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Citrate synthase is a crucial enzyme in the TCA cycle and represents a potential therapeutic target. However, knowledge about this enzyme in Leishmania parasites remains limited. In this study, we have successfully cloned, expressed, and purified citrate synthase from Leishmania donovani (LdCS) using a bacterial system, and characterized it through various biophysical and biochemical methods. Circular dichroism analysis at physiological pH indicates that LdCS is properly folded. Further investigation into its tertiary structure using a quencher reveals that most tryptophan residues are located within the protein's hydrophobic core. Biochemical assays show that the recombinant enzyme is catalytically active, with optimal activity at pH 7.0. Kinetic studies provided parameters such as Km and Vmax. Enzyme inhibition assays revealed that LdCS activity is competitively inhibited by FDA-approved compounds-Abemaciclib, Bazedoxifene, Vorapaxar, and Imatinib-with Ki values ranging from 2 to 3 μM, demonstrating significant binding affinity. This research paves the way for exploring LdCS as a potential drug target for treating leishmaniasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Natural autoantibodies (natAAbs) react with evolutionarily conserved antigens but they do not lead to pathological tissue destruction, contrary to pathological autoantibodies (pathAAbs). NatAAbs usually belong to the IgM isotype, and their network, also known as the "immunological homunculus", is thought to play a role in immunological tolerance. NatAAbs are produced by B1 cells found mostly on the serosa surfaces or the spleen. The exact relation between natAAbs and pathAAbs is still not completely understood. The recombinant human proteoglycan (PG) aggrecan G1 domain (rhG1)-induced arthritis (GIA) is an excellent mouse model for rheumatoid arthritis because it represents most of the clinical, immunological and laboratory parameters of the corresponding human pathology. Recently, we studied the role of the spleen in GIA, and found that a splenectomy modified the development of autoimmunity. To further characterize the possible role of the nAAb levels in tolerance and autoimmunity, in the present study, we set out to measure the nat- and pathAAb levels in GIA. We analyzed the natAAb levels in the serum against cartilage PG aggrecan, Hsp60 and Hsp70, and the mitochondrial citrate synthase (CS) antigens in healthy control and arthritic mice. Furthermore, we studied whether the splenectomy influenced the production of nat- and pathAAbs in mice with GIA. Our results show that the natAAb levels against PG aggrecan, Hsp60, Hsp70 and CS showed age-related variations in healthy BALB/c mice. The induction of autoimmune arthritis did not change the levels of the measured natAAbs significantly. Splenectomy, on the other hand, clearly decreased the levels of all the measured natAAbs. Interestingly, the levels of the pathAAbs showed the opposite change: they were higher in the splenectomized group than in the control arthritic mice. Based on these results, we conclude that the spleen plays a role in setting the balance between nat- and pathAAbs in autoimmune arthritis.

This review discusses the intriguing yet controversial concept of metabolons, focusing on the malate dehydrogenase-citrate synthase (MDH-CISY) metabolon as a model. Metabolons are multienzyme complexes composed of enzymes that catalyze sequential reactions in metabolic pathways. Metabolons have been proposed to enhance metabolic pathway efficiency by facilitating substrate channeling. However, there is skepticism about the presence of metabolons and their functionality in physiological conditions in vivo. We address the skepticism by reviewing compelling evidence supporting the existence of the MDH-CISY metabolon and highlighting its potential functions in cellular metabolism. The electrostatic interaction between MDH and CISY and the intermediate oxaloacetate, channeled within the metabolon, has been demonstrated using various experimental techniques, including protein-protein interaction assays, isotope dilution studies, and enzyme coupling assays. Regardless of the wealth of in vitro evidence, further validation is required to elucidate the functionality of MDH-CISY metabolons in living systems using advanced structural and spatial analysis techniques., (© 2024 The Author(s).)

Purpose: We investigated the effects of low- and high-volume speed endurance training (SET), with a reduced training volume, on sprint ability, short- and long-term exercise capacity, muscle mitochondrial properties, ion transport proteins, and maximal enzyme activity in highly trained athletes., Methods: Highly trained male cyclists (maximal oxygen consumption (V̇O 2max ): 68.3 ± 5.0 mL·min -1 ·kg -1 , n = 24) completed 6 wk of either low (SET-L; 6 × 30-s intervals, n = 8) or high (SET-H; 12 × 30-s intervals, n = 8) volume SET twice per week with a 30% reduction in training volume. A control group (CON; n = 8) maintained their training. Exercise performance was evaluated by i) 6-s sprinting, ii) a 4-min time trial, and iii) a 60-min preload at 60% V̇O 2max followed by a 20-min time trial. A biopsy of m. vastus lateralis was collected before and after the training intervention., Results: In SET-L, 4-min time trial performance was improved ( P < 0.05) by 3.8%, with no change in SET-H and CON. Sprint ability, prolonged endurance exercise capacity, V̇O 2max , muscle mitochondrial respiratory capacity, maximal citrate synthase activity, fiber type-specific mitochondrial proteins (complexes I-V), and phosphofructokinase (PFK) content did not change in any of the groups. In SET-H, maximal activity of muscle PFK and abundance of Na + -K + pump-subunit α 1 , α 2 , β 1 , and phospholemman (FXYD1) were 20%, 50%, 19%, 24%, and 42% higher ( P < 0.05), respectively after compared with before the intervention, with no changes in SET-L or CON., Conclusions: Low SET volume combined with a reduced aerobic low- and moderate-intensity training volume does improve short-duration intense exercise performance and maintain sprinting ability, V̇O 2max , endurance exercise performance, and muscle oxidative capacity, whereas, high volume of SET seems necessary to upregulate muscle ion transporter content and maximal PFK activity in highly trained cyclists., (Copyright © 2024 by the American College of Sports Medicine.)

In pancreatic ductal adenocarcinoma (PDAC), metabolic rewiring and resistance to standard therapy are closely associated. PDAC cells show enormous requirements for glucose-derived citrate, the first rate-limiting metabolite in the synthesis of new lipids. Both the expression and activity of citrate synthase (CS) are extraordinarily upregulated in PDAC. However, no previous relationship between gemcitabine response and citrate metabolism has been documented in pancreatic cancer. Here, we report for the first time that pharmacological doses of vitamin C are capable of exerting an inhibitory action on the activity of CS, reducing glucose-derived citrate levels. Moreover, ascorbate targets citrate metabolism towards the de novo lipogenesis pathway, impairing fatty acid synthase (FASN) and ATP citrate lyase (ACLY) expression. Lowered citrate availability was found to be directly associated with diminished proliferation and, remarkably, enhanced gemcitabine response. Moreover, the deregulated citrate-derived lipogenic pathway correlated with a remarkable decrease in extracellular pH through inhibition of lactate dehydrogenase (LDH) and overall reduced glycolytic metabolism. Modulation of citric acid metabolism in highly chemoresistant pancreatic adenocarcinoma, through molecules such as vitamin C, could be considered as a future clinical option to improve patient response to standard chemotherapy regimens., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Introduction: Chronic hyperglycemia affects neutrophil functions, leading to reduced pathogen killing and increased morbidity. This impairment has been directly linked to increased glycemia, however, how this specifically affects neutrophils metabolism and their differentiation in the bone marrow is unclear and difficult to study., Research Design and Methods: We used high-resolution respirometry to investigate the metabolism of resting and activated donor neutrophils, and flow cytometry to measure surface CD15 and CD11b expression. We then used HL-60 cells differentiated towards neutrophil-like cells in standard media and investigated the effect of doubling glucose concentration on differentiation metabolism. We measured the oxygen consumption rate (OCR), and the enzymatic activity of carnitine palmitoyl transferase 1 (CPT1) and citrate synthase during neutrophil-like differentiation. We compared the surface phenotype, functions, and OCR of neutrophil-like cells differentiated under both glucose concentrations., Results: Donor neutrophils showed significant instability of CD11b and OCR after phorbol 12-myristate 13-acetate stimulation at 3 hours post-enrichment. During HL-60 neutrophil-like cell differentiation, there was a significant increase in surface CD15 and CD11b expression together with the loss of mitochondrial mass. Differentiated neutrophil-like cells also exhibited higher CD11b expression and were significantly more phagocytic. In higher glucose media, we measured a decrease in citrate synthase and CPT1 activities during neutrophil-like differentiation., Conclusions: HL-60 neutrophil-like differentiation recapitulated known molecular and metabolic features of human neutrophil differentiation. Increased glucose concentrations correlated with features described in hyperglycemic donor neutrophils including increased CD11b and phagocytosis. We used this model to describe metabolic features of neutrophil-like cell differentiation in hyperglycemia and show for the first time the downregulation of CPT1 and citrate synthase activity, independently of mitochondrial mass., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

