Metabolic reprogramming of the retinal pigment epithelium by cytokines associated with age-related macular degeneration.

The complex metabolic relationship between the retinal pigment epithelium (RPE) and photoreceptors is essential for maintaining retinal health. Recent evidence indicates the RPE acts as an adjacent lactate sink, suppressing glycolysis in the epithelium in order to maximize glycolysis in the photoreceptors. Dysregulated metabolism within the RPE has been implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of vision loss. In the present study, we investigate the effects of four cytokines associated with AMD, TNFα, TGF-ß2, IL-6, and IL-1ß, as well as a cocktail containing all four cytokines, on RPE metabolism using ARPE-19 cells, primary human RPE cells, and ex vivo rat eyecups. Strikingly, we found cytokine-specific changes in numerous metabolic markers including lactate production, glucose consumption, extracellular acidification rate, and oxygen consumption rate accompanied by increases in total mitochondrial volume and ATP production. Together, all four cytokines could potently override the constitutive suppression of glycolysis in the RPE, through a mechanism independent of PI3K/AKT, MEK/ERK, or NF-κB. Finally, we observed changes in glycolytic gene expression with cytokine treatment, including in lactate dehydrogenase subunit and glucose transporter expression. Our findings provide new insights into the metabolic changes in the RPE under inflammatory conditions and highlight potential therapeutic targets for AMD.

Single-B cell analysis correlates high-lactate secretion with stress and increased apoptosis.

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSRhigh B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSRhigh cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

Large-scale animal model study uncovers altered brain pH and lactate levels as a transdiagnostic endophenotype of neuropsychiatric disorders involving cognitive impairment.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Enhanced glycolysis in the myometrium with ectopic endometrium of patients with adenomyosis: a preliminary study.

OBJECTIVES: The objective of this study was to investigate the glycolytic activity of adenomyosis, which is characterized by malignant biological behaviors including abnormal cell proliferation, migration, invasion, cell regulation, and epithelial-mesenchymal transition. METHODS: From January 2021 to August 2022, a total of 15 patients who underwent total hysterectomy for adenomyosis and 14 patients who had non-endometrial diseases, specifically with cervical squamous intraepithelial neoplasia and uterine myoma, were included in this study. Myometrium with ectopic endometrium from patients with adenomyosis while normal myometrium from patients in the control group were collected. All samples were confirmed by a histopathological examination. The samples were analyzed by liquid chromatography-mass spectrometry (LC-MS), real-time quantitative PCR, NAD+/NADH assay kit as well as the glucose and lactate assay kits. RESULTS: Endometrial stroma and glands could be observed within the myometrium of patients in the adenomyosis group. We found that the mRNA expressions of HK1, PFKFB3, glyceraldehyde-3-phospate dehydrogenase (GAPDH), PKM2, and PDHA as well as the protein expressions of PFKFB3 were elevated in ectopic endometrial tissues of the adenomyosis group as compared to normal myometrium of the control group. The level of fructose 1,6-diphosphate was increased while NAD + and NAD+/NADH ratio were decreased compared with the control group. Besides, increased glucose consumption and lactate production were observed in myometrium with ectopic endometrium. CONCLUSIONS: We concluded that altered glycolytic phenotype of the myometrium with ectopic endometrium in women with adenomyosis may contribute the development of adenomyosis.

A BMP-controlled metabolic/epigenetic signaling cascade directs midfacial morphogenesis.

Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients and are associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that bone morphogenetic protein (BMP) signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53-dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling-dependent lactate generation drove histone lactylation levels to alter essential genes of Pdgfra, thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein BMP signaling controls a metabolic/epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation.

MLKL overexpression leads to Ca2+ and metabolic dyshomeostasis in a neuronal cell model.

