Glycolytic Activity Research Articles

#1211 IGFBP-5/TGF-β1-mediated cell crosstalk between endothelia and tubular epithelia promotes renal fibrosis

Abstract Background and Aims Renal fibrosis is a common pathophysiological characteristic of chronic kidney disease (CKD) for which there is no effective treatment. The mechanism of renal fibrosis is complicated and remains unclear. Our previous study revealed that Insulin-like growth factor binding protein 5 (IGFBP-5) induces glycolytic activation in ECs and ultimately aggravates renal inflammation in diabetic kidney disease (DKD). This study aims to discover the relationship between IGFBP-5 and renal fibrosis. Method HK-2 and HUVEC cells were included in this study. UUO and ANN model were obstructed by Wild-type, IGFBP-5−/− and IGFBP-5flox/flox, cre mice. IGFBP-5, TGF-β and fibrosis makers were detected to explore the mechanism of IGFBP-5 in fibrosis. Results We found that the serum IGFBP-5 levels were significantly increased in CKD, that IGFBP-5 was dominantly localized in vascular endothelial cells (ECs) of kidney tissue, and that IGFBP-5 deficiency relieved renal fibrosis in CKD model mice. In vitro experiments indicated that IGFBP-5 exacerbated the fibrosis phenotypic alteration of tubular epithelial cells (TECs) by the TGF-β1/p-Smad3 signaling pathway. In turn, TGF-β1 could facilitate the synthesis of IGFBP-5 in ECs. The crosstalk between TECs and ECs mediated by IGFBP-5 and TGF-β1 was confirmed in a coculture system and endothelial-specific IGFBP-5-deficient mice. Conclusion Renal fibrosis is exacerbated by TEC-EC cellular crosstalk through the feedback loop consisting of IGFBP-5 and TGF-β1. These findings suggest that IGFBP-5 may be a new pathogenic factor in renal fibrosis and a potential new therapeutic target in CKD.

Relationship between wooden breast severity in broiler chicken, antioxidant enzyme activity and markers of energy metabolism

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.

A lactate-responsive gene signature predicts the prognosis and immunotherapeutic response of patients with triple-negative breast cancer.

Increased glycolytic activity and lactate production are characteristic features of triple-negative breast cancer (TNBC). The aim of this study was to determine whether a subset of lactate-responsive genes (LRGs) could be used to classify TNBC subtypes and predict patient outcomes. Lactate levels were initially measured in different breast cancer (BC) cell types. Subsequently, MDA-MB-231 cells treated with 2-Deoxy-d-glucose or l-lactate were subjected to RNA sequencing (RNA-seq). The gene set variation analysis algorithm was utilized to calculate the lactate-responsive score, conduct a differential analysis, and establish an association with the extent of immune infiltration. Consensus clustering was then employed to classify TNBC patients. Tumor immune dysfunction and exclusion, cibersort, single-sample gene set enrichment analysis, and EPIC, were used to compare the tumor-infiltrating immune cells between TNBC subtypes and predict the response to immunotherapy. Furthermore, a prognostic model was developed by combining 98 machine learning algorithms, to assess the predictive significance of the LRG signature. The predictive value of immune infiltration and the immunotherapy response was also assessed. Finally, the association between lactate and various anticancer drugs was examined based on expression profile similarity principles. We found that the lactate levels of TNBC cells were significantly higher than those of other BC cell lines. Through RNA-seq, we identified 14 differentially expressed LRGs in TNBC cells under varying lactate levels. Notably, this LRG signature was associated with interleukin-17 signaling pathway dysregulation, suggesting a link between lactate metabolism and immune impairment. Furthermore, the LRG signature was used to categorize TNBC into two distinct subtypes, whereby Subtype A was characterized by immunosuppression, whereas Subtype B was characterized by immune activation. We identified an LRG signature in TNBC, which could be used to predict the prognosis of patients with TNBC and gauge their response to immunotherapy. Our findings may help guide the precision treatment of patients with TNBC.

The effect of near-infrared low-level light on the in vitro quality of platelets during storage.