This study aims to provide new insight on the association between the development of wooden breast myopathy and mitochondrial and glycolytic activity under oxidative stress. Myopathic muscle had higher oxidative stress together with altered glycolytic metabolism and tricarboxylic acid (TCA) cycle. This was evidenced by significantly elevated antioxidant enzyme activities (catalase, superoxide dismutase, and glutathione peroxidase), decreased citrate synthase activity and postmortem glycolytic potential with increasing wooden breast severity. In addition, affected muscles also exhibited higher initial and ultimate pH values as well as reduced total glucose and lactate contents. Citrate synthase activity was negatively correlated to antioxidant enzyme activities. Taken together, we propose that the development of the wooden breast lesion is a chronic process that may be related to the failure of muscle fibers to defend against the excessively generated oxidative products promoted by mitochondrial damage accompanied by impaired TCA cycle. Furthermore, there was a positive correlation between citrate synthase activity and glycolytic potential, which suggests that the wooden breast condition is linked to the overall altered energy metabolism of the muscle, including the oxidative phosphorylation and glycolytic pathways., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Citrate synthase catalyzes the first and the rate-limiting reaction of the tricarboxylic acid (TCA) cycle, producing citrate from the condensation of oxaloacetate and acetyl-coenzyme A. The parasitic protozoan Toxoplasma gondii has full TCA cycle activity, but its physiological roles remain poorly understood. In this study, we identified three proteins with predicted citrate synthase (CS) activities two of which were localized in the mitochondrion, including the 2-methylcitrate synthase (PrpC) that was thought to be involved in the 2-methylcitrate cycle, an alternative pathway for propionyl-CoA detoxification. Further analyses of the two mitochondrial enzymes showed that both had citrate synthase activity, but the catalytic efficiency of CS1 was much higher than that of PrpC. Consistently, the deletion of CS1 resulted in a significantly reduced flux of glucose-derived carbons into TCA cycle intermediates, leading to decreased parasite growth. In contrast, disruption of PrpC had little effect. On the other hand, simultaneous disruption of both CS1 and PrpC resulted in more severe metabolic changes and growth defects than a single deletion of either gene, suggesting that PrpC does contribute to citrate production under physiological conditions. Interestingly, deleting Δcs1 and Δprpc individually or in combination only mildly or negligibly affected the virulence of parasites in mice, suggesting that both enzymes are dispensable in vivo. The dispensability of CS1 and PrpC suggests that either the TCA cycle is not essential for the asexual reproduction of tachyzoites or there are other routes of citrate supply in the parasite mitochondrion., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To identify therapies for combined D, L-2-hydroxyglutaric aciduria (C-2HGA), a rare genetic disorder caused by recessive variants in the SLC25A1 gene., Methods: Patients C-2HGA were identified and diagnosed by whole exome sequencing and biochemical genetic testing. Patient derived fibroblasts were then treated with phenylbutyrate and the functional effects assessed by metabolomics and RNA-sequencing., Results: In this study, we demonstrated that C-2HGA patient derived fibroblasts exhibited impaired cellular bioenergetics. Moreover, Fibroblasts form one patient exhibited worsened cellular bioenergetics when supplemented with citrate. We hypothesized that treating patient cells with phenylbutyrate (PB), an FDA approved pharmaceutical drug that conjugates glutamine for renal excretion, would reduce mitochondrial 2-ketoglutarate, thereby leading to improved cellular bioenergetics. Metabolomic and RNA-seq analyses of PB-treated fibroblasts demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of PB, an increased level of phenylacetylglutamine in patient cells was consistent with the drug acting as 2-ketoglutarate sink., Conclusion: Our pre-clinical studies suggest that citrate supplementation has the possibility exacerbating energy metabolism in this condition. However, improvement in cellular bioenergetics suggests phenylbutyrate might have interventional utility for this rare disease., Competing Interests: Declaration of competing interest Authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Introduction: Data suggest malfunctioning mitochondria reduce oxidation and adenosine triphosphate (ATP) production, disrupting insulin signalling. Cytochrome c (CC), acylcarnitine (AC) and citrate synthase (CS) are essential components of the mitochondria machinery and can be used as reliable biomarkers of mitochondrial dysfunction. This study aimed to determine whether mitochondrial biomarkers (AC, CS and CC) are altered in individuals with type 2 diabetes mellitus (T2DM) and to examine the association between these biomarkers and insulin resistance., Methodology: A cross-sectional observational study that recruited 170 participants (88 with T2DM and 82 without DM) was conducted. Blood samples were collected from the recruits and analysed for levels of fasting glucose (FBG), AC, CS, CC, insulin, total cholesterol, triglycerides (TG), glycated haemoglobin (HbA1c) and magnesium. Blood pressure (BP) and anthropometric characteristics of participants were also taken. Appropriate formulas were used to determine %body fat, body mass index (BMI), waist-to-hip ratio (WHR), the homeostatic model assessment for insulin resistance (HOMA-IR) and insulin sensitivity (HOMA-β)., Results: Patients with T2DM had higher levels of CC, %body fat, FBG, TG, HbA1c, BMI and HOMA-IR than controls (p < 0.05, respectively). Results showed a significant relationship between circulating CC levels versus HOMA-β (r = -0.40, p = 0.001), CS (r = -0.70, p = 0.001) and AC (r = -0.72, p = 0.001) levels in patients with T2DM. The adjusted odds increased in the T2DM patients for VLDL (OR = 6.66, p = 0.002), HbA1c (OR = 6.50, p = 0.001), FPG (OR = 3.17, p = 0.001), TG (OR = 2.36, p = 0.010), being female (OR = 2.09, p = 0.020) and CC (OR = 1.14, p = 0.016)., Conclusion: Overall, alterations in mitochondrial biomarkers, measured by AC, CC and CS, were observed in people with T2DM and showed a direct relationship with insulin resistance. These findings are potentially significant in Africa, although additional confirmation from a larger cohort is necessary., (© 2024 The Author(s). Endocrinology, Diabetes & Metabolism published by John Wiley & Sons Ltd.)

Purpose: We previously showed that offspring delivered to baboons in which levels of estradiol (E 2 ) were suppressed during the second half of gestation exhibit insulin resistance. Mitochondria are essential for the production of ATP as the main source of energy for intracellular metabolic pathways, and skeletal muscle of type 2 diabetics exhibit mitochondrial abnormalities. Mitochondria express estrogen receptor β and E 2 enhances mitochondrial function in adults. Therefore, the current study ascertained whether exposure of the fetus to E 2 is essential for mitochondrial development., Methods: Levels of ATP synthase and citrate synthase and the morphology of mitochondria were determined in fetal skeletal muscle obtained near term from baboons untreated or treated daily with the aromatase inhibitor letrozole or letrozole plus E 2 ., Results: Specific activity and amount of ATP synthase were 2-fold lower (P < 0.05) in mitochondria from skeletal muscle of E 2 suppressed letrozole-treated fetuses and restored to normal by treatment with letrozole plus E 2 . Immunocytochemistry showed that in contrast to the punctate formation of mitochondria in myocytes of untreated and letrozole plus E 2 treated animals, mitochondria appeared to be diffuse in myocytes of estrogen-suppressed fetuses. However, citrate synthase activity and levels of proteins that control mitochondrial fission/fusion were similar in estrogen replete and suppressed animals., Conclusion: We suggest that estrogen is essential for fetal skeletal muscle mitochondrial development and thus glucose homeostasis in adulthood., (© 2024. The Author(s).)

Background: The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme., Results: Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA [A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source., Conclusions: This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation., (© 2024. The Author(s).)