The necroptotic effector molecule MLKL accumulates in neurons over the lifespan of mice, and its downregulation has the potential to improve cognition through neuroinflammation, and changes in the abundance of synaptic proteins and enzymes in the central nervous system. Notwithstanding, direct evidence of cell-autonomous effects of MLKL expression on neuronal physiology and metabolism are lacking. Here, we tested whether the overexpression of MLKL in the absence of cell death in the neuronal cell line Neuro-2a recapitulates some of the hallmarks of aging at the cellular level. Using genetically-encoded fluorescent biosensors, we monitored the cytosolic and mitochondrial Ca2+ levels, along with the cytosolic concentrations of several metabolites involved in energy metabolism (lactate, glucose, ATP) and oxidative stress (oxidized/reduced glutathione). We found that MLKL overexpression marginally decreased cell viability, however, it led to reduced cytosolic and mitochondrial Ca2+ elevations in response to Ca2+ influx from the extracellular space. On the contrary, Ca2+ signals were elevated after mobilizing Ca2+ from the endoplasmic reticulum. Transient elevations in cytosolic Ca2+, mimicking neuronal stimulation, lead to higher lactate levels and lower glucose concentrations in Neuro-2a cells when overexpressing MLKL, which suggest enhanced neuronal glycolysis. Despite these alterations, energy levels and glutathione redox state in the cell bodies remained largely preserved after inducing MLKL overexpression for 24-48 h. Taken together, our proof-of-concept experiments are consistent with the hypothesis that MLKL overexpression in the absence of cell death contributes to both Ca2+ and metabolic dyshomeostasis, which are cellular hallmarks of brain aging.

Exosomal or follicular FNDC3A decreases FOLR1 mRNA abundance and progesterone and lactate synthesis in bovine granulosa cells.

In brief: Dairy cattle experience a period of infertility postpartum that is caused in part by the development of IGF1/insulin resistance. This study suggests that an adipokine, FNDC3A, reduces IGF1-dependent glycolysis and may contribute to postpartum infertility. Abstract: Dairy cows go through a period of subfertility after parturition, triggered in part by a disruption of energy homeostasis. The mobilization of body fat alters the secretion of adipokines, which have been shown to impact ovarian function. Fibronectin type III domain-containing 3A (FNDC3A) is a recently discovered adipokine-myokine, and FNDC3A mRNA abundance in subcutaneous adipose tissue is increased postpartum in cattle. In this study, we hypothesized that FNDC3A may compromise granulosa cell function in cattle and investigated this using a well-established in vitro cell culture model. Here, we demonstrate the presence of FNDC3A protein associated with extracellular vesicles in follicular fluid and in plasma, suggesting an endocrine role for this adipokine. FNDC3A protein and mRNA was also detected in the bovine ovary (cortex, granulosa and theca cells, cumulus, oocyte and corpus luteum). Abundance of FNDC3A mRNA in granulosa cells from small follicles was increased by in vitro treatment with the adipokines leptin and TNF but not by visfatin, resistin, adiponectin, chemerin or IGF1. Addition of recombinant FNDC3A at physiological doses (10 ng/mL) to granulosa cells decreased IGF1-dependent progesterone but not estradiol secretion and IGF1-dependent lactate secretion and abundance of GLUT3 and GLUT4 mRNA. This concentration of FNDC3A increased cell viability, abundance of mRNA encoding a putative receptor FOLR1, and increased phosphorylation of Akt. Collectively, these data suggest that FNDC3A may regulate folliculogenesis in cattle by modulating IGF1-dependent granulosa cell steroidogenesis and glucose metabolism.

Cold Ambient Temperature Does Not Alter Subcutaneous Abdominal Adipose Tissue Lipolysis and Blood Flow in Endurance-Trained Cyclists.

This study sought to investigate the effect of cold ambient temperature on subcutaneous abdominal adipose tissue (SCAAT) lipolysis and blood flow during steady-state endurance exercise in endurance-trained cyclists. Ten males (age: 23 ± 3 years; peak oxygen consumption: 60.60 ± 4.84 ml·kg-1·min-1; body fat: 18.4% ± 3.5%) participated in baseline lactate threshold (LT) and peak oxygen consumption testing, two familiarization trials, and two experimental trials. Experimental trials consisted of cycling in COLD (3 °C; 42% relative humidity) and neutral (NEU; 19 °C; 39% relative humidity) temperatures. Exercise consisted of 25 min cycling at 70% LT and 25 min at 90% LT. In situ SCAAT lipolysis and blood flow were measured via microdialysis. Heart rate, core temperature, carbohydrate and fat oxidation, blood glucose, and blood lactate were also measured. Heart rate, core temperature, oxygen consumption, and blood lactate increased with exercise but were not different between COLD and NEU. SCAAT blood flow did not change from rest to exercise or between COLD and NEU. Interstitial glycerol increased during exercise (p < .001) with no difference between COLD and NEU. Fat oxidation increased (p < .001) at the onset of exercise and remained elevated thereafter with no difference between COLD and NEU. Carbohydrate oxidation increased with increasing exercise intensity and was greater at 70% LT in COLD compared to NEU (p = .030). No differences were observed between conditions for any other variable. Cycling exercise increased SCAAT lipolysis but not blood flow. Ambient temperature did not alter SCAAT metabolism, SCAAT blood flow, or fat oxidation in well-trained cyclists, though cold exposure increased whole-body carbohydrate oxidation at lower exercise intensities.

