Effects of endurance training under calorie restriction on energy substrate metabolism in mouse skeletal muscle and liver.

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.

Physiological and muscle histochemical assessment of men and women 10 km runners matched for race performance or training volume.

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 V̇o2max 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 %HRmax (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 V̇o2max 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% HRmax zone may explain the higher % PTS and %HRmax 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, V̇o2max, 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.

Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions.

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.

HBV suppresses macrophage immune responses by impairing the TCA cycle through the induction of CS/PDHC hyperacetylation.

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.

Tissue Expressions of Regulatory Enzymes of the Krebs Cycle in Low- and High-grade Gliomas.

AIM: To compare tissue levels of the regulatory enzymes related to the Krebs cycle between low, and high-grade supratentorial gliomas. MATERIAL AND METHODS: Forty patients who underwent surgery for supratentorial gliomas (19 with low-grade and 21 with high-grade gliomas) were evaluated. The regulatory enzymes directly involved in the Krebs cycle, namely pyruvate dehydrogenase, citrate synthase, ?-ketoglutarate dehydrogenase, and isocitrate dehydrogenase, and two enzymes that indirectly regulate the Krebs cycle, namely glutamate dehydrogenase and glutaminase, were quantitatively studied in tumor tissues using ELISA. The results were compared between the two groups. RESULTS: The levels of all enzymes were higher in the high-grade glioma group but only pyruvate dehydrogenase, citrate synthase, and isocitrate dehydrogenase levels showed statistical significance. Moreover, all enzymes showed higher tissue levels in grade- II compared to grade-I gliomas, but only two enzymes, glutamate dehydrogenase and glutaminase, reached significantly higher levels. In the high-grade glioma group, all enzymes again showed higher tissue levels in grade-IV gliomas than in grade-III gliomas, but none showed statistical significance. CONCLUSION: Regulatory enzymes of the Krebs cycle are increased in high-grade gliomas compared to low-grade gliomas. Glutaminolysis enzymes, namely glutamate dehydrogenase and glutaminase, which are required for resupplying the Krebs cycle, are also increased in order to meet the high energy demand in high-grade gliomas.

Dapagliflozin Attenuates Heart Failure With Preserved Ejection Fraction Remodeling and Dysfunction by Elevating ß-Hydroxybutyrate-activated Citrate Synthase.

ABSTRACT: Heart failure with preserved ejection fraction (HFpEF) is highly prevalent, accounting for 50% of all heart failure patients, and is associated with significant mortality. Sodium-glucose cotransporter subtype inhibitor (SGLT2i) is recommended in the AHA and ESC guidelines for the treatment of HFpEF, but the mechanism of SGLT2i to prevent and treat cardiac remodeling and dysfunction is currently unknown, hindering the understanding of the pathophysiology of HFpEF and the development of novel therapeutics. HFpEF model was induced by a high-fat diet (60% calories from lard) + N [w] -nitro- l -arginine methyl ester ( l -NAME-0.5 g/L) (2 Hit) in male Sprague Dawley rats to effectively recapture the myriad phenotype of HFpEF. This study's results showed that administration of dapagliflozin (DAPA, SGLT2 inhibitor) significantly limited the 2-Hit-induced cardiomyocyte hypertrophy, apoptosis, inflammation, oxidative stress, and fibrosis. It also improved cardiac diastolic and systolic dysfunction in a late-stage progression of HFpEF. Mechanistically, DAPA influences energy metabolism associated with fatty acid intake and mitochondrial dysfunction in HFpEF by increasing ß-hydroxybutyric acid (ß-OHB) levels, directing the activation of citrate synthase, reducing acetyl coenzyme A (acetyl-CoA) pools, modulating adenosine 5'-triphosphate production, and increasing the expression of mitochondrial oxidative phosphorylation system complexes I-V. In addition, following clinical DAPA therapy, the blood levels of ß-OHB and citrate synthase increased and the levels of acetyl-CoA in the blood of HFpEF patients decreased. SGLT2i plays a beneficial role in the prevention and treatment of cardiac remodeling and dysfunction in HFpEF model by attenuating cardiometabolic dysregulation.

Novel chemical scaffold as potential drug against Leishmania donovani: Integrated computational and experimental approaches.

