3-oxoacid CoA-transferase Research Articles

The first report of submandibular lymph node infection with Flavobacterium ceti in a horse and its complete genome sequence.

This is the first report describing the isolation and genome sequence of Flavobacterium ceti (F. ceti IJFC025) from a draining submandibular lymph node abscess in a 6-year-old mixed-breed mare in Korea. Since F. ceti is not a usual infectious pathogen that causes equine health problems, this study presents the complete genome sequence of F. ceti isolated from a submandibular lymph node abscess sample in Jeju, South Korea, in 2022. The entire genome sequence of F. ceti IJFC025 consisted of a 3,144,426-bp chromosome with 35.6% G+C content. Gene prediction revealed that this strain possesses 2,909 coding sequences (CDSs), 74 tRNAs, and five rRNAs. Average nucleotide identity analysis of the whole-genome sequence revealed 98.6% to 98.8% nucleotide identity between F. ceti IJFC025 and F. ceti CECT7184, which was previously isolated from beaked whales. PathogenFinder revealed four different virulence genes in the genome that could potentially cause infections in horses. F. ceti CECT7184 contained genes encoding propionyl-CoA carboxylase, 3-oxoacid CoA-transferase A subunit, and the translation initiation factor IF-1, whereas F. ceti IJFC025 does not. Instead, F. ceti IJFC025 harbors genes encoding deoxyhypusine synthase, whereas F. ceti CECT7184 does not. Antimicrobial susceptibility tests revealed that F. ceti IJFC025 is a multidrug-resistant strain that is resistant to gentamicin, ceftazidime, nalidixic acid, ciprofloxacin, and colistin. However, no resistance genes were detected in the whole-genome sequence. Because of the similarity of the clinical signs with those of strangles, this pathogen needs to be included in the differential diagnosis of submandibular lymphadenopathy in horses.

SCOT Deficiency Mimicking Sepsis: An Unusual Cause of Increased Anion Gap Metabolic Acidosis

Introduction: Succinyl-CoA:3-oxoacid CoA transferase (SCOT, EC 2.8.3.5) deficiency is a rare autosomal recessive inborn error of metabolism (IEM). We report here an infant admitted to intensive care unit with the diagnosis of sepsis. Case Presentation: A five-month-old female patient was admitted to the intensive care unit with lethargy and respiratory distress. She had severe high anion gap metabolic acidosis. The IEM was screened by plasma amino acid analysis, showing no abnormalities, and by acylcarnitine analysis, showing low-normal levels of free carnitine. Urine organic acid analysis revealed massive ketonuria and elevated levels of dicarboxylic acids. Fatty acid oxidation disorder-targeted gene panel revealed a homozygous splice site variant (c.78+1_78+6 del) in the OXCT1 gene. Discussion and Conclusion: SCOT deficiency should be considered when massive ketosis is detected in increased anion gap metabolic acidosis with sepsis-like manifestation. Supportive therapy should be initiated quickly to prevent irreversible neurological damage.

The ketone body β-Hydroxybutyrate as a fuel source of chondrosarcoma cells

Chondrosarcoma (CS) is a malignant bone tumor arising from cartilage-producing cells. The conventional subtype of CS typically develops within a dense cartilaginous matrix, creating an environment deficient in oxygen and nutrients, necessitating metabolic adaptation to ensure proliferation under stress conditions. Although ketone bodies (KBs) are oxidized by extrahepatic tissue cells such as the heart and brain, specific cancer cells, including CS cells, can undergo ketolysis. In this study, we found that KBs catabolism is activated in CS cells under nutrition-deprivation conditions. Interestingly, cytosolic β-hydroxybutyrate dehydrogenase 2 (BDH2), rather than mitochondrial BDH1, is expressed in these cells, indicating a specific metabolic adaptation for ketolysis in this bone tumor. The addition of the KB, β-Hydroxybutyrate (β-HB) in serum-starved CS cells re-induced the expression of BDH2, along with the key ketolytic enzyme 3-oxoacid CoA-transferase 1 (OXCT1) and monocarboxylate transporter-1 (MCT1). Additionally, internal β-HB production was quantified in supplied and starved cells, suggesting that CS cells are also capable of ketogenesis alongside ketolysis. These findings unveil a novel metabolic adaptation wherein nutrition-deprived CS cells utilize KBs for energy supply and proliferation.