Disruption of hepatic mitochondrial pyruvate and amino acid metabolism impairs gluconeogenesis and endurance exercise capacity in mice.

Exercise robustly increases the glucose demands of skeletal muscle. This demand is met by not only muscle glycogenolysis but also accelerated liver glucose production from hepatic glycogenolysis and gluconeogenesis to fuel mechanical work and prevent hypoglycemia during exercise. Hepatic gluconeogenesis during exercise is dependent on highly coordinated responses within and between muscle and liver. Specifically, exercise increases the rate at which gluconeogenic precursors such as pyruvate/lactate or amino acids are delivered from muscle to the liver, extracted by the liver, and channeled into glucose. Herein, we examined the effects of interrupting hepatic gluconeogenic efficiency and capacity on exercise performance by deleting mitochondrial pyruvate carrier 2 (MPC2) and/or alanine transaminase 2 (ALT2) in the liver of mice. We found that deletion of MPC2 or ALT2 alone did not significantly affect time to exhaustion or postexercise glucose concentrations in treadmill exercise tests, but mice lacking both MPC2 and ALT2 in hepatocytes (double knockout, DKO) reached exhaustion faster and exhibited lower circulating glucose during and after exercise. Use of 2H/1³C metabolic flux analyses demonstrated that DKO mice exhibited lower endogenous glucose production owing to decreased glycogenolysis and gluconeogenesis at rest and during exercise. Decreased gluconeogenesis was accompanied by lower anaplerotic, cataplerotic, and TCA cycle fluxes. Collectively, these findings demonstrate that the transition of the liver to the gluconeogenic mode is critical for preventing hypoglycemia and sustaining performance during exercise. The results also illustrate the need for interorgan cross talk during exercise as described by the Cahill and Cori cycles.NEW & NOTEWORTHY Martino and colleagues examined the effects of inhibiting hepatic gluconeogenesis on exercise performance and systemic metabolism during treadmill exercise in mice. Combined inhibition of gluconeogenesis from lactate/pyruvate and alanine impaired exercise endurance and led to hypoglycemia during and after exercise. In contrast, suppressing either pyruvate-mediated or alanine-mediated gluconeogenesis alone had no effect on these parameters. These findings provide new insight into the molecular nodes that coordinate the metabolic responses of muscle and liver during exercise.

Comparative study of 1H-NMR metabolomic profile of canine synovial fluid in patients affected by four progressive stages of spontaneous osteoarthritis.

The study aimed to assess the metabolomic profile of the synovial fluid (SF) of dogs affected by spontaneous osteoarthritis (OA) and compare any differences based on disease progression. Sixty client-owned dogs affected by spontaneous OA underwent clinical, radiographic, and cytologic evaluations to confirm the diagnosis. The affected joints were divided into four study groups based on the Kallgreen-Lawrence classification: OA1 (mild), OA2 (moderate), OA3 (severe), and OA4 (extremely severe/deforming). The osteoarthritic joint's SF was subjected to cytologic examination and 1H-NMR analysis. The metabolomic profiles of the study groups' SF samples were statistically compared using one-way ANOVA. Sixty osteoarthritic joints (45 stifles, 10 shoulders and 5 elbows) were included in the study. Fourteen, 28, and 18 joints were included in the OA1, OA2, and OA3 groups, respectively (0 joints in the OA4 group). Metabolomic analysis identified 48 metabolites, five of which were significantly different between study groups: Mannose and betaine were elevated in the OA1 group compared with the OA2 group, and the 2-hydroxyisobutyrate concentration decreased with OA progression; in contrast, isoleucine was less concentrated in mild vs. moderate OA, and lactate increased in severe OA. This study identified different 1H-NMR metabolomic profiles of canine SF in patients with progressive degrees of spontaneous OA, suggesting 1H-NMR metabolomic analysis as a potential alternative method for monitoring OA progression. In addition, the results suggest the therapeutic potentials of the metabolomic pathways that involve mannose, betaine, 2-hydroxyisobutyrate, isoleucine, and lactate.