We investigated whether calorie restriction (CR) enhances metabolic adaptations to endurance training (ET). Ten-week-old male Institute of Cancer Research (ICR) mice were fed ad libitum or subjected to 30% CR. The mice were subdivided into sedentary and ET groups. The ET group performed treadmill running (20-25 m/min, 30 min, 5 days/week) for 5 weeks. We found that CR decreased glycolytic enzyme activity and monocarboxylate transporter (MCT) 4 protein content, while enhancing glucose transporter 4 protein content in the plantaris and soleus muscles. Although ET and CR individually increased citrate synthase activity in the plantaris muscle, the ET-induced increase in respiratory chain complex I protein content was counteracted by CR. In the soleus muscle, mitochondrial enzyme activity and protein levels were increased by ET, but decreased by CR. It has been suggested that CR partially interferes with skeletal muscle adaptation to ET., (© 2024. The Author(s).)

Fetuses affected by intrauterine growth restriction have an increased risk of developing heart disease and failure in adulthood. Compared with controls, late gestation intrauterine growth-restricted (IUGR) fetal sheep have fewer binucleated cardiomyocytes, reflecting a more immature heart, which may reduce mitochondrial capacity to oxidize substrates. We hypothesized that the late gestation IUGR fetal heart has a lower capacity for mitochondrial oxidative phosphorylation. Left (LV) and right (RV) ventricles from IUGR and control (CON) fetal sheep at 90% gestation were harvested. Mitochondrial respiration (states 1-3, Leak Omy , and maximal respiration) in response to carbohydrates and lipids, citrate synthase (CS) activity, protein expression levels of mitochondrial oxidative phosphorylation complexes (CI-CV), and mRNA expression levels of mitochondrial biosynthesis regulators were measured. The carbohydrate and lipid state 3 respiration rates were lower in IUGR than CON, and CS activity was lower in IUGR LV than CON LV. However, relative CII and CV protein levels were higher in IUGR than CON; CV expression level was higher in IUGR than CON. Genes involved in lipid metabolism had lower expression in IUGR than CON. In addition, the LV and RV demonstrated distinct differences in oxygen flux and gene expression levels, which were independent from CON and IUGR status. Low mitochondrial respiration and CS activity in the IUGR heart compared with CON are consistent with delayed cardiomyocyte maturation, and CII and CV protein expression levels may be upregulated to support ATP production. These insights will provide a better understanding of fetal heart development in an adverse in utero environment. KEY POINTS: Growth-restricted fetuses have a higher risk of developing and dying from cardiovascular diseases in adulthood. Mitochondria are the main supplier of energy for the heart. As the heart matures, the substrate preference of the mitochondria switches from carbohydrates to lipids. We used a sheep model of intrauterine growth restriction to study the capacity of the mitochondria in the heart to produce energy using either carbohydrate or lipid substrates by measuring how much oxygen was consumed. Our data show that the mitochondria respiration levels in the growth-restricted fetal heart were lower than in the normally growing fetuses, and the expression levels of genes involved in lipid metabolism were also lower. Differences between the right and left ventricles that are independent of the fetal growth restriction condition were identified. These results indicate an impaired metabolic maturation of the growth-restricted fetal heart associated with a decreased capacity to oxidize lipids postnatally., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

This study presents an in-depth analysis of mitochondrial enzyme activities in Friedreich's ataxia (FA) patients, focusing on the Electron Transport Chain complexes I, II, and IV, the Krebs Cycle enzyme Citrate Synthase, and Coenzyme Q10 levels. It examines a cohort of 34 FA patients, comparing their mitochondrial enzyme activities and clinical parameters, including disease duration and cardiac markers, with those of 17 healthy controls. The findings reveal marked reductions in complexes II and, specifically, IV, highlighting mitochondrial impairment in FA. Additionally, elevated Neurofilament Light Chain levels and cardiomarkers were observed in FA patients. This research enhances our understanding of FA pathophysiology and suggests potential biomarkers for monitoring disease progression. The study underscores the need for further clinical trials to validate these findings, emphasizing the critical role of mitochondrial dysfunction in FA assessment and treatment., (© 2024. The Author(s).)

In prokaryotes and lower eukaryotes, 2-methylcitrate cycle (2-MCC) is the main pathway for propionate decomposition and transformation, but little is known about the 2-MCC pathway of Eimeria tenella. The analysis of genomic data found that the coding gene of 2- methylcitrate synthase (EC 2.3.3.5, PrpC) exists in E. tenella, which is a key enzyme of 2-MCC pathway. Through the search analysis of the database (ToxoDB), it was found that ETH&#95; 00026655 contains the complete putative sequence of EtprpC. In this study, we amplified the ORF sequence of EtprpC based on putative sequence. Then, prokaryotic expression, enzyme activity and kinetic analysis was performed. The results showed that the EtprpC ORF sequence was 1272 bp, encoding a 46.3 kDa protein comprising 424 amino acids. Enzyme activity assays demonstrate linearity between the initial reaction rate (OD/min) and EtPrpC concentration (ranging from 1.5 to 9 µg/reaction), with optimal enzyme activity observed at 41°C and pH 8.0. The results of enzymatic kinetic analysis showed that the Km of EtPrpC for propionyl-CoA, oxaloacetic acid, and acetyl-CoA was 5.239 ± 0.17 mM, 1.102 ± 0.08 μM, and 5.999 ± 1.24 μM, respectively. The Vmax was 191.11 ± 19.1 nmol/min/mg, 225.48 ± 14.4 nmol/min/mg, and 370.02 ± 25.8 nmol/min/mg when EtPrpC concentration at 4, 6, and 8 μg, respectively. Although the ability of EtPrpC to catalyze acetyl-CoA is only 0.11% of its ability to catalyze propionyl-CoA, it indicates that the 2-MCC pathway in E. tenella is similar to that in bacteria and may have a bypass function in the TCA cycle. This study can provide the theoretical foundation for the new drug targets and the development of new anticoccidial drugs., Competing Interests: Declaration of Competing Interest Authors (Zhenxing Gong, Zigang Qu, and Jianping Cai) declare that there are no conflict of interests either financially or otherwise as relating the publication of the manuscript or whatever benefits that may result thereafter., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Women have a disadvantage for performance in long-distance running compared with men. To elaborate on inherent characteristics, 12 subelite women were matched with 12 men for training volume (M-Tm) (56.6 ± 18 vs. 55.7 ± 17 km/wk). The women were also matched to other men for a 10 km staged outdoor time trial (M-Pm) (42:36 min:s) to determine which factors could explain equal running performance. Anthropometry and treadmill tests were done. Fiber type (% Type I and Type IIA) and citrate synthase activities were analyzed in muscle biopsy samples. Consistent sex differences for both comparisons included height, weight, % body fat ( P < 0.01), and hematocrit ( P < 0.05). Women had lower V̇o 2max and peak treadmill speed (PTS) compared with both M-Tm and M-Pm ( P < 0.01). Training matched pairs had no sex difference in % PTS at race pace but compared with M-Pm women ran at a higher % PTS ( P < 0.05) and %HR max ( P < 0.01) at race pace. On average, the women trained 22.9 km/wk more than M-Pm (+67.5%, P < 0.01). This training was not associated with higher V̇o 2max or better running economy. Muscle morphology and oxidative capacity did not differ between groups. Percentage body fat remained significantly higher in women. In conclusion, women matched to men for training volume had slower 10 km performance (-10.5% P < 0.05). Higher training volume, more high-intensity sessions/wk, and time spent training in the 95%-100% HR max zone may explain the higher % PTS and %HR max at race pace in women compared with performance-matched men. NEW & NOTEWORTHY When subelite women 10 km runners were matched with male counterparts for 10 km race performance, inherent differences in % body fat, V̇o 2max , Hct, and peak treadmill speed were counteracted by significantly higher training volume, more time training at higher %HRmax and consequently, higher %HRmax and %PTS at race pace. Citrate synthase activity and muscle fiber types did not differ. When women and men matched for training, 10 km performance of men was 10.5% faster.

Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000 km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Fractals are patterns that are self-similar across multiple length-scales 1 . Macroscopic fractals are common in nature 2-4 ; however, so far, molecular assembly into fractals is restricted to synthetic systems 5-12 . Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpiński triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution., (© 2024. The Author(s).)