Rosemary leaf powder improves egg quality, antioxidant status, gut barrier function, and cecal microbiota and metabolites of late-phase laying hens

This study sought to determine the effects of rosemary leaf powder (RP) on laying performance, egg quality, serum indices, gut barrier function, and cecal microbiota and metabolites of late-phase laying hens. A total of 84 “Jing Tint 6” laying hens at 65-week old were randomly divided into 2 groups and fed either a basal diet (CON) or a basal diet supplemented with 0.3% RP. Our study revealed that RP improved the Haugh unit and decreased yolk n-6/n-3 polyunsaturated fatty acid (PUFA) ratio of laying hens, increased serum superoxide dismutase (SOD), jejunal activities of SOD and catalase (CAT), and jejunal zonula occludens-1 (ZO-1) expression, as well as decreased serum tumor necrosis factor-α (TNF-α) level and jejunal TNF-α mRNA expression. Rosemary leaf powder markedly enhanced (P < 0.05) cecal abundances of Rikenellaceae, Rikenellaceae_RC9_gut_group, and Turicibacter, tended to promote (P = 0.076) butyrate concentration, and reduced (P < 0.05) cecal abundances of Erysipelatoclostridiaceae, Sutterellaceae, Fusobacteriaceae, Campylobacteraceae, Sutterella, Campylobacter, and Fusobacterium, which were closely linked with Haugh unit, yolk n-6/n-3 PUFA ratio, serum SOD and TNF-α. In addition, RP altered the metabolic functions of cecal microbiota and enhanced the abundances of butyrate-synthesizing enzymes, including lysine 2,3-aminomutase, β-lysine 5,6-aminomutase, and 3-oxoacid CoA-transferase. Together, 0.3% RP has the potential to enhance egg quality by partially modulating serum antioxidant status, jejunal barrier function, and cecal microbiota structure and metabolites, indicating that RP could be considered a promising feed additive to promote the production performance of late-phase laying hens.

OXCT1 regulates hippocampal neurogenesis and alleviates cognitive impairment via the Akt/GSK-3β/β-catenin pathway after subarachnoid hemorrhage

BackgroundSubarachnoid hemorrhage (SAH) is a life-threatening neurological disease that usually has a poor prognosis. Neurogenesis is a potential therapeutic target for brain injury. Ketone metabolism also plays neuroprotective roles in many neurological disorders. OXCT1 (3-Oxoacid CoA-Transferase 1) is the rate-limiting enzyme of ketone body oxidation. In this study, we explored whether increasing ketone oxidation by upregulating OXCT1 in neurons could promote neurogenesis after SAH, and evaluated the potential mechanism involved in this process. MethodsThe β-hydroxybutyrate content was measured using an enzymatic colorimetric assay. Adeno-associated virus targeting neurons was injected to overexpress OXCT1, and the expression and localization of proteins were evaluated by western blotting and immunofluorescence staining. Adult hippocampal neurogenesis was evaluated by dual staining with doublecortin and 5-Ethynyl-2′-Deoxyuridine. LY294002 was intracerebroventricularly administered to inhibit Akt activity. The Morris water maze and Y-maze tests were employed to assess cognitive function after SAH. ResultsThe results showed that OXCT1 expression and hippocampal neurogenesis significantly decreased in the early stage of SAH. Overexpression of OXCT1 successfully increased hippocampal neurogenesis via activation of Akt/GSK-3β/β-catenin signaling and improved cognitive function, both of which were reversed by administration of LY294002. ConclusionsOXCT1 regulated hippocampal ketone body metabolism and increased neurogenesis through mechanisms mediated by the Akt/GSK-3β/β-catenin pathway, improving cognitive impairment after SAH.