Near-infrared (NIR) light has been successfully applied to improve the quality of mouse platelets during storage. Because it is suspected that the mitochondria contain the primary photon acceptor, we hypothesized that human platelets for transfusion may be affected similarly and could benefit from NIR light treatment. The optimal light dose was determined using portions of platelet concentrates (PCs) in PAS-E. A pool-and-split design was used to prepare PCs in PAS-E or plasma (n = 6). On day 1, one unit of both pairs was illuminated with 830 nm light (light-emitting diodes, 15 J/cm2). PCs were stored at 22°C and sampled regularly for analysis. Data were compared with their corresponding controls with a paired two-sided t-test. Illuminated platelets in PAS-E were less activated with significantly lower CD62P expression (day 8: 10.8 ± 1.8 vs. 12.2 ± 2.6, p < 0.05) and lower Annexin A5 binding (day 8: 11.8 ± 1.9 vs. 13.1 ± 2.4, ns). They produced significantly less lactate resulting in a higher pH (days 6-10). ATP content and mitochondrial membrane potential were not affected. Although these trends were also observed for PCs in plasma, the differences did not reach statistical significance as compared with the control group. Our study demonstrates that the glycolysis rate of human platelets can be modulated through the use of NIR, possibly through mitochondrial aerobic metabolism, but this requires confirmation. If NIR illumination can be further optimized, it may potentially become a useful tool in situations in which glycolysis and platelet activation are exacerbated.

Small Molecule‐Inducible and Photoactivatable Cellular RNA N1‐Methyladenosine Editing

AbstractN1‐methyladenosine (m1A) modification is one of the most prevalent epigenetic modifications on RNA. Given the vital role of m1A modification in RNA processing such as splicing, stability and translation, developing a precise and controllable m1A editing tool is pivotal for in‐depth investigating the biological functions of m1A. In this study, we developed an abscisic acid (ABA)‐inducible and reversible m1A demethylation tool (termed AI‐dm1A), which targets specific transcripts by combining the chemical proximity‐induction techniques with the CRISPR/dCas13b system and ALKBH3. We successfully employed AI‐dm1A to selectively demethylate the m1A modifications at A8422 of MALAT1 RNA, and this demethylation process could be reversed by removing ABA. Furthermore, we validated its demethylation function on various types of cellular RNAs including mRNA, rRNA and lncRNA. Additionally, we used AI‐dm1A to specifically demethylate m1A on ATP5D mRNA, which promoted ATP5D expression and enhanced the glycolysis activity of tumor cells. Conversely, by replacing the demethylase ALKBH3 with methyltransferase TRMT61A, we also developed a controllable m1A methylation tool, namely AI‐m1A. Finally, we caged ABA by 4,5‐dimethoxy‐2‐nitrobenzyl (DMNB) to achieve light‐inducible m1A methylation or demethylation on specific transcripts. Collectively, our m1A editing tool enables us to flexibly study how m1A modifications on specific transcript influence biological functions and phenotypes.

Small Molecule-Inducible and Photoactivatable Cellular RNA N1-Methyladenosine Editing.

N1-methyladenosine (m1A) modification is one of the most prevalent epigenetic modifications on RNA. Given the vital role of m1A modification in RNA processing such as splicing, stability and translation, developing a precise and controllable m1A editing tool is pivotal for in-depth investigating the biological functions of m1A. In this study, we developed an abscisic acid (ABA)-inducible and reversible m1A demethylation tool (termed AI-dm1A), which targets specific transcripts by combining the chemical proximity-induction techniques with the CRISPR/dCas13b system and ALKBH3. We successfully employed AI-dm1A to selectively demethylate the m1A modifications at A8422 of MALAT1 RNA, and this demethylation process could be reversed by removing ABA. Furthermore, we validated its demethylation function on various types of cellular RNAs including mRNA, rRNA and lncRNA. Additionally, we used AI-dm1A to specifically demethylate m1A on ATP5D mRNA, which promoted ATP5D expression and enhanced the glycolysis activity of tumor cells. Conversely, by replacing the demethylase ALKBH3 with methyltransferase TRMT61A, we also developed a controllable m1A methylation tool, namely AI-m1A. Finally, we caged ABA by 4,5-dimethoxy-2-nitrobenzyl (DMNB) to achieve light-inducible m1A methylation or demethylation on specific transcripts. Collectively, our m1A editing tool enables us to flexibly study how m1A modifications on specific transcript influence biological functions and phenotypes.

Oxidative Stress in Breast Cancer: A Biochemical Map of Reactive Oxygen Species Production.