We discuss the role of epigenetic changes at the level of promoter methylation of the key enzymes of carbon metabolism in the regulation of respiration by light. While the direct regulation of enzymes via modulation of their activity and post-translational modifications is fast and readily reversible, the role of cytosine methylation is important for providing a prolonged response to environmental changes. In addition, adenine methylation can play a role in the regulation of transcription of genes. The mitochondrial and extramitochondrial forms of several enzymes participating in the tricarboxylic acid cycle and associated reactions are regulated via promoter methylation in opposite ways. The mitochondrial forms of citrate synthase, aconitase, fumarase, NAD-malate dehydrogenase are inhibited while the cytosolic forms of aconitase, fumarase, NAD-malate dehydrogenase, and the peroxisomal form of citrate synthase are activated. It is concluded that promoter methylation represents a universal mechanism of the regulation of activity of respiratory enzymes in plant cells by light. The role of the regulation of the mitochondrial and cytosolic forms of respiratory enzymes in the operation of malate and citrate valves and in controlling the redox state and balancing the energy level of photosynthesizing plant cells is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Aims: This study aimed to investigate the relationship between microglial metabolism and neuroinflammation by examining the impact of citrate accumulation in microglia and its potential regulation through Cs K215 hypoacetylation., Methods: Experimental approaches included assessing Cs enzyme activity through Cs K215Q mutation and investigating the inhibitory effects of hesperidin, a natural flavanone glycoside, on citrate synthase. Microglial phagocytosis and expression of pro-inflammatory cytokines were also examined in relation to Cs K215Q mutation and hesperidin treatment., Results: Cs K215Q mutation and hesperidin exhibited significant inhibitory effects on Cs enzyme activity, microglial citrate accumulation, phagocytosis, and pro-inflammatory cytokine expression. Interestingly, Sirt3 knockdown aggravated microglial pro-inflammatory functions during neuroinflammation, despite its proven role in Cs deacetylation., Conclusion: Cs K215Q mutation and hesperidin effectively inhibited microglial pro-inflammatory functions without reversing the metabolic reprogramming. These findings suggest that targeting Cs K215 hypoacetylation and utilizing hesperidin may hold promise for modulating neuroinflammation in microglia., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N∆Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N∆Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N∆Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N∆Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCFUcc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Objective: To evaluate the effect of monocarbonyl analogues of curcumin on changes in the processes of mitophagy and mitochondrial biogenesis in the cerebral cortex of rats with experimental Alzheimer's disease., Material and Methods: Alzheimer's disease was modeled in Wistar rats of both sexes by injection of β-amyloid fragments into the hippocampus of the animal. Compounds (1E, 4E)-1.5-bis (3.4.5-trimethoxyphenyl) penta-1.4-diene-3-one (AZBAX4 code) and (1E, 4E)-1.5-bis (2.4.6-trimethoxyphenyl) penta-1.4-diene-3-one (AZBAX6 code) at a dose of 20 mg/ kg (orally) and the reference drug donepezil at a dose of 50 mg/kg (orally) were administered for 30 days, after which changes in the activity of succinate dehydrogenase, cytochrome-c oxidase and citrate synthase as enzymatic biomarkers of mitochondrial biogenesis and mitophagy, respectively, were evaluated in the mitochondrial fraction of the cerebral cortex., Results: The administration of AZBAX4 and AZBAX6 compounds led to an increase in the activity of succinate dehydrogenase; cytochrome-c oxidase, as well as citrate synthase in relation to the same indicators of the group of untreated animals. The use of the analyzed compounds was equally effective in both female and male rats. At the same time, it should be noted that the analyzed compounds significantly exceeded the activity level of the reference donepezil., Conclusion: AZBAX4 and AZBAX6 contribute to an increase in the intensity of mitochondrial biogenesis and mitophagy reactions in the cerebral cortex of rats with Alzheimer's disease, which makes them potentially effective neuroprotective compounds.

Following the parasitic juvenile phase of their life cycle, sea lamprey (Petromyzon marinus) mature into a reproductive but rapidly aging and deteriorating adult, and typically die shortly after spawning in May or June. However, pre-spawning upstream migrant sea lamprey can be maintained for several months beyond their natural lifespan when held in cold water (∼4-8 °C) under laboratory conditions. We exploited this feature to investigate the interactions between senescence, oxidative stress, and metabolic function in this phylogenetically ancient fish. We investigated how life history traits and mitochondria condition, as indicated by markers of oxidative stress (catalase activity, lipid peroxidation) and aerobic capacity (citrate synthase activity), changed in adult sea lamprey from June to December after capture during their upstream spawning migration. Body mass but not liver mass declined with age, resulting in an increase in hepatosomatic index. Both effects were most pronounced in males, which also tended to have larger livers than females. Lamprey experienced greater oxidative stress with age, as reflected by increasing activity of the antioxidant enzyme catalase and increasing levels of lipid peroxidation in liver mitochondrial isolates over time. Surprisingly, the activity of citrate synthase also increased with age in both sexes. These observations implicate mitochondrial dysfunction and oxidative stress in the senescence of sea lamprey. Due to their unique evolutionary position and the technical advantage of easily delaying the onset of senescence in lampreys using cold water, these animals could represent an evolutionary unique and tractable model to investigate senescence in vertebrates., Competing Interests: Declaration of Competing Interest We declare no competing or financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The obesity pandemic has worsened global disease burden, including type 2 diabetes, cardiovascular disease, and cancer. Metabolic/bariatric surgery (MBS) is the most effective and durable obesity treatment, but the mechanisms underlying its long-term weight loss efficacy remain unclear. MBS drives substrate oxidation that has been linked to improvements in metabolic function and improved glycemic control that are potentially mediated by mitochondria-a primary site of energy production. As such, augmentation of intestinal mitochondrial function may drive processes underlying the systemic metabolic benefits of MBS. Herein, we applied a highly sensitive technique to evaluate intestinal mitochondrial function ex vivo in a mouse model of MBS., Methods: Mice were randomized to surgery, sham, or non-operative control. A simplified model of MBS, ileal interposition, was performed by interposition of a 2-cm segment of terminal ileum into the proximal bowel 5 mm from the ligament of Treitz. After a four-week recovery period, intestinal mucosa of duodenum, jejunum, ileum, and interposed ileum were assayed for determination of mitochondrial respiratory function. Citrate synthase activity was measured as a marker of mitochondrial content., Results: Ileal interposition was well tolerated and associated with modest body weight loss and transient hypophagia relative to controls. Mitochondrial capacity declined in the native duodenum and jejunum of animals following ileal interposition relative to controls, although respiration remained unchanged in these segments. Similarly, ileal interposition lowered citrate synthase activity in the duodenum and jejunum following relative to controls but ileal function remained constant across all groups., Conclusion: Ileal interposition decreases mitochondrial volume in the proximal intestinal mucosa of mice. This change in concentration with preserved respiration suggests a global mucosal response to segment specific nutrition signals in the distal bowel. Future studies are required to understand the causes underlying these mitochondrial changes., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: It is now understood that HBV can induce innate and adaptive immune response disorders by affecting immunosuppressive macrophages, resulting in chronic HBV infection. However, the underlying mechanism is not fully understood. Dysregulated protein acetylation can reportedly influence the differentiation and functions of innate immune cells by coordinating metabolic signaling. This study aims to assess whether HBV suppresses macrophage-mediated innate immune responses by affecting protein acetylation and to elucidate the underlying mechanisms of HBV immune escape., Methods: We investigated the effect of HBV on the acetylation levels of human THP-1 macrophages and identified potential targets of acetylation that play a role in glucose metabolism. Metabolic and immune phenotypes of macrophages were analyzed using metabolomic and flow cytometry techniques. Western blot, immunoprecipitation, and immunofluorescence were performed to measure the interactions between deacetylase and acetylated targets. Chronic HBV persistent infected mice were established to evaluate the role of activating the tricarboxylic acid (TCA) cycle in macrophages for HBV clearance., Results: Citrate synthase/pyruvate dehydrogenase complex hyperacetylation in macrophages after HBV stimulation inhibited their enzymatic activities and was associated with impaired TCA cycle and M2-like polarization. HBV downregulated Sirtuin 3 (SIRT3) expression in macrophages by means of the toll-like receptor 2 (TLR2)-NF-κB- peroxisome proliferatoractivated receptor γ coactivator 1α (PGC-1α) axis, resulting in citrate synthase/pyruvate dehydrogenase complex hyperacetylation. In vivo administration of the TCA cycle agonist dichloroacetate inhibited macrophage M2-like polarization and effectively reduced the number of serum HBV DNA copies., Conclusions: HBV-induced citrate synthase/pyruvate dehydrogenase complex hyperacetylation negatively modulates the innate immune response by impairing the TCA cycle of macrophages. This mechanism represents a potential therapeutic target for controlling HBV infection., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