Multiomic analysis in fibroblasts of patients with inborn errors of cobalamin metabolism reveals concordance with clinical and metabolic variability

The high variability in clinical and metabolic presentations of inborn errors of cobalamin (cbl) metabolism (IECM), such as the cblC/epicblC types with combined deficits in methylmalonyl-coA mutase (MUT) and methionine synthase (MS), are not well understood. They could be explained by the impaired expression/activity of enzymes from other metabolic pathways. We performed metabolomic, genomic, proteomic, and post-translational modification (PTM) analyses in fibroblasts from three cblC cases and one epi-cblC case compared with three cblG cases with specific MS deficits and control fibroblasts. CblC patients had metabolic profilings consistent with altered urea cycle, glycine, and energy mitochondrial metabolism. Metabolomic analysis showed partial disruption and increased glutamate/ketoglutarate anaplerotic pathway of the tricarboxylic acid cycle (TCA), in patient fibroblasts. RNA-seq analysis showed decreased expression of MT-TT (mitochondrial tRNA threonine), MT-TP (mitochondrial tRNA proline), OXCT1 (succinyl CoA:3-oxoacid CoA transferase deficiency), and MT-CO1 (cytochrome C oxidase subunit 1). Proteomic changes were observed for key mitochondrial enzymes, including NADH:ubiquinone oxidoreductase subunit A8 (NDUFA8), carnitine palmitoyltransferase 2 (CPT2), and ubiquinol-cytochrome C reductase, complex III subunit X (UQCR10). Propionaldehyde addition in ornithine aminotransferase was the predominant PTM in cblC cells and could be related with the dramatic cellular increase in propionate and methylglyoxalate. It is consistent with the decreased concentration of ornithine reported in 3 cblC cases. Whether the changes detected after multi-omic analyses underlies clinical features in cblC and cblG types of IECM, such as peripheral and central neuropathy, cardiomyopathy, pulmonary hypertension, development delay, remains to be investigated. The omics-related effects of IECM on other enzymes and metabolic pathways are consistent with the diversity and variability of their age-related metabolic and clinical manifestations. PTMs are expected to produce cumulative effects, which could explain the influence of age on neurological manifestations. French Agence Nationale de la Recherche (Projects PREDICTS and EpiGONE) and Inserm.

Rosemary extract improves egg quality by altering gut barrier function, intestinal microbiota and oviductal gene expressions in late-phase laying hens

BackgroundRosemary extract (RE) has been reported to exert antioxidant property. However, the application of RE in late-phase laying hens on egg quality, intestinal barrier and microbiota, and oviductal function has not been systematically studied. This study was investigated to detect the potential effects of RE on performance, egg quality, serum parameters, intestinal heath, cecal microbiota and metabolism, and oviductal gene expressions in late-phase laying hens. A total of 210 65-week-old “Jing Tint 6” laying hens were randomly allocated into five treatments with six replicates and seven birds per replicate and fed basal diet (CON) or basal diet supplemented with chlortetracycline at 50 mg/kg (CTC) or RE at 50 mg/kg (RE50), 100 mg/kg (RE100), and 200 mg/kg (RE200).ResultsOur results showed that RE200 improved (P < 0.05) Haugh unit and n-6/n-3 of egg yolk, serum superoxide dismutase (SOD) compared with CON. No significant differences were observed for Haugh unit and n-6/n-3 of egg yolk among CTC, RE50, RE100 and RE200 groups. Compared with CTC and RE50 groups, RE200 increased serum SOD activity on d 28 and 56. Compared with CON, RE supplementation decreased (P < 0.05) total cholesterol (TC) level. CTC, RE100 and RE200 decreased (P < 0.05) serum interleukin-6 (IL-6) content compared with CON. CTC and RE200 increased jejunal mRNA expression of ZO-1 and Occludin compared with CON. The biomarkers of cecal microbiota and metabolite induced by RE 200, including Firmicutes, Eisenbergiella, Paraprevotella, Papillibacter, and butyrate, were closely associated with Haugh unit, n-6/n-3, SOD, IL-6, and TC. PICRUSt2 analysis indicated that RE altered carbohydrate and amino acid metabolism of cecal microbiota and increased butyrate synthesizing enzymes, including 3-oxoacid CoA-transferase and butyrate-acetoacetate CoA-transferase. Moreover, transcriptomic analysis revealed that RE200 improved gene expressions and functional pathways related to immunity and albumen formation in the oviductal magnum.ConclusionsDietary supplementation with 200 mg/kg RE could increase egg quality of late-phase laying hens via modulating intestinal barrier, cecal microbiota and metabolism, and oviductal function. Overall, RE could be used as a promising feed additive to improve egg quality of laying hens at late stage of production.Graphical

Recurrent metabolic acidosis: is it by chance?