In this study, we have screened a large number of Food and Drug Administration-approved compounds for novel anti-leishmanial molecules targeting the citrate synthase enzyme of the parasite. Based on their docking and molecular dynamic simulation statistics, five compounds were selected. These compounds followed Lipinski's rule of five. Additionally, in vitro, antileishmanial and cytotoxicity studies were performed. The three compounds, Abemaciclib, Bazedoxifene, and Vorapaxar, had shown effective anti-leishmanial activities with IC50 values of 0.92 ± 0.02, 0.65 ± 0.09, and 6.1 ± 0.91 against Leishmania donovani promastigote and with EC50 values of 1.52 ± 0.37, 2.11 ± 0.38, 10.4 ± 1.27 against intramacrophagic amastigote without significantly harming macrophage cells. Among them, from in silico and antileishmanial activities studies, Abemaciclib had been selected based on their less binding energy, good antileishmanial activities, and also a significant difference in their binding energy with human citrate synthase for cell death mechanistic studies using flow cytometry and a DNA fragmentation assay. The action of this compound resulted in an increased reactive oxygen species production, depolarization of mitochondrial membrane potential, DNA damage, and an increase in the sub-G1 cell population. These properties are the hallmarks of apoptosis which were further confirmed by apoptotic assay. Based on the above result, this anticancer compound Abemaciclib could be employed as a potential treatment option for leishmaniasis after further confirmation.

Bariatric surgery alters mitochondrial function in gut mucosa.

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.

The implications of exercise in Drosophila melanogaster: insights into Akt/p38 MAPK/Nrf2 pathway associated with Hsp70 regulation in redox balance maintenance.

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.

Hepatic citrate synthase suppression in the freeze-tolerant wood frog (Rana sylvatica).

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 Vmax 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.

Aerobic exercise training-induced alteration of gut microbiota composition affects endurance capacity.

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.

MAEL facilitates metabolic reprogramming and breast cancer progression by promoting the degradation of citrate synthase and fumarate hydratase via chaperone-mediated autophagy.

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 NH4 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.

Decreased propionyl-CoA metabolism facilitates metabolic reprogramming and promotes hepatocellular carcinoma.

BACKGROUND & AIMS: Alterations of multiple metabolites characterize distinct features of metabolic reprograming in hepatocellular carcinoma (HCC). However, the role of most metabolites, including propionyl-CoA (Pro-CoA), in metabolic reprogramming and hepatocarcinogenesis remains elusive. In this study, we aimed to dissect how Pro-CoA metabolism affects these processes. METHODS: TCGA data and HCC samples were used to analyze ALDH6A1-mediated Pro-CoA metabolism and its correlation with HCC. Multiple metabolites were assayed by targeted mass spectrometry. The role of ALDH6A1-generated Pro-CoA in HCC was evaluated in HCC cell lines as well as xenograft nude mouse models and primary liver cancer mouse models. Non-targeted metabolomic and targeted energy metabolomic analyses, as well as multiple biochemical assays, were performed. RESULTS: Decreases in Pro-CoA and its derivative propionyl-L-carnitine due to ALDH6A1 downregulation were tightly associated with HCC. Functionally, ALDH6A1-mediated Pro-CoA metabolism suppressed HCC proliferation in vitro and impaired hepatocarcinogenesis in mice. The aldehyde dehydrogenase activity was indispensable for this function of ALDH6A1, while Pro-CoA carboxylases antagonized ALDH6A1 function by eliminating Pro-CoA. Mechanistically, ALDH6A1 caused a signature enrichment of central carbon metabolism in cancer and impaired energy metabolism: ALDH6A1-generated Pro-CoA suppressed citrate synthase activity, which subsequently reduced tricarboxylic acid cycle flux, impaired mitochondrial respiration and membrane potential, and decreased ATP production. Moreover, Pro-CoA metabolism generated 2-methylcitric acid, which mimicked the inhibitory effect of Pro-CoA on citrate synthase and dampened mitochondrial respiration and HCC proliferation. CONCLUSIONS: The decline of ALDH6A1-mediated Pro-CoA metabolism contributes to metabolic remodeling and facilitates hepatocarcinogenesis. Pro-CoA, propionyl-L-carnitine and 2-methylcitric acid may serve as novel metabolic biomarkers for the diagnosis and treatment of HCC. Pro-CoA metabolism may provide potential targets for development of novel strategies against HCC. IMPACT AND IMPLICATIONS: Our study presents new insights on the role of propionyl-CoA metabolism in metabolic reprogramming and hepatocarcinogenesis. This work has uncovered potential diagnostic and predictive biomarkers, which could be used by physicians to improve clinical practice and may also serve as targets for the development of therapeutic strategies against HCC.