The role of GPR81-cAMP-PKA pathway in endurance training-induced intramuscular triglyceride accumulation and mitochondrial content changes in rats.

The athlete's paradox phenomenon involves the accumulation of intramuscular triglycerides (IMTG) in both insulin-resistant and insulin-sensitive endurance athletes. Nevertheless, a complete understanding of this phenomenon is yet to be achieved. Recent research indicates that lactate, a common byproduct of physical activity, may increase the accumulation of IMTG in skeletal muscle. This is achieved through the activation of G protein-coupled receptor 81 (GPR81) leads to the suppression of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway. The mechanism accountable for the increase in mitochondrial content in skeletal muscle triggered by lactate remains incomprehensible. Based on current research, our objective is to explore the role of the GPR81-inhibited cAMP-PKA pathway in the aggregation of IMTG and the increase in mitochondrial content as a result of prolonged exercise. The GPR81-cAMP-PKA-signaling pathway regulates the buildup of IMTG caused by extended periods of endurance training (ET). This is likely due to a decrease in proteins related to fat breakdown and an increase in proteins responsible for fat production. It is possible that the GPR81-cAMP-PKA pathway does not contribute to the long-term increase in mitochondrial biogenesis and content, which is induced by chronic ET. Additional investigation is required to explore the possible hindrance of the mitochondrial biogenesis and content process during physical activity by the GPR81-cAMP-PKA signal.

A genome-reduced Corynebacterium glutamicum derivative discloses a hidden pathway relevant for 1,2-propanediol production.

BACKGROUND: 1,2-propanediol (1,2-PDO) is widely used in the cosmetic, food, and drug industries with a worldwide consumption of over 1.5 million metric tons per year. Although efforts have been made to engineer microbial hosts such as Corynebacterium glutamicum to produce 1,2-PDO from renewable resources, the performance of such strains is still improvable to be competitive with existing petrochemical production routes. RESULTS: In this study, we enabled 1,2-PDO production in the genome-reduced strain C. glutamicum PC2 by introducing previously described modifications. The resulting strain showed reduced product formation but secreted 50 ± 1 mM D-lactate as byproduct. C. glutamicum PC2 lacks the D-lactate dehydrogenase which pointed to a yet unknown pathway relevant for 1,2-PDO production. Further analysis indicated that in C. glutamicum methylglyoxal, the precursor for 1,2-PDO synthesis, is detoxified with the antioxidant native mycothiol (MSH) by a glyoxalase-like system to lactoylmycothiol and converted to D-lactate which is rerouted into the central carbon metabolism at the level of pyruvate. Metabolomics of cell extracts of the empty vector-carrying wildtype, a 1,2-PDO producer and its derivative with inactive D-lactate dehydrogenase identified major mass peaks characteristic for lactoylmycothiol and its precursors MSH and glucosaminyl-myo-inositol, whereas the respective mass peaks were absent in a production strain with inactivated MSH synthesis. Deletion of mshA, encoding MSH synthase, in the 1,2-PDO producing strain C. glutamicum ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) improved the product yield by 56% to 0.53 ± 0.01 mM1,2-PDO mMglucose-1 which is the highest value for C. glutamicum reported so far. CONCLUSIONS: Genome reduced-strains are a useful basis to unravel metabolic constraints for strain engineering and disclosed in this study the pathway to detoxify methylglyoxal which represents a precursor for 1,2-PDO production. Subsequent inactivation of the competing pathway significantly improved the 1,2-PDO yield.

OPTIMIZATION OF EXPERIMENTAL MODEL SYSTEMS FOR EVALUATING RECIPROCAL INFLUENCE OF BIFIDOBACTERIUM ANIMALIS AND HUMAN BREAST CANCER CELLS IN VITRO.

