are largely unknown. In the present study, we found that several enzymes involved in ß-oxidation are associated with CLPX, the AAA+ unfoldase that is a component of the mitochondrial matrix protease ClpXP. The suppression of CLPX expression increased ß-oxidation activity in the HepG2 cell line and in primary human hepatocytes without glucagon treatment. However, the protein levels of enzymes involved in ß-oxidation did not significantly increase in CLPX-deleted HepG2 cells (CLPX-KO cells). Coimmunoprecipitation experiments revealed that the protein level in the immunoprecipitates of each antibody changed after the treatment of WT cells with glucagon, and a part of these changes was also observed in the comparison of WT and CLPX-KO cells without glucagon treatment. Although the exogenous expression of WT or ATP-hydrolysis mutant CLPX suppressed ß-oxidation activity in CLPX-KO cells, glucagon treatment induced ß-oxidation activity only in CLPX-KO cells expressing WT CLPX. These results suggest that the dissociation of CLPX from its target proteins is essential for the induction of ß-oxidation in HepG2 cells. Moreover, specific phosphorylation of AMP-activated protein kinase and a decrease in the expression of acetyl-CoA carboxylase 2 were observed in CLPX-KO cells, suggesting that CLPX might participate in the regulation of the cytosolic signaling pathway for ß-oxidation. The mechanism for AMP-activated protein kinase phosphorylation remains elusive; however, our results uncovered the hitherto unknown role of CLPX in mitochondrial ß-oxidation in human liver cells.