Systems-Wide Dissection of Organic Acid Assimilation in Pseudomonas aeruginosa Reveals a Novel Path To Underground Metabolism.

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 13C-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."

Transcriptome and Metabolome Analysis Revealing the Improved ε-Poly-l-Lysine Production Induced by a Microbial Call from Botrytis cinerea.

ε-Poly-l-lysine (ε-PL) is a wide-spectrum antimicrobial agent, while its biosynthesis-inducing signals are rarely reported. This study found that Botrytis cinerea extracts could act as a microbial call to induce a physiological modification of Streptomyces albulus for ε-PL efficient biosynthesis and thereby resulted in ε-PL production (34.2 g/liter) 1.34-fold higher than control. The elicitors could be primary isolated by ethanol and butanol extraction, which resulted in more vibrant, aggregate and stronger mycelia. The elicitor-derived physiological changes focused on three aspects: ε-PL synthase, energy metabolism, and lysine biosynthesis. After elicitor addition, upregulated sigma factor hrdD and improved transcription and expression of pls directly contributed to the high ε-PL productivity; upregulated genes in tricarboxylic acid (TCA) cycle and energy metabolism promoted activities of citrate synthase and the electron transport system; in addition, pool enlargements of ATP, ADP, and NADH guaranteed the ATP provision for ε-PL assembly. Lysine biosynthesis was also increased based on enhancements of gene transcription, key enzyme activities, and intracellular metabolite pools related to carbon source utilization, the Embden-Meyerhof pathway (EMP), the diaminopimelic acid pathway (DAP), and the replenishment pathway. Interestingly, the elicitors stimulated the gene transcription for the quorum-sensing system and resulted in upregulation of genes for other antibiotic production. These results indicated that the Botrytis cinerea could produce inducing signals to change the Streptomyces mycelial physiology and accelerate the ε-PL biosynthesis. IMPORTANCE This work identified the role of microbial elicitors on ε-PL production and disclosed the underlying mechanism through analysis of gene transcription, key enzyme activities, and intracellular metabolite pools, including transcriptome and metabolome analysis. It was the first report for the inducing effects of the "microbial call" to Streptomyces albulus and ε-PL biosynthesis, and these elicitors could be potentially obtained from decayed fruits infected by Botrytis cinerea; hence, this may be a way of turning a biohazard into bioproduct wealth. This study provided a reference for application of microbial signals in secondary metabolite production, which is of theoretical and practical significance in industrial antibiotic production.

Cardiac Mitochondria Dysfunction in Dyslipidemic Mice.

Dyslipidemia triggers many severe pathologies, including atherosclerosis and chronic inflammation. Several lines of evidence, including our studies, have suggested direct effects of dyslipidemia on cardiac energy metabolism, but details of these effects are not clear. This study aimed to investigate how mild dyslipidemia affects cardiac mitochondria function and vascular nucleotide metabolism. The analyses were performed in 3- and 6-month-old knock-out mice for low-density lipoprotein receptor (Ldlr-/-) and compared to wild-type C57Bl/6J mice (WT). Cardiac isolated mitochondria function was analyzed using Seahorse metabolic flux analyzer. The mechanical function of the heart was measured using echocardiography. The levels of fusion, fission, and mitochondrial biogenesis proteins were determined by ELISA kits, while the cardiac intracellular nucleotide concentration and vascular pattern of nucleotide metabolism ecto-enzymes were analyzed using reverse-phase high-performance liquid chromatography. We revealed the downregulation of mitochondrial complex I, together with a decreased activity of citrate synthase (CS), reduced levels of nuclear respiratory factor 1 and mitochondrial fission 1 protein, as well as lower intracellular adenosine and guanosine triphosphates' pool in the hearts of 6-month Ldlr-/- mice vs. age-matched WT. The analysis of vascular ecto-enzyme pattern revealed decreased rate of extracellular adenosine monophosphate hydrolysis and increased ecto-adenosine deaminase activity (eADA) in 6-month Ldlr-/- vs. WT mice. No changes were observed in echocardiography parameters in both age groups of Ldlr-/- mice. Younger hyperlipidemic mice revealed no differences in cardiac mitochondria function, CS activity, intracellular nucleotides, mitochondrial biogenesis, and dynamics but exhibited minor changes in vascular eADA activity vs. WT. This study revealed that dysfunction of cardiac mitochondria develops during prolonged mild hyperlipidemia at the time point corresponding to the formation of early vascular alterations.