BACKGROUND: The development of human breast cancer (BC) is known to be closely related to disturbances in the mammary gland microbiota. Bacteria of the genus Bifidobacterium are an important component of normal breast microbiota and exert antitumor activity. The molecular-biological mechanisms of interaction between BC cells and microbiota members remain poorly studied yet. The aim of this study was to develop and optimize an experimental model system for the co-cultivation of BC cells with Bifidobacterium animalis in vitro. MATERIALS AND METHODS: Human ВС cells of the MCF-7, T47D, and MDA-MB-231 lines, as well as live and heat-inactivated bacteria of Bifidobacterium animalis subsp. lactis (B. animalis) were used as research objects. The growth kinetics and viability of B. animalis in the presence of different ВС cell lines and without them were determined by both the turbidimetry method and seeding on an elective nutrient medium. Glucose consumption and lactate production by bifidobacteria were assessed by biochemical methods. The viability of BC cells was determined by a standard colorimetric method. RESULTS: The growth kinetics of B. animalis in the complete DMEM nutrient medium showed standard patterns. The indicators of glucose consumption and lactate production of B. animalis confirm its physiological metabolic activity under the growth conditions. The presence of BC cells in the model system did not affect the duration of the growth phases of the B. animalis cells' population but contributed to the increase in their counts. A significant decrease in the number of live BC cells of all studied lines was observed only after 48 h of co-cultivation with live B. animalis. To achieve similar suppression of the BC cell viability, 10-30-fold higher counts of heatinactivated bacteria were required compared to live ones. CONCLUSIONS: The optimal conditions for co-cultivation of human BC cells and living B. animalis cells in vitro have been identified.

Harnessing Fermentation May Enhance the Performance of Biological Sulfate-Reducing Bioreactors.

Biological sulfate reduction (BSR) represents a promising strategy for bioremediation of sulfate-rich waste streams, yet the impact of metabolic interactions on performance is largely unexplored. Here, genome-resolved metagenomics was used to characterize 17 microbial communities in reactors treating synthetic sulfate-contaminated solutions. Reactors were supplemented with lactate or acetate and a small amount of fermentable substrate. Of the 163 genomes representing all the abundant bacteria, 130 encode 321 NiFe and FeFe hydrogenases and all genomes of the 22 sulfate-reducing microorganisms (SRM) encode genes for H2 uptake. We observed lactate oxidation solely in the first packed bed reactor zone, with propionate and acetate oxidation in the middle and predominantly acetate oxidation in the effluent zone. The energetics of these reactions are very different, yet sulfate reduction kinetics were unaffected by the type of electron donor available. We hypothesize that the comparable rates, despite the typically slow growth of SRM on acetate, are a result of the consumption of H2 generated by fermentation. This is supported by the sustained performance of a predominantly acetate-supplemented stirred tank reactor dominated by diverse fermentative bacteria encoding FeFe hydrogenase genes and SRM capable of acetate and hydrogen consumption and CO2 assimilation. Thus, addition of fermentable substrates to stimulate syntrophic relationships may improve the performance of BSR reactors supplemented with inexpensive acetate.

Combined functional genomic and metabolomic approaches identify new genes required for growth in human urine by multidrug-resistant Escherichia coli ST131.

Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, with ~400 million cases across the globe each year. Uropathogenic Escherichia coli (UPEC) is the major cause of UTI and increasingly associated with antibiotic resistance. This scenario has been worsened by the emergence and spread of pandemic UPEC sequence type 131 (ST131), a multidrug-resistant clone associated with extraordinarily high rates of infection. Here, we employed transposon-directed insertion site sequencing in combination with metabolomic profiling to identify genes and biochemical pathways required for growth and survival of the UPEC ST131 reference strain EC958 in human urine (HU). We identified 24 genes required for growth in HU, which mapped to diverse pathways involving small peptide, amino acid and nucleotide metabolism, the stringent response pathway, and lipopolysaccharide biosynthesis. We also discovered a role for UPEC resistance to fluoride during growth in HU, most likely associated with fluoridation of drinking water. Complementary nuclear magnetic resonance (NMR)-based metabolomics identified changes in a range of HU metabolites following UPEC growth, the most pronounced being L-lactate, which was utilized as a carbon source via the L-lactate dehydrogenase LldD. Using a mouse UTI model with mixed competitive infection experiments, we demonstrated a role for nucleotide metabolism and the stringent response in UPEC colonization of the mouse bladder. Together, our application of two omics technologies combined with different infection-relevant settings has uncovered new factors required for UPEC growth in HU, thus enhancing our understanding of this pivotal step in the UPEC infection pathway. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) cause ~80% of all urinary tract infections (UTIs), with increasing rates of antibiotic resistance presenting an urgent threat to effective treatment. To cause infection, UPEC must grow efficiently in human urine (HU), necessitating a need to understand mechanisms that promote its adaptation and survival in this nutrient-limited environment. Here, we used a combination of functional genomic and metabolomic techniques and identified roles for the metabolism of small peptides, amino acids, nucleotides, and L-lactate, as well as the stringent response pathway, lipopolysaccharide biosynthesis, and fluoride resistance, for UPEC growth in HU. We further demonstrated that pathways involving nucleotide metabolism and the stringent response are required for UPEC colonization of the mouse bladder. The UPEC genes and metabolic pathways identified in this study represent targets for the development of innovative therapeutics to prevent UPEC growth during human UTI, an urgent need given the rapidly rising rates of global antibiotic resistance.