Succinyl CoA 3 oxoacid CoA transferase deficiency (SCOTD) is an autosomal recessive disorder of ketone body utilization which present with recurrent episodes of metabolic acidosis. We have a 23-month old child who presented with 3 episodes of metabolic acidosis. Each time the toxicology profile came positive for ethyl alcohol. We couldn’t get any history suggestive of poisoning. So we were in a great dilemma and considered the possibility of Munchausen syndrome by proxy and a metabolic cause. Even though the initial genetic result was nonspecific when we reanalyzed the sample for genetic analysis it came positive as Succinyl CoA 3 oxoacid CoA transferase (SCOT) deficiency. The child required intensive care support in all 3 times. We were able to manage the child with supportive measures each time. Now the child is under regular follow up and doing well. SCOTD should be considered in any children presenting as recurrent episodes of metabolic acidosis. The rarity of this disorder will lead to the diagnostic dilemma that we face as like in this case.

Ketolysis is required for the proper development and function of the somatosensory nervous system

Ketogenic diets are emerging as protective interventions in preclinical and clinical models of somatosensory nervous system disorders. Additionally, dysregulation of succinyl-CoA 3-oxoacid CoA-transferase 1 (SCOT, encoded by Oxct1), the fate-committing enzyme in mitochondrial ketolysis, has recently been described in Friedreich's ataxia and amyotrophic lateral sclerosis. However, the contribution of ketone metabolism in the normal development and function of the somatosensory nervous system remains poorly characterized. We generated sensory neuron-specific, Advillin-Cre knockout of SCOT (Adv-KO-SCOT) mice and characterized the structure and function of their somatosensory system. We used histological techniques to assess sensory neuronal populations, myelination, and skin and spinal dorsal horn innervation. We also examined cutaneous and proprioceptive sensory behaviors with the von Frey test, radiant heat assay, rotarod, and grid-walk tests. Adv-KO-SCOT mice exhibited myelination deficits, altered morphology of putative Aδ soma from the dorsal root ganglion, reduced cutaneous innervation, and abnormal innervation of the spinal dorsal horn compared to wildtype mice. Synapsin 1-Cre-driven knockout of Oxct1 confirmed deficits in epidermal innervation following a loss of ketone oxidation. Loss of peripheral axonal ketolysis was further associated with proprioceptive deficits, yet Adv-KO-SCOT mice did not exhibit drastically altered cutaneous mechanical and thermal thresholds. Knockout of Oxct1 in peripheral sensory neurons resulted in histological abnormalities and severe proprioceptive deficits in mice. We conclude that ketone metabolism is essential for the development of the somatosensory nervous system. These findings also suggest that decreased ketone oxidation in the somatosensory nervous system may explain the neurological symptoms of Friedreich's ataxia.

Neuroprotective mechanisms of OXCT1 via the SIRT3-SOD2 pathway after traumatic brain injury

BackgroundKetones are not only utilized to produce energy but also play a neuroprotective role in many neurodegenerative diseases. However, whether this process has an impact on secondary brain damage after traumatic brain injury (TBI) remains unknown. OXCT1 (3-Oxoacid CoA-Transferase 1) is the rate-limiting enzyme in the intra-neuronal utilization of ketones. In this study, we investigated whether reduced expression of OXCT1 after TBI could impact neuroprotective mechanisms and exacerbate neurological dysfunction. Materials and methodsExperimental TBI was induced by a modified version of the weight drop model, it is a model of severe head trauma. Expression of OXCT1 in the injured hippocampus of mice was measured at different time points using immunoblotting assays. The release of abnormal mitochondrial cytochrome c from neurons of the mouse injured lateral hippocampus was measured 1 week after TBI using immunoblotting assays. Neuronal death was assessed by Nissl staining and the level of reactive oxygen species (ROS) within the neurons of the injured lateral hippocampus was assessed by Dihydroethidium staining. ResultsOXCT1 was overexpressed in hippocampal neurons by injection of adeno-associated virus into the lateral ventricle. OXCT1 expression levels decreased significantly 1 week post-TBI. After comparing the data obtained from different groups of mice, OXCT1 was found to significantly increase the expression of SIRT3 and reduce the proportion of acetylated SOD2, thus decreasing the production of ROS in the injured hippocampal neurons, reducing neuronal death, and improving cognitive function. ConclusionsOXCT1 has a critical previously unappreciated protective role in neurological impairment following TBI via the SIR3-SOD2 pathway. These findings highlight the potential of OXCT1 as a simple treatment for patients with TBI.