The domestic dog (Canis lupus familiaris) species comprises hundreds of breeds, each differing in physical characteristics, behavior, strength, and running capability. Very little is known about the skeletal muscle composition and metabolism between the different breeds, which may explain disease susceptibility. Muscle samples from the triceps brachii (TB) and vastus lateralis (VL) were collected post mortem from 35 adult dogs, encompassing 16 breeds of varying ages and sex. Samples were analyzed for fiber type composition, fiber size, oxidative, and glycolytic metabolic capacity (citrate synthase [CS], 3-hydroxyacetyl-coA dehydrogenase [3HAD], creatine kinase [CK], and lactate dehydrogenase [LDH] enzyme activities). There was no significant difference between the TB and VL in any of the measurements. However, there were large intra species variation, with some variables confirming the physical attributes of a specific breed. Collectively, type IIA was the predominant fiber type followed by type I and type IIX. The cross-sectional areas (CSA) of the fibers were all smaller when compared to humans and similar to other wild animals. There was no difference in the CSA between the fiber types and muscle groups. Metabolically, the muscle of the dog displayed high oxidative capacity with high activities for CS and 3HAD. Lower CK and higher LDH activities than humans indicate a lower and higher flux through the high energy phosphate and glycolytic pathways, respectively. The high variability found across the different breeds may be attributed to genetics, function or lifestyle which have largely been driven through human intervention. This data may provide a foundation for future research into the role of these parameters in disease susceptibility, such as insulin resistance and diabetes, across breeds., (© 2023 The Authors. The Anatomical Record published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

This study investigated the potential effects of exercise on the responses of energy metabolism, redox balance maintenance, and apoptosis regulation in Drosophila melanogaster to shed more light on the mechanisms underlying the increased performance that this emerging exercise model provides. Three groups were evaluated for seven days: the control (no exercise or locomotor limitations), movement-limited flies (MLF) (no exercise, with locomotor limitations), and EXE (with exercise, no locomotor limitations). The EXE flies demonstrated greater endurance-like tolerance in the swimming test, associated with increased citrate synthase activity, lactate dehydrogenase activity and lactate levels, and metabolic markers in exercise. Notably, the EXE protocol regulated the Akt/p38 MAPK/Nrf2 pathway, which was associated with decreased Hsp70 activation, culminating in glutathione turnover regulation. Moreover, reducing the locomotion environment in the MLF group decreased endurance-like tolerance and did not alter citrate synthase activity, lactate dehydrogenase activity, or lactate levels. The MLF treatment promoted a pro-oxidant effect, altering the Akt/p38 MAPK/Nrf2 pathway and increasing Hsp70 levels, leading to a poorly-regulated glutathione system. Lastly, we demonstrated that exercise could modulate major metabolic responses in Drosophila melanogaster aerobic and anaerobic metabolism, associated with apoptosis and cellular redox balance maintenance in an emergent exercise model., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Acute respiratory distress syndrome (ARDS) is a leading cause of respiratory failure and death in patients in the intensive care unit. Experimentally, acute lung injury resolution depends on the repair of mitochondrial oxidant damage by the mitochondrial quality control (MQC) pathways, mitochondrial biogenesis, and mitophagy, but nothing is known about this in the human lung. In a case-control autopsy study, we compared the lungs of subjects dying of ARDS (n = 8; cases) and age-/gender-matched subjects dying of nonpulmonary causes (n = 7; controls). Slides were examined by light microscopy and immunofluorescence confocal microscopy, randomly probing for co-localization of citrate synthase with markers of oxidant stress, mitochondrial DNA damage, mitophagy, and mitochondrial biogenesis. ARDS lungs showed diffuse alveolar damage with edema, hyaline membranes, and neutrophils. Compared with controls, a high degree of mitochondrial oxidant damage was seen in type 2 epithelial (AT2) cells and alveolar macrophages by 8-hydroxydeoxyguanosine and malondialdehyde co-staining with citrate synthase. In ARDS, antioxidant protein heme oxygenase-1 and DNA repair enzyme N-glycosylase/DNA lyase (Ogg1) were found in alveolar macrophages but not in AT2 cells. Moreover, MAP1 light chain-3 (LC3) and serine/threonine-protein kinase (Pink1) staining were absent in AT2 cells, suggesting a mitophagy failure. Nuclear respiratory factor-1 staining was missing in the alveolar region, suggesting impaired mitochondrial biogenesis. Widespread hyperproliferation of AT2 cells in ARDS could suggest defective differentiation into type 1 cells. ARDS lungs show profuse mitochondrial oxidant DNA damage but little evidence of MQC activity in AT2 epithelium. Because these pathways are important for acute lung injury resolution, our findings support MQC as a novel pharmacologic target for ARDS resolution., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Carbon metabolism is critical for microbial physiology and remarkably affects the outcome of secondary metabolite production. The production of 2,4-diacetylphloroglucinol (2,4-DAPG), a bacterial secondary metabolite with a broad spectrum of antibiotic activity, is a major mechanism used by the soil bacterium Pseudomonas fluorescens 2P24 to inhibit the growth of plant pathogens and control disease occurrence. Strain 2P24 has evolved a complex signaling cascade to regulate the production of 2,4-DAPG. However, the role of the central carbon metabolism in modulating 2,4-DAPG production has not been fully determined. In this study, we report that the gltA gene, which encodes citrate synthase, affects the expression of the 2,4-DAPG biosynthesis gene and is essential for the biocontrol capacity of strain 2P24. Our data showed that the mutation of gltA remarkably decreased the biosynthesis of 2,4-DAPG. Consistent with this result, the addition of citrate in strain 2P24 resulted in increased 2,4-DAPG production and decreased levels of RsmA and RsmE. In comparison with the wild-type strain, the gltA mutant was severely impaired in terms of biocontrol activity against the bacterial wilt disease of tomato plants caused by Ralstonia solanacearum . Moreover, the gltA mutant exhibited increased antioxidant activity, and the expression of oxidative, stress-associated genes, including ahpB , katB , and oxyR , was significantly upregulated in the gltA mutant compared to the wild-type strain. Overall, our data indicate that the citrate synthase GltA plays an important role in the production of 2,4-DAPG and oxidative stress and is required for biocontrol capacity.

The selection and utilization of ornamental plants that are highly tolerant to salt are helpful for landscape construction and the ecological protection of coastal and arid areas. To evaluate salt tolerance, one of the most used methods is the observation of seed germination under salt stress. Therefore, this work aimed to evaluate the influence of different concentrations of NaCl in water absorption, germination, and respiratory metabolism in seeds of different Flueggea suffruticosa genotypes. P2 and P27, salt-sensitive and salt-tolerant line s of F . suffruticosa , were chosen for treatment with 0, 40, 80, 120, 160, 200, and 240 mM NaCl. F . suffruticosa under salt stress exhibited inhibition of seed germination. The seeds of F . suffruticosa have different times for the physiological phases of water absorption with different NaCl concentrations. Salt stress retarded the seed water absorption process, and it depended on seed genotypes for F . suffruticosa . Soluble sugars accumulated in both P2 and P27 under salt stress. Meanwhile, the activities of hexokinase, 6-phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase, citrate synthase, and glucose-6-phosphate dehydrogenase were overall increased in P27 after salt treatment, which caused increases in pyruvic acid and citric acid. The citrate synthase and glucose-6-phosphate dehydrogenase activities decreased in P2. These results suggest that the respiratory metabolism of salt-tolerant F . suffruticosa was enhanced, compared with the salt-sensitive line, to ameliorate the repression of seed germination under salt stress. The different changes in respiratory metabolism could influence the degree of salt tolerance., Competing Interests: The authors declare there are no competing interests., (©2023 Xu et al.)