Histone lactylation inhibits RARγ expression in macrophages to promote colorectal tumorigenesis through activation of TRAF6-IL-6-STAT3 signaling.

Macrophages are phenotypically and functionally diverse in the tumor microenvironment (TME). However, how to remodel macrophages with a protumor phenotype and how to manipulate them for therapeutic purposes remain to be explored. Here, we show that in the TME, RARγ is downregulated in macrophages, and its expression correlates with poor prognosis in patients with colorectal cancer (CRC). In macrophages, RARγ interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6), which prevents TRAF6 oligomerization and autoubiquitination, leading to inhibition of nuclear factor κB signaling. However, tumor-derived lactate fuels H3K18 lactylation to prohibit RARγ gene transcription in macrophages, consequently enhancing interleukin-6 (IL-6) levels in the TME and endowing macrophages with tumor-promoting functions via activation of signal transducer and activator of transcription 3 (STAT3) signaling in CRC cells. We identified that nordihydroguaiaretic acid (NDGA) exerts effective antitumor action by directly binding to RARγ to inhibit TRAF6-IL-6-STAT3 signaling. This study unravels lactate-driven macrophage function remodeling by inhibition of RARγ expression and highlights NDGA as a candidate compound for treating CRC.

Distinguishing metabolic signals of liver tumors from surrounding liver cells using hyperpolarized 13 C MRI and gadoxetate.

PURPOSE: To use the hepatocyte-specific gadolinium-based contrast agent gadoxetate combined with hyperpolarized (HP) [1-13 C]pyruvate MRI to selectively suppress metabolic signals from normal hepatocytes while preserving the signals arising from tumors. METHODS: Simulations were performed to determine the expected changes in HP 13 C MR signal in liver and tumor under the influence of gadoxetate. CC531 colon cancer cells were implanted into the livers of five Wag/Rij rats. Liver and tumor metabolism were imaged at 3 T using HP [1-13 C] pyruvate chemical shift imaging before and 15 min after injection of gadoxetate. Area under the curve for pyruvate and lactate were measured from voxels containing at least 75% of normal-appearing liver or tumor. RESULTS: Numerical simulations predicted a 36% decrease in lactate-to-pyruvate (L/P) ratio in liver and 16% decrease in tumor. In vivo, baseline L/P ratio was 0.44 ± 0.25 in tumors versus 0.21 ± 0.08 in liver (p = 0.09). Following administration of gadoxetate, mean L/P ratio decreased by an average of 0.11 ± 0.06 (p < 0.01) in normal-appearing liver. In tumors, mean L/P ratio post-gadoxetate did not show a statistically significant change from baseline. Compared to baseline levels, the relative decrease in L/P ratio was significantly greater in liver than in tumors (-0.52 ± 0.16 vs. -0.19 ± 0.25, p < 0.05). CONCLUSIONS: The intracellular hepatobiliary contrast agent showed a greater effect suppressing HP 13 C MRI metabolic signals (through T1 shortening) in normal-appearing liver when compared to tumors. The combined use of HP MRI with selective gadolinium contrast agents may allow more selective imaging in HP 13 C MRI.

Transient Synaptic Enhancement Triggered by Exogenously Supplied Monocarboxylate in Drosophila Motoneuron Synapse.