Swimming Endurance Capacity of Mice is Increased by Chronic Consumption of Medium-Chain Triglycerides

The effect of chronic administration of medium-chain triglycerides (MCT) on swimming endurance (swim capacity) was investigated in male Std ddY mice. The mice were fed a diet containing 80 g MCT + 20 g long-chain triglycerides (LCT)/kg diet for 6 wk; mice fed diet containing 100 g LCT/kg diet were used as controls. After being accustomed to swimming, the mice were subjected to forced swimming every 2 d in the current water pool that we had developed, and the total swimming period until exhaustion was measured. The total swimming period was used as the index of swim capacity. The group fed MCT showed significantly greater swim capacity than the control group (89.5 ± 2.5 vs. 80.2 ± 2.0 min). In another experiment, after 4 wk of MCT diet consumption, significantly greater swim capacity was found in untrained mice. The major metabolic consequences of the adaptations of muscle to prolonged MCT administration during endurance training were higher activities of 3-oxo acid CoA-transferase (P < 0.01), citrate synthase (P < 0.1) and malate dehydrogenase (P < 0.1). These findings suggest that increases in the enzyme activities of the tricarboxylic acid cycle and ketone body utilization associated with the chronic administration of an MCT-containing diet enhance swim capacity in mice.

CircRNA OXCT1 promotes the malignant progression and glutamine metabolism of non-small cell lung cancer by absorbing miR-516b-5p and upregulating SLC1A5

ABSTRACT Previous study has demonstrated the high expression of circular RNA 3-oxoacid CoA-transferase 1 (circ-OXCT1) in lung adenocarcinoma tumor tissues. However, the role and possible mechanism of circ-OXCT1 in non-small cell lung cancer (NSCLC) progression was unclear.Quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) staining assay were performed to detect the expression of circ-OXCT1, microRNA-516b-5p (miR-516b-5p), solute carrier family 1 member 5 (SLC1A5) and other indicated protein markers. Cell proliferation was measured by Cell counting kit 8 (CCK8), colony formation and 5-Ethynyl-2’-deoxyuridine (EdU) assays. Flow cytometry was employed to detect the rate of apoptotic cells. Cell migration and invasion were measured using transwell assay. The relative glutamine uptake and α-ketoglutarate (α-KG) production was determined using commercial kits. Interaction between miR-516b-5p and circ-OXCT1 or SLC1A5 was predicted by bioinformatics analysis and confirmed via luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo assay was implemented to demonstrate the effect of circ-OXCT1 in tumor growth.Circ-OXCT1 and SLC1A5 were upregulated and miR-516b-5p was downregulated in NSCLC tissues and cells. Functional experiments revealed that circ-OXCT1 silencing suppressed cell proliferation, migration and invasion, but promoted cell apoptosis in vitro. Circ-OXCT1 knockdown repressed tumor formation in vivo. Besides, miR-516b-5p was a target of circ-OXCT1, and miR-516b-5p inhibitor could relieve circ-OXCT1 absence-mediated effects in NSCLC cells. SLC1A5 was identified as a target of miR-516b-5p. Circ-OXCT1 promoted SLC1A5 expression by target binding with miR-516b-5p.Circ-OXCT1 facilitated NSCLC progression via miR-516b-5p-dependent regulation of SLC1A5, which provided a possible circRNA-targeted therapy for NSCLC.

Cancer Metabolism: Fasting Reset, the Keto-Paradox and Drugs for Undoing

In tumor cells, ketolysis "via" succinyl-CoA: 3-oxoacid-CoAtransferase (SCOT) and acetyl-CoA acetyltransferase 1 (ACAT1) is a major source of mitochondrial acetyl-CoA. Active ACAT1 tetramers stabilize by tyrosine phosphorylation, which facilitates the SCOT reaction and ketolysis. Tyrosine phosphorylation of pyruvate kinase PK M2 has the opposite effect, stabilizing inactive dimers, while pyruvate dehydrogenase (PDH), which is already inhibited by phosphorylation, is acetylated by ACAT1 and is doubly locked. This closes the glycolytic supply of acetyl-CoA. In addition, since tumor cells must synthesize fatty acids to create new membranes, they automatically turn off the degradation of fatty acids into acetyl-CoA ("via" the malonyl-CoA brake for the fatty acid carnityl transporter). Thus, inhibiting SCOT the specific ketolytic enzyme and ACAT1 should hold back tumor progression. However, tumor cells are still able to take up external acetate and convert it into acetyl-CoA in their cytosol "via" an acetyl-CoA synthetase, which feeds the lipogenic pathway; additionally, inhibiting this enzyme would make it difficult for tumor cells to form new lipid membrane and survive.