The wood frog, Rana sylvatica endures whole body freezing for weeks/months while overwintering at subzero temperatures. Survival of long-term freezing requires not only cryoprotectants but also strong metabolic rate depression (MRD) and reorganization of essential processes in order to maintain a balance between ATP-producing and ATP-consuming processes. Citrate synthase (CS) (E.C. 2.3.3.1) is an important irreversible enzyme of the tricarboxylic acid (TCA) cycle and forms a crucial checkpoint for many metabolic processes. Present study investigated the regulation of CS from wood frog liver during freezing. CS was purified to homogeneity by a two-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and, notably, demonstrated a significant decrease in the V max of the purified form of CS from frozen frogs as compared to controls when assayed at both 22 °C and 5 °C. This was further supported by a decrease in the maximum activity of CS from liver of frozen frogs. Immunoblotting also showed changes in posttranslational modifications with a significant decrease in threonine phosphorylation (by 49 %) for CS from frozen frogs. Taken together, these results suggest that CS is suppressed and TCA flux is inhibited during freezing, likely to support MRD survival of harsh winters., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Metabolic reprogramming is a hallmark of cancer. Several studies have shown that inactivation of Krebs cycle enzymes, such as citrate synthase (CS) and fumarate hydratase (FH), facilitates aerobic glycolysis and cancer progression. MAEL has been shown to play an oncogenic role in bladder, liver, colon, and gastric cancers, but its role in breast cancer and metabolism is still unknown. Here, we demonstrated that MAEL promoted malignant behaviours and aerobic glycolysis in breast cancer cells. Mechanistically, MAEL interacted with CS/FH and HSAP8 via its MAEL domain and HMG domain, respectively, and then enhanced the binding affinity of CS/FH with HSPA8, facilitating the transport of CS/FH to the lysosome for degradation. MAEL-induced degradation of CS and FH could be suppressed by the lysosome inhibitors leupeptin and NH 4 Cl, but not by the macroautophagy inhibitor 3-MA or the proteasome inhibitor MG132. These results suggested that MAEL promoted the degradation of CS and FH via chaperone-mediated autophagy (CMA). Further studies showed that the expression of MAEL was significantly and negatively correlated with CS and FH in breast cancer. Moreover, overexpression of CS or/and FH could reverse the oncogenic effects of MAEL. Taken together, MAEL promotes a metabolic shift from oxidative phosphorylation to glycolysis by inducing CMA-dependent degradation of CS and FH, thereby promoting breast cancer progression. These findings have elucidated a novel molecular mechanism of MAEL in cancer., (© 2023 Federation of European Biochemical Societies.)

This study aimed to clarify whether aerobic exercise training-induced alterations in the gut microbiota affect physiological adaptation with endurance exercise capacity. In study 1, ICR mice were randomly divided into three groups: vehicle intake + sedentary (V+S), vehicle intake + exercise training (V+Ex) and antibiotic intake + exercise training (AB+Ex). In the exercise training groups, treadmill running was performed for 8 weeks. During the exercise training intervention, the antibiotic-intake group freely drank water containing antibiotics. In study 2, ICR mice were randomly divided into three groups: Sham, transplantation of caecum microbiota from sedentary mice (Sed-CMT) and exercise training mice (Ex-CMT). In study 1, the treadmill running time to exhaustion, an index of maximal aerobic capacity, after aerobic exercise training in the V+Ex group was significantly longer than that in the V+S and AB+Ex groups. Gastrocnemius muscle citrate synthase (CS) activity and PGC-1α protein levels in the V+Ex group were significantly higher than in the V+S and AB+Ex groups. The bacterial Erysipelotrichaceae and Alcaligenaceae families were positively correlated with treadmill running time to exhaustion. In study 2, the treadmill running time to exhaustion after transplantation was significantly higher in the Ex-CMT group than in the Sham and Sed-CMT groups. Furthermore, CS activity and PGC-1α protein levels in the gastrocnemius muscle were significantly higher in the Ex-CMT group than in the Sham and Sed-CMT groups. Thus, gut microbiota altered by aerobic exercise training may be involved in the augmentation of endurance capacity and muscle mitochondrial energy metabolism. KEY POINTS: Aerobic exercise training changes gut microbiota composition, and the Erysipelotrichaceae and Alcaligenaceae families were among the altered gut bacteria. The gut microbiota was associated with endurance performance and metabolic regulator levels in skeletal muscle after aerobic exercise training. Continuous antibiotic treatment attenuated the increase in endurance performance, citrate synthase activity and PGC-1α levels in skeletal muscle induced by aerobic exercise training. Gut microbiota transplantation from exercise-trained mice improved endurance performance and metabolic regulator levels in recipient skeletal muscle, despite the absence of aerobic exercise training., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L -1 resveratrol from supplemented p-coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

ATP citrate lyase (ACLY) is the predominant nucleocytosolic source of acetyl-CoA and is aberrantly regulated in many diseases making it an attractive therapeutic target. Structural studies of ACLY reveal a central homotetrameric core citrate synthase homology (CSH) module flanked by acyl-CoA synthetase homology (ASH) domains, with ATP and citrate binding the ASH domain and CoA binding the ASH-CSH interface to produce acetyl-CoA and oxaloacetate products. The specific catalytic role of the CSH module and an essential D1026A residue contained within it has been a matter of debate. Here, we report biochemical and structural analysis of an ACLY-D1026A mutant demonstrating that this mutant traps a (3S)-citryl-CoA intermediate in the ASH domain in a configuration that is incompatible with the formation of acetyl-CoA, is able to convert acetyl-CoA and OAA to (3S)-citryl-CoA in the ASH domain, and can load CoA and unload acetyl-CoA in the CSH module. Together, this data support an allosteric role for the CSH module in ACLY catalysis., (© 2023. The Author(s).)

Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCF Ucc1 . Unexpectedly, our structural and genetic analyses revealed that nonimported citrate synthase appears to form an enzymatically active conformation in the cytosol. Its excess accumulation caused ectopic citrate synthesis, which, in turn, led to an imbalance in carbon flux of sugar, a reduction of the pool of amino acids and nucleotides, and a growth defect. Under these conditions, translation repression is induced and acts as a protective mechanism that mitigates the growth defect. We propose that the consequence of mitochondrial import failure is not limited to proteotoxic insults, but that the accumulation of a nonimported metabolic enzyme elicits ectopic metabolic stress.

Mitochondrial RTG (an acronym for ReTroGrade) signaling plays a cytoprotective role under various intracellular or environmental stresses. We have previously shown its contribution to osmoadaptation and capacity to sustain mitochondrial respiration in yeast. Here, we studied the interplay between RTG2 , the main positive regulator of the RTG pathway, and HAP4 , encoding the catalytic subunit of the Hap2-5 complex required for the expression of many mitochondrial proteins that function in the tricarboxylic acid (TCA) cycle and electron transport, upon osmotic stress. Cell growth features, mitochondrial respiratory competence, retrograde signaling activation, and TCA cycle gene expression were comparatively evaluated in wild type and mutant cells in the presence and in the absence of salt stress. We showed that the inactivation of HAP4 improved the kinetics of osmoadaptation by eliciting both the activation of retrograde signaling and the upregulation of three TCA cycle genes: citrate synthase 1 ( CIT1 ), aconitase 1 ( ACO1 ), and isocitrate dehydrogenase 1 ( IDH1 ). Interestingly, their increased expression was mostly dependent on RTG2 . Impaired respiratory competence in the HAP4 mutant does not affect its faster adaptive response to stress. These findings indicate that the involvement of the RTG pathway in osmostress is fostered in a cellular context of constitutively reduced respiratory capacity. Moreover, it is evident that the RTG pathway mediates peroxisomes-mitochondria communication by modulating the metabolic function of mitochondria in osmoadaptation.