Kombucha tea improves glucose tolerance and reduces hepatic steatosis in obese mice.

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.

SARS-CoV-2 infection and replication kinetics in different human cell types: The role of autophagy, cellular metabolism and ACE2 expression.

AIMS: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication. MAIN METHODS: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated. KEY FINDINGS: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. SIGNIFICANCE: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.

Dexamethasone enhances glucose uptake by SGLT1 and GLUT1 and boosts ATP generation through the PPP-TCA cycle in bovine neutrophils.

BACKGROUND: Clinical dexamethasone (DEX) treatment or stress in bovines results in extensive physiological changes with prominent hyperglycemia and neutrophils dysfunction. OBJECTIVES: To elucidate the effects of DEX treatment in vivo on cellular energy status and the underlying mechanism in circulating neutrophils. METHODS: We selected eight-month-old male bovines and injected DEX for 3 consecutive days (1 time/d). The levels of glucose, total protein (TP), total cholesterol (TC), and the proinflammatory cytokines interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α in blood were examined, and we then detected glycogen and adenosine triphosphate (ATP) content, phosphofructosekinase-1 (PFK1) and glucose-6-phosphate dehydrogenase (G6PDH) activity, glucose transporter (GLUT)1, GLUT4, sodium/glucose cotransporter (SGLT)1 and citrate synthase (CS) protein expression and autophagy levels in circulating neutrophils. RESULTS: DEX injection markedly increased blood glucose, TP and TC levels, the Ca2+/P5+ ratio and the neutrophil/lymphocyte ratio and significantly decreased blood IL-1ß, IL-6 and TNF-α levels. Particularly in neutrophils, DEX injection inhibited p65-NFκB activation and elevated glycogen and ATP contents and SGLT1, GLUT1 and GR expression while inhibiting PFK1 activity, enhancing G6PDH activity and CS expression and lowering cell autophagy levels. CONCLUSIONS: DEX induced neutrophils glucose uptake by enhancing SGLT1 and GLUT1 expression and the transformation of energy metabolism from glycolysis to pentose phosphate pathway (PPP)-tricarboxylic acid (TCA) cycle. This finding gives us a new perspective on deeper understanding of clinical anti-inflammatory effects of DEX on bovine.

Transcriptome Sequencing and Metabolome Analysis Reveals the Molecular Mechanism of Drought Stress in Millet.

As one of the oldest agricultural crops in China, millet (Panicum miliaceum) has powerful drought tolerance. In this study, transcriptome and metabolome analyses of 'Hequ Red millet' (HQ) and 'Yanshu No.10' (YS10) millet after 6 h of drought stress were performed. Transcriptome characteristics of drought stress in HQ and YS10 were characterized by Pacbio full-length transcriptome sequencing. The pathway analysis of the differentially expressed genes (DEGs) showed that the highly enriched categories were related to starch and sucrose metabolism, pyruvate metabolism, metabolic pathways, and the biosynthesis of secondary metabolites when the two millet varieties were subjected to drought stress. Under drought stress, 245 genes related to energy metabolism were found to show significant changes between the two strains. Further analysis showed that 219 genes related to plant hormone signal transduction also participated in the drought response. In addition, numerous genes involved in anthocyanin metabolism and photosynthesis were confirmed to be related to drought stress, and these genes showed significant differential expression and played an important role in anthocyanin metabolism and photosynthesis. Moreover, we identified 496 transcription factors related to drought stress, which came from 10 different transcription factor families, such as bHLH, C3H, MYB, and WRKY. Further analysis showed that many key genes related to energy metabolism, such as citrate synthase, isocitrate dehydrogenase, and ATP synthase, showed significant upregulation, and most of the structural genes involved in anthocyanin biosynthesis also showed significant upregulation in both strains. Most genes related to plant hormone signal transduction showed upregulated expression, while many JA and SA signaling pathway-related genes were downregulated. Metabolome analysis was performed on 'Hequ red millet' (HQ) and 'Yanshu 10' (YS10), a total of 2082 differential metabolites (DEMs) were identified. These findings indicate that energy metabolism, anthocyanins, photosynthesis, and plant hormones are closely related to the drought resistance of millet and adapt to adversity by precisely regulating the levels of various molecular pathways.