A Novel Mutation in the OXCT1 Gene Causing Succinyl-CoA:3-Ketoacid CoA Transferase (SCOT) Deficiency Starting with Neurologic Manifestations.

Succinyl-CoA:3-oxoacid CoA-transferase (SCOT) deficiency is an inborn error of ketone body utilization characterized by intermittent ketoacidosis crises. This study reports the first Iranian patient with SCOT deficiency who presented with seizure and hypotonia at birth. Accordingly, she was consequently re-hospitalized due to hypotonia and respiratory distress. Laboratory tests revealed hyperammonemia, ketonuria, and metabolic acidosis. Besides, the plasma glucose level was normal without any other abnormality. Despite treatment with high-dose bicarbonate, severe acidosis persisted. Poor response to treatment raised a significant diagnostic challenge among specialists until genetic investigation identified a homozygous nonsense mutation (c.79G>T; p.Gly27*) in the OXCT1 gene (NM_000436), causing SCOT deficiency. Genetic studies help clinicians achieve a definite diagnosis of such metabolic disorders. In this case, the accurate and early diagnosis of SCOT deficiency opened new therapeutic possibilities, including frequent carbohydrate-rich meals and low fat and protein diet. Moreover, our findings expand the mutational and clinical spectrum of SCOT deficiency.

Frataxin controls ketone body metabolism through regulation of OXCT1.

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the deficiency of mitochondrial protein frataxin, which plays a crucial role in iron–sulphur cluster formation and ATP production. The cellular function of frataxin is not entirely known. Here, we demonstrate that frataxin controls ketone body metabolism through regulation of 3-Oxoacid CoA-Transferase 1 (OXCT1), a rate limiting enzyme catalyzing the conversion of ketone bodies to acetoacetyl-CoA that is then fed into the Krebs cycle. Biochemical studies show a physical interaction between frataxin and OXCT1 both in vivo and in vitro. Frataxin overexpression also increases OXCT1 protein levels in human skin fibroblasts while frataxin deficiency decreases OXCT1 in multiple cell types including cerebellum and skeletal muscle both acutely and chronically, suggesting that frataxin directly regulates OXCT1. This regulation is mediated by frataxin-dependent suppression of ubiquitin–proteasome system (UPS)-dependent OXCT1 degradation. Concomitantly, plasma ketone bodies are significantly elevated in frataxin deficient knock-in/knockout (KIKO) mice with no change in the levels of other enzymes involved in ketone body production. In addition, ketone bodies fail to be metabolized to acetyl-CoA accompanied by increased succinyl-CoA in vitro in frataxin deficient cells, suggesting that ketone body elevation is caused by frataxin-dependent reduction of OXCT1 leading to deficits in tissue utilization of ketone bodies. Considering the potential role of metabolic abnormalities and deficiency of ATP production in FRDA, our results suggest a new role for frataxin in ketone body metabolism and also suggest modulation of OXCT1 may be a potential therapeutic approach for FRDA.

Abstract 3016: Beta-hydroxybutyrate alters lipid metabolism affecting oncogenicity of MCF7 cells