Carbohydrate metabolism plays essential roles in energy generation and providing carbon skeletons for amino acid syntheses. In addition, carbohydrate metabolism has been shown to influence bacterial susceptibility to antibiotics and virulence. In this study, we demonstrate that citrate synthase gltA mutation can increase the expression of the type III secretion system (T3SS) genes and antibiotic tolerance in Pseudomonas aeruginosa. The stringent response is activated in the gltA mutant, and deletion of the (p)ppGpp synthetase gene relA restores the antibiotic tolerance and expression of the T3SS genes to wild-type level. We further demonstrate that the intracellular level of cAMP is increased by the stringent response in the gltA mutant, which increases the expression of the T3SS master regulator gene exsA . Overall, our results reveal an essential role of GltA in metabolism, antibiotic tolerance, and virulence, as well as a novel regulatory mechanism of the stringent response-mediated regulation of the T3SS in P. aeruginosa. IMPORTANCE Rising antimicrobial resistance imposes a severe threat to human health. It is urgent to develop novel antimicrobial strategies by understanding bacterial regulation of virulence and antimicrobial resistance determinants. The stringent response plays an essential role in virulence and antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. The bacterium produces an arsenal of virulence factors and is highly resistant to a variety of antibiotics. In this study, we provide evidence that citrate synthase GltA plays a critical role in P. aeruginosa metabolism and influences the antibiotic tolerance and virulence. We further reveal a role of the stringent response in the regulation of the antibiotic tolerance and virulence. The significance of this work is in elucidation of novel regulatory pathways that control both antibiotic tolerance and virulence in P. aeruginosa.

Purpose: Angiotensin-converting enzyme (ACE) inhibitor treatment is widely applied, but the fact that plasma ACE activity is a potential determinant of training-induced local muscular adaptability is often neglected. Thus, we investigated the hypothesis that ACE inhibition modulates the response to systematic aerobic exercise training on leg and arm muscular adaptations., Methods: Healthy, untrained, middle-aged participants (40 ± 7 yrs) completed a randomized, double-blinded, placebo-controlled trial. Participants were randomized to placebo (PLA: CaCO 3 ) or ACE inhibitor (ACE i : enalapril) for 8 weeks and completed a supervised, high-intensity exercise training program. Muscular characteristics in the leg and arm were extensively evaluated pre and post-intervention., Results: Forty-eight participants (n ACEi  = 23, n PLA  = 25) completed the trial. Exercise training compliance was above 99%. After training, citrate synthase, 3-hydroxyacyl-CoA dehydrogenase and phosphofructokinase maximal activity were increased in m. vastus lateralis in both groups (all P < 0.05) without statistical differences between them (all time × treatment P > 0.05). In m. deltoideus, citrate synthase maximal activity was upregulated to a greater extent (time × treatment P < 0.05) in PLA (51 [33;69] %) than in ACE i (28 [13;43] %), but the change in 3-hydroxyacyl-CoA dehydrogenase and phosphofructokinase maximal activity was similar between groups. Finally, the training-induced changes in the platelet endothelial cell adhesion molecule-1 protein abundance, a marker of capillary density, were similar in both groups in m. vastus lateralis and m. deltoideus., Conclusion: Eight weeks of high-intensity whole-body exercise training improves markers of skeletal muscle mitochondrial oxidative capacity, glycolytic capacity and angiogenesis, with no overall effect of pharmacological ACE inhibition in healthy adults., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The underlying mechanism of the role of mitochondria in color changing of tilapia fillet during 0-4 d storage is not completely clear. A total of 209 differentially significant expressed proteins (DSEPs) were identified by using label-free mitochondrial proteomics, with 56 proteins up-regulated in T2 and 61 proteins (up-regulated) in T3. Protein-Protein interaction reveled proteins which participate in TCA cycles (Citrate synthase (cs)), Oxidoreductase (Malate dehydrogenase (mdh1, mdh2), Succinyl-CoA (Oxct1), Hydroxyacyl-coenzyme a dehydrogenase (hadh), Dehydrogenase/reductase (SDR family) member 1 (dhrs1)) interacted strongly with each other. In turn, they can increase the level of mitochondrial respiration and mitochondrial function, leading to color changing of tilapia fillet. The heat shock 60kD protein 1 (chaperonin, hspd1) interacted with metabolic enzymes (cs and mdh2) and had important effects on color. These results could help researchers better understand the color changing mechanism on the surface of tilapia fillet during the storage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Citrate synthase is a key mitochondrial enzyme that utilizes acetyl-CoA and oxaloacetate to form citrate in the mitochondrial membrane, which participates in energy production in the TCA cycle and linked to the electron transport chain. Citrate transports through a citrate malate pump and synthesizes acetyl-CoA and acetylcholine (ACh) in neuronal cytoplasm. In a mature brain, acetyl-CoA is mainly utilized for ACh synthesis and is responsible for memory and cognition. Studies have shown low citrate synthase in different regions of brain in Alzheimer's disease (AD) patients, which reduces mitochondrial citrate, cellular bioenergetics, neurocytoplasmic citrate, acetyl-CoA, and ACh synthesis. Reduced citrate mediated low energy favors amyloid-β (Aβ) aggregation. Citrate inhibits Aβ25-35 and Aβ1-40 aggregation in vitro. Hence, citrate can be a better therapeutic option for AD by improving cellular energy and ACh synthesis, and inhibiting Aβ aggregation, which prevents tau hyperphosphorylation and glycogen synthase kinase-3 beta. Therefore, we need clinical studies if citrate reverses Aβ deposition by balancing mitochondrial energy pathway and neurocytoplasmic ACh production. Furthermore, in AD's silent phase pathophysiology, when neuronal cells are highly active, they shift ATP utilization from oxidative phosphorylation to glycolysis and prevent excessive generation of hydrogen peroxide and reactive oxygen species (oxidative stress) as neuroprotective action, which upregulates glucose transporter-3 (GLUT3) and pyruvate dehydrogenase kinase-3 (PDK3). PDK3 inhibits pyruvate dehydrogenase, which decreases mitochondrial-acetyl-CoA, citrate, and cellular bioenergetics, and decreases neurocytoplasmic citrate, acetyl-CoA, and ACh formation, thus initiating AD pathophysiology. Therefore, GLUT3 and PDK3 can be biomarkers for silent phase of AD.

Exercise training can increase both mitochondrial content and mitochondrial respiration. Despite its popularity, high-intensity exercise can be accompanied by mild acidosis (also present in certain pathological states), which may limit exercise-induced adaptations to skeletal muscle mitochondria. The aim of this study was to determine if administration of ammonium chloride (0.05 g/kg) to Wistar rats before each individual exercise session (5 high-intensity exercise sessions/wk for 8 wk) reduced training-induced increases in mitochondrial content (measured by citrate synthase activity and protein content of electron transport system complexes) and respiration (measured in permeabilized muscle fibers). In the soleus muscle, the exercise-training-induced increase in mitochondrial respiration was reduced in rats administered ammonium chloride compared to control animals, but mitochondrial content was not altered. These effects were not present in the white gastrocnemius muscle. In conclusion, ammonium chloride administration before each exercise session over 8 wk reduced improvements in mitochondrial respiration in the soleus muscle but did not alter mitochondrial content. This suggests that mild acidosis may affect training-induced improvements in the respiration of mitochondria in some muscles.

The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive under diverse environmental conditions and ecological niches; yet, little is known about the central metabolic pathways that sustain its growth during infection or precisely how these pathways operate. In this work, we used a combination of 'omics approaches (transcriptomics, proteomics, metabolomics, and 13 C-fluxomics) and reverse genetics to provide systems-level insight into how the infection-relevant organic acids succinate and propionate are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (2-MCS; PrpC) and its paralogue citrate (CIT) synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then derepresses prpC expression. Our findings provide a clear example of how "underground metabolism," facilitated by enzyme substrate promiscuity, "rewires" Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that, due to its unrivalled resistance to antibiotics, ubiquity in the built environment, and aggressiveness in infection scenarios, has acquired the somewhat dubious accolade of being designated a "critical priority pathogen" by the WHO. In this work, we uncover the pathways and mechanisms used by P. aeruginosa to grow on a substrate that is abundant at many infection sites: propionate. We found that if the organism is prevented from metabolizing propionate, the substrate turns from being a convenient nutrient source into a potent poison, preventing bacterial growth. We further show that one of the enzymes involved in these reactions, 2-methylcitrate synthase (PrpC), is promiscuous and can moonlight for another essential enzyme in the cell (citrate synthase). Indeed, mutations that abolish citrate synthase activity (which would normally prevent the cell from growing) can be readily overcome if the cell acquires additional mutations that increase the expression of PrpC. This is a nice example of the evolutionary utility of so-called "underground metabolism."