Current evidence suggests that glial cells provide C3 carbon sources to fuel neuronal activity; however, this notion has become challenged by biosensor studies carried out in acute brain slices or in vivo, showing that neuronal activity does not rely on the import of astrocyte-produced L-lactate. Rather, stimulated neurons become net lactate exporters, as it was also shown in Drosophila neurons, in which astrocyte-provided lactate returns as lipid droplets to be stored in glial cells. In this view, we investigate whether exogenously supplied monocarboxylates can support Drosophila motoneuron neurotransmitter release (NTR). By assessing the excitatory post-synaptic current (EPSC) amplitude under voltage-clamp as NTR indicative, we found that both pyruvate and L-lactate, as the only carbon sources in the synapses bathing-solution, cause a large transient NTR enhancement, which declines to reach a synaptic depression state, from which the synapses do not recover. The FM1-43 pre-synaptic loading ability, however, is maintained under monocarboxylate, suggesting that SV cycling should not contribute to the synaptic depression state. The NTR recovery was reached by supplementing the monocarboxylate medium with sucrose. However, monocarboxylate addition to sucrose medium does not enhance NTR, but it does when the disaccharide concentration becomes too reduced. Thus, when pyruvate concentrations become too reduced, exogenously supplied L-lactate could be converted to pyruvate and metabolized by the neural mitochondria, triggering the NTR enhancement. SIGNIFICANCE STATEMENT: The question of whether monocarboxylic acids can fuel the Drosophila motoneuron NTR was challenged. Our findings show that exogenously supplied monocarboxylates trigger a large transient synaptic enhancement just under extreme glycolysis reduction but fail to maintain NTR under sustained synaptic demand, still at low frequency stimulation, driven to the synapses to a synaptic depression state. Glycolysis activation, by adding sucrose to the monocarboxylate bath solution, restores the motoneuron NTR ability, giving place to a hexoses role in SV recruitment. Moreover these results suggest exogenously supplied C3 carbon sources could have an additional role beyond providing energetic support for neural activity.

The immune checkpoint TIGIT/CD155 promotes the exhaustion of CD8 + T cells in TNBC through glucose metabolic reprogramming mediated by PI3K/AKT/mTOR signaling.

OBJECTIVE: The CD155/TIGIT axis has attracted considerable interest as an emerging immune checkpoint with potential applications in cancer immunotherapy. Our research focused on investigating the role of CD155/TIGIT checkpoints in the progression of triple-negative breast cancer (TNBC). METHODS: We evaluated CD155 and TIGIT expression in TNBC tissues using both immunohistochemistry (IHC) and gene expression profiling. Our experiments, both in vivo and in vitro, provided evidence that inhibiting the CD155/TIGIT pathway reinstates the ability of CD8 + T cells to generate cytokines. To assess the impact of CD155/TIGIT signaling blockade, we utilized Glucose Assay Kits and Lactate Assay Kits to measure alterations in glucose and lactate levels within CD8 + T cells. We employed western blotting (WB) to investigate alterations in glycolytic-related proteins within the PI3K/AKT/mTOR pathways following the inhibition of CD155/TIGIT signaling. RESULTS: CD155 exhibits heightened expression within TNBC tissues and exhibits a negative correlation with the extent of infiltrating CD8 + T cells. Furthermore, patients with TNBC demonstrate elevated levels of TIGIT expression. Our findings indicate that the interaction between CD155 and TIGIT disrupts the glucose metabolism of CD8 + T cells by suppressing the activation of the PI3K/AKT/mTOR signaling pathway, ultimately leading to the reduced production of cytokines by CD8 + T cells. Both in vivo and in vitro experiments have conclusively demonstrated that the inhibition of CD155/TIGIT interaction reinstates the capacity of CD8 + T cells to generate cytokines. Moreover, in vivo administration of the blocking antibody against TIGIT not only inhibits tumor growth but also augments the functionality of CD8 + T lymphocytes. CONCLUSIONS: Our research findings strongly suggest that CD155/TIGIT represents a promising therapeutic target for treating TNBC.

Oligodendrocyte-axon metabolic coupling is mediated by extracellular K+ and maintains axonal health.

The integrity of myelinated axons relies on homeostatic support from oligodendrocytes (OLs). To determine how OLs detect axonal spiking and how rapid axon-OL metabolic coupling is regulated in the white matter, we studied activity-dependent calcium (Ca2+) and metabolite fluxes in the mouse optic nerve. We show that fast axonal spiking triggers Ca2+ signaling and glycolysis in OLs. OLs detect axonal activity through increases in extracellular potassium (K+) concentrations and activation of Kir4.1 channels, thereby regulating metabolite supply to axons. Both pharmacological inhibition and OL-specific inactivation of Kir4.1 reduce the activity-induced axonal lactate surge. Mice lacking oligodendroglial Kir4.1 exhibit lower resting lactate levels and altered glucose metabolism in axons. These early deficits in axonal energy metabolism are associated with late-onset axonopathy. Our findings reveal that OLs detect fast axonal spiking through K+ signaling, making acute metabolic coupling possible and adjusting the axon-OL metabolic unit to promote axonal health.