Abstract The ketogenic diet, based on high fat (over 70% of daily calories), low carbohydrate, and adequate protein intake, has become popular due to its potential therapeutic benefits for several diseases including cancer. Recent evidence suggests ketogenic metabolic therapy as a complementary or alternative approach to breast cancer treatment. Under ketogenic conditions, as in starvation, the levels of glucose, insulin, and insulin-like growth factors in the blood decrease and stabilize, and the liver produces ketone bodies, acetone, acetoacetate (AcA) and β-hydroxybutyrate (ΒHb) by beta-oxidation of fatty acids, as an alternative energy source. Due to the volatile nature of acetone and its instability in the blood, acetone has no effect on cancer cells under physiological conditions. AcA alone is unstable as well, and is used in research as a sodium or lithium salt. Despite the widely accepted use of lithium AcA as a ketone body source, we and others recently demonstrated that its cytotoxic effects result from the lithium and not from acetoacetate.In this study, we aimed to elucidate the effect of the third ketone body, ΒHb, on breast cancer cells in vitro. Using two cancer (MCF7 and MDA-MB-231) and one non-cancer (HB2) breast cell lines, we evaluated the effect of β-hydroxybutyrate treatment on cell growth parameters and assessed the effect of BHb on metabolism and the gene expression profile. We found that ΒHb increases viability and proliferation of MCF7, but has no affect MDA-MB-231 and non-cancer HB2 cell viability. We observed no changes in glucose intake or lactate output following BHb treatment, but an increase in ROS level was detected. Gene expression analysis revealed a differential effect of BHb treatment on changes in genes involved in lipid metabolism and oxidative phosphorylation, among the tested cell lines. While genes involved in lipid metabolism were downregulated in MCF7 cells, ΒHb mitochondrial monocarboxylate transporter-4 (MCT4) expression was found to be significantly upregulated in both cancer cell lines with 10mM BHb but not affected in non-cancer breast cells. 3-hydroxybutyrate dehydrogenase 1 (BDH1) and 3-oxoacid CoA-transferase 1 (OXCT1), enzymes involved in BHb oxidation in the mitochondria, were significantly increased in MCF7 with a non-significant trend toward upregulation in MDA-MB-231 cells. Treatment of MCF7 cells with 10mM of BHb also significantly downregulated AMP-activated protein kinase (AMPK) and upregulated HDAC1, both known to be involved in BHb metabolism. Based on our results, we conclude that differential response of breast cell lines to BHb treatment, as alternative energy source or signal to altered lipid metabolism and oncogenicity, supports the need for a personalized approach to breast cancer patient treatment. Citation Format: Elimelech Nesher, Hadas Fulman-Levy, Igor Koman, Raichel Cohen-Harazi. Beta-hydroxybutyrate alters lipid metabolism affecting oncogenicity of MCF7 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3016.

OXCT1 Enhances Gemcitabine Resistance Through NF-κB Pathway in Pancreatic Ductal Adenocarcinoma.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a type of malignant tumor with a five-year survival rate of less than 10%. Gemcitabine (GEM) is the most commonly used drug for PDAC chemotherapy. However, a vast majority of patients with PDAC develop resistance after GEM treatment.MethodsWe screened for GEM resistance genes through bioinformatics analysis. We used immunohistochemistry to analyze 3-oxoacid CoA-transferase 1 (OXCT1) expression in PDAC tissues. The survival data were analyzed using the Kaplan–Meier curve. The expression levels of the genes related to OXCT1 and the NF-κB signaling pathway were quantified using real−time quantitative PCR and western blot analyses. We performed flow cytometry to detect the apoptosis rate. Colony formation assay was performed to measure the cell proliferation levels. The cytotoxicity assays of cells were conducted using RTCA. The downstream pathway of OXCT1 was identified via the Gene Set Enrichment Analysis. Tumor growth response to GEM in vivo was also determined in mouse models.ResultsBioinformatics analysis revealed that OXCT1 is the key gene leading to GEM resistance. Patients with high OXCT1 expression exhibited short relapse-free survival under GEM treatment. OXCT1 overexpression in PDAC cell lines exerted inhibitory effect on apoptosis after GEM treatment. However, the down-regulation of OXCT1 showed the opposite effect. Blocking the NF-κB signaling pathway also reduced GEM resistance of PDAC cells. Tumor growth inhibition induced by GEM in vivo reduced after OXCT1 overexpression. Moreover, the effect of OXCT1 on GEM refractoriness in PDAC cell lines was reversed through using an NF-κB inhibitor.ConclusionOXCT1 promoted GEM resistance in PDAC via the NF-κB signaling pathway both in vivo and in vitro. Our results suggest that OXCT1 could be used as a potential therapeutic target for patients with PDAC.

Differential Response of Hippocampal and Cerebrocortical Autophagy and Ketone Body Metabolism to the Ketogenic Diet.