Notothenioidei fishes have evolved under stable cold temperatures; however, ocean conditions are changing globally, with polar regions poised to experience the greatest changes in environmental factors, such as warming. These stressors have the potential to dramatically affect energetic demands, and the persistence of the notothenioids will be dependent on metabolic capacity, or the ability to match energy supply with energy demand, to restore homeostasis in the face of changing climate conditions. In this study we examined aerobic metabolic capacity in three species, Trematomus bernacchii, T. pennellii and T. newnesi, and between two life stages, juvenile and adult, by assessing mitochondrial function of permeabilized cardiac fibers. Respiratory capacity differed among the adult notothenioids in this study, with greater oxidative phosphorylation (OXPHOS) respiration in the pelagic T. newnesi than the benthic T. bernacchii and T. pennellii. The variation in mitochondrial respiratory capacity was likely driven by differences in the mitochondrial content, as measured by citrate synthase activity, which was the highest in T. newnesi. In addition to high OXPHOS, T. newnesi exhibited lower LEAK respiration, resulting in greater mitochondrial efficiency than either T. bernacchii or T. pennellii. Life stage largely had an effect on mitochondrial efficiency and excess complex IV capacity, but there were little differences in OXPHOS respiration and electron transfer capacity, pointing to a lack of significant differences in the metabolic capacity between juveniles and adults. Overall, these results demonstrate species-specific differences in cardiac metabolic capacity, which may influence the acclimation potential of notothenioid fishes to changing environmental conditions., (© 2022. The Author(s).)

Aims: Iron (Fe) deficiency in soil is a continuing problem for soybean (Glycine max L.) production, partly as a result of continuing climate change. This study elucidates how Trichoderma harzianum strain T22 (TH) mitigates growth retardation associated with Fe-deficiency in a highly sensitive soybean cultivar., Methods and Results: Soil TH supplementation led to mycelial colonization and the presence of UAOX1 gene in roots that caused substantial improvement in chlorophyll score, photosynthetic efficiency and morphological parameters, indicating a positive influence on soybean health. Although rhizosphere acidification was found to be a common feature of Fe-deficient soybean, the upregulation of Fe-reductase activity (GmFRO2) and total phenol secretion were two of the mechanisms that substantially increased the Fe availability by TH. Heat-killed TH applied to soil caused no improvement in photosynthetic attributes and Fe-reductase activity, confirming the active role of TH in mitigating Fe-deficiency. Consistent increases in tissue Fe content and increased Fe-transporter (GmIRT1, GmNRAMP2a, GmNRAMP2b and GmNRAMP7) mRNA levels in roots following TH supplementation were observed only under Fe-deprivation. Root cell death, electrolyte leakage, superoxide (O 2 •- ) and hydrogen peroxide (H 2 O 2 ) substantially declined due to TH in Fe-deprived plants. Further, the elevation of citrate and malate concentration along with the expression of citrate synthase (GmCs) and malate synthase (GmMs) caused by TH suggest improved chelation of Fe in Fe-deficient plants. Results also suggest that TH has a role in triggering antioxidant defence by increasing the activity of glutathione reductase (GR) along with elevated S-metabolites (glutathione and methionine) to stabilize redox status under Fe-deficiency., Conclusions: TH increases the availability and mobilization of Fe by inducing Fe-uptake pathways, which appears to help provide resistance to oxidative stress associated with Fe-shortage in soybean., Significance and Impact of the Study: These findings indicate that while Fe deficiency does not affect the rate or degree of TH hyphal association in soybean roots, the beneficial effects of TH alone may be Fe deficiency-dependent., (© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one billion individuals and may be present in approximately 25% of the population globally. NAFLD is viewed as a hepatic manifestation of metabolic syndrome, with humans and animal models presenting dyslipidemia, hypertension, and diabetes. The gut-liver axis has been considered the main pathogenesis branch for NAFLD development. Considering that foods or beverages could modulate the gastrointestinal tract, immune system, energy homeostasis regulation, and even the gut-liver axis, we conducted an exploratory study to analyze the effects of kombucha probiotic on hepatic steatosis, glucose tolerance, and hepatic enzymes involved in carbohydrate and fat metabolism using a pre-clinical model. The diet-induced obese mice presented glucose intolerance, hyperinsulinemia, hepatic steatosis, increased collagen fiber deposition in liver vascular spaces, and upregulated TNF-alpha and SREBP-1 gene expression. Mice receiving the kombucha supplement displayed improved glucose tolerance, reduced hyperinsulinemia, decreased citrate synthase and phosphofructokinase-1 enzyme activities, downregulated G-protein-coupled bile acid receptor, also known as TGR5, and farnesol X receptor gene expression, and attenuated steatosis and hepatic collagen fiber deposition. The improvement in glucose tolerance was accompanied by the recovery of acute insulin-induced liver AKT serine phosphorylation. Thus, it is possible to conclude that this probiotic drink has a beneficial effect in reducing the metabolic alterations associated with diet-induced obesity. This probiotic beverage deserves an extension of studies to confirm or refute its potentially beneficial effects., Competing Interests: Conflict of interest statement The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Masson SAS.)

Histone deacetylase 4/5 (HDAC4/5) are essential for regulating metabolic gene expression; AMPKα2 regulates HDAC4/5 activity and the expression of MuRF1 during exercise. In this study, we used wild-type and AMPKα2 -/- mice to explore the potential regulatory relationship between AMPKα2 and HDAC4/5 expression during exercise. Firstly, we fed C57BL/6J mice with high-fat diet for 8 weeks to assess the effects of high-fat diet on skeletal muscle metabolism and HDAC4/5 expression. We then performed a 6-week treadmill exercise on both wild-type and AMPKα2 -/- mice. After exercise, the expressions of HDAC4/5 were examined in both gastrocnemius and soleus. The citrate synthase activity and proteins involved in skeletal muscle oxidative process were assessed. To determine the relationship of HDAC4/5 and skeletal muscle oxidative capacity, citrate synthase activity was assessed after silencing HDAC4/5. Moreover, HDAC5 ubiquitination and the association of MuRF1 to HDAC5 were also investigated. Our results showed that 6-week exercise increased the skeletal muscle oxidative capacity and decreased HDAC4/5 expression only in soleus. HDAC5 silencing increased C2C12 cell oxidative capacity. Proteasome inhibition by MG132 abolished exercise-induced HDAC5 degradation mediated by MuRF1-ubiquitin-proteasome system. However, the ubiquitin-proteasome system (UPS) did not dominantly account for exercise-induced HDAC4 degradation. Exercise upregulated MuRF1-HDAC5 association in wild-type mice but not in AMPKα2 -/- mice. Our results revealed that 6-week exercise increased the skeletal muscle oxidative capacity and promoted HDAC5 degradation in soleus through the UPS, MuRF1-mediated HDAC5 ubiquitination. Although AMPKα2 played a partial role in regulating MuRF1 expression and HDAC5 ubiquitination, exercise-induced HDAC5 degradation did not fully depend on AMPKα2.

Selective breeding and discipline specific training has led to equine breeds adept at various athletic disciplines. Breed-specific skeletal muscle adaptations have been studied in many breeds but not Warmbloods (WB). We evaluated gluteal muscle contractile muscle fiber types and citrate synthase activity (CS), a marker for mitochondrial volume density, in WB trained for dressage (second level-Grand Prix) contrasted with Quarter Horses (QH). Gluteus medius muscle biopsies from 14 unfit/18 fit dressage-trained WB and 20 unfit/16 fit reining/working cow QH were analyzed fluorometrically and fiber types determined by ATPase activity. Comparisons were made by one-way ANOVA. Unfit and fit WB had significantly higher % type 1 and lower % type 2X fibers than QH. Unfit WB had significantly higher CS than unfit QH but CS did not differ between fit WB and fit QH. CS was only significantly higher in fit versus unfit QH, not fit versus unfit WB. In conclusion, WB gluteal muscle has an inherently high % type 1/low % type 2X fibers and high mitochondrial content whether unfit or trained for dressage, contrasting QH with an inherently low % type 1/high % type 2X and low mitochondrial content, that was enhanced in fit QH. Similar CS activity in fit WB versus QH despite a two-fold difference in % type 2X fibers indicates that mitochondrial volume density cannot accurately be predicted from contractile fiber type composition., Competing Interests: Declaration of Competing Interest None, (Copyright © 2022. Published by Elsevier Inc.)