Experimental and clinical data support the neuroprotective properties of the ketogenic diet and ketone bodies, but there is still a lot to discover to comprehensively understand the underlying mechanisms. Autophagy is a key mechanism for maintaining cell homeostasis, and therefore its proper function is necessary for preventing accelerated brain aging and neurodegeneration. Due to many potential interconnections, it is possible that the stimulation of autophagy may be one of the mediators of the neuroprotection afforded by the ketogenic diet. Recent studies point to possible interconnections between ketone body metabolism and autophagy. It has been shown that autophagy is essential for hepatic and renal ketogenesis in starvation. On the other hand, exogenous ketone bodies modulate autophagy both in vitro and in vivo. Many regional differences occur between brain structures which concern i.e., metabolic responses and autophagy dynamics. The aim of the present study was to evaluate the influence of the ketogenic diet on autophagic markers and the ketone body utilizing and transporting proteins in the hippocampus and frontal cortex. C57BL/6N male mice were fed with two ketogenic chows composed of fat of either animal or plant origins for 4 weeks. Markers of autophagosome formation as well as proteins associated with ketolysis (BDH1—3-hydroxybutyrate dehydrogenase 1, SCOT/OXCT1—succinyl CoA:3-oxoacid CoA transferase), ketone transport (MCT1—monocarboxylate transporter 1) and ketogenesis (HMGCL, HMGCS2) were measured. The hippocampus showed a robust response to nutritional ketosis in both changes in the markers of autophagy as well as the levels of ketone body utilizing and transporting proteins, which was also accompanied by increased concentrations of ketone bodies in this brain structure, while subtle changes were observed in the frontal cortex. The magnitude of the effects was dependent on the type of ketogenic diet used, suggesting that plant fats may exert a more profound effect on the orchestrated upregulation of autophagy and ketone body metabolism markers. The study provides a foundation for a deeper understanding of the possible interconnections between autophagy and the neuroprotective efficacy of nutritional ketosis.

Conversion of dietary inositol into propionate and acetate by commensal Anaerostipes associates with host health

We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [13C]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [13C]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health.

Changes in Metabolite and Protein Expression Profiles of Atypical Dark-cutting and Normal-pH Beef

Abstract The color of meat is an important deciding factor in consumers’ assessment of meat quality. To meat buyers, the bright cherry-red color of meat indicates freshness and wholesomeness. However, atypical dark-cutting beef represents dark-colored meat with a muscle pH between 5.6 and 5.8. Although previous studies have indicated that the ultimate pH of atypical dark-cutting beef is greater than normal, the mechanistic basis for the occurrence is not clear. Therefore, the objective of this study was to identify protein and metabolite profiles of normal and atypical dark-cutting beef. Longissimus thoracic (LT) muscles from 12 different animals (6 atypical dark-cutters and 6 normal-pH beef) were analyzed by comparing changes in metabolites and protein profiles using GC-MS non-targeted metabolomics and LC-MS/MS-based proteomics, respectively. Metabolomics analysis showed down-regulation of glycolytic metabolites such as glucose-6-phosphate, lactose, and glucose, indicating a reduced capacity to accumulate lactate postmortem in atypical dark-cutters compared with normal-pH beef. Mass spectrometry analysis identified 66 proteins with significant changes in protein expression profiles (P &amp;lt; 0.05) between atypical and normal-pH beef. Of these, 22 proteins were up-regulated while 14 were down-regulated in atypical dark-cutting beef. Functional annotation and protein-protein interaction network analyses revealed that up-regulated proteins such as NADH ubiquinone oxidoreductase subunit A7, ATPase Na+/K+ transporting subunit alpha 2, 3-oxoacid CoA-transferase 1, cytochrome b5 reductase 3, actinin alpha 4, tropomyosin 2, heat shock protein beta-7, and DnaJ heat shock protein family (Hsp40) member B4 are involved in energy synthesis, muscle contraction and stress responses. By contrast, down-regulated proteins, including phosphofructokinase, glycogen phosphorylase, amylo-alpha-1,6-glucosidase, phosphorylase kinase regulatory subunit alpha and gamma are involved in glycogen metabolism. In summary, lower glycolytic metabolites and glycogen degradation enzymes are associated with differences in muscle pH and color of atypical dark-cutting beef.