This review systematizes information about the metabolic features of breast cancer directly related to oxidative stress. It has been shown those redox changes occur at all levels and affect many regulatory systems in the human body. The features of the biochemical processes occurring in breast cancer are described, ranging from nonspecific, at first glance, and strictly biochemical to hormone-induced reactions, genetic and epigenetic regulation, which allows for a broader and deeper understanding of the principles of oncogenesis, as well as maintaining the viability of cancer cells in the mammary gland. Specific pathways of the activation of oxidative stress have been studied as a response to the overproduction of stress hormones and estrogens, and specific ways to reduce its negative impact have been described. The diversity of participants that trigger redox reactions from different sides is considered more fully: glycolytic activity in breast cancer, and the nature of consumption of amino acids and metals. The role of metals in oxidative stress is discussed in detail. They can act as both co-factors and direct participants in oxidative stress, since they are either a trigger mechanism for lipid peroxidation or capable of activating signaling pathways that affect tumorigenesis. Special attention has been paid to the genetic and epigenetic regulation of breast tumors. A complex cascade of mechanisms of epigenetic regulation is explained, which made it possible to reconsider the existing opinion about the triggers and pathways for launching the oncological process, the survival of cancer cells and their ability to localize.

Angiotensin II participates in mitochondrial thermogenic functions via the activation of glycolysis in chemically induced human brown adipocytes

Brown adipocytes are potential therapeutic targets for the prevention of obesity-associated metabolic diseases because they consume circulating glucose and fatty acids for heat production. Angiotensin II (Ang II) peptide is involved in the pathogenesis of obesity- and cold-induced hypertension; however, the mechanism underlying the direct effects of Ang II on human brown adipocytes remains unclear. Our transcriptome analysis of chemical compound-induced brown adipocytes (ciBAs) showed that the Ang II type 1 receptor (AGTR1), but not AGTR2 and MAS1 receptors, was expressed. The Ang II/AGTR1 axis downregulated the expression of mitochondrial uncoupling protein 1 (UCP1). The simultaneous treatment with β-adrenergic receptor agonists and Ang II attenuated UCP1 expression, triglyceride lipolysis, and cAMP levels, although cAMP response element-binding protein (CREB) phosphorylation was enhanced by Ang II mainly through the protein kinase C pathway. Despite reduced lipolysis, both coupled and uncoupled mitochondrial respiration was enhanced in Ang II-treated ciBAs. Instead, glycolysis and glucose uptake were robustly activated upon treatment with Ang II without a comprehensive transcriptional change in glucose metabolic genes. Elevated mitochondrial energy status induced by Ang II was likely associated with UCP1 repression. Our findings suggest that the Ang II/AGTR1 axis participates in mitochondrial thermogenic functions via glycolysis.

Cancer-associated fibroblast-derived gene signature discriminates distinct prognoses by integrated single-cell and bulk RNA-seq analyses in breast cancer.

Cancer-associated fibroblasts (CAFs) are one of the most predominant cellular subpopulations in the tumor stroma and play an integral role in cancer occurrence and progression. However, the prognostic role of CAFs in breast cancer remains poorly understood. We identified a number of CAF-related biomarkers in breast cancer by combining single-cell and bulk RNA-seq analyses. Based on univariate Cox regression as well as Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis, a novel CAF-associated prognostic model was developed. Breast cancer patients were grouped according to the median risk score and further analyzed for outcome, clinical characteristic, pathway activity, genomic feature, immune landscape, and drug sensitivity. A total of 341 CAF-related biomarkers were identified from single-cell and bulk RNA-seq analyses. We eventually screened eight candidate prognostic genes, including CERCAM, EMP1, SDC1, PRKG1, XG, TNN, WLS, and PDLIM4, and constructed the novel CAF-related prognostic model. Grouped by the median risk score, high-risk patients showed a significantly worse prognosis and exhibited distinct pathway activities such as uncontrolled cell cycle progression, angiogenesis, and activation of glycolysis. In addition, the combined risk score and tumor mutation burden significantly improved the ability to predict patient prognosis. Importantly, patients in the high-risk group had a higher infiltration of M2 macrophages and a lower infiltration of CD8+ T cells and activated NK cells. Finally, we calculated the IC50 for a range of anticancer drugs and personalized the treatment regimen for each patient. Integrating single-cell and bulk RNA-seq analyses, we identified a list of compositive CAF-associated biomarkers and developed a novel CAF-related prognostic model for breast cancer. This robust CAF-derived gene signature acts as an excellent predictor of patient outcomes and treatment responses in breast cancer.

Acetylation and Phosphorylation Regulate the Role of Pyruvate Kinase as a Glycolytic Enzyme or a Protein Kinase in Lamb.

Protein post-translational modifications (PTMs) play an essential role in meat quality development. However, the effect of specific PTM sites on meat proteins has not been investigated yet. The characteristics of pyruvate kinase M (PKM) were found to exhibit a close correlation with final meat quality, and thus, serine 99 (S99) and lysine 137 (K137) in PKM were mutated to study their effect on PKM function. The structural and functional properties of five lamb PKM variants, including wild-type PKM (wtPKM), PKM_S99D (S99 phosphorylation), PKM_S99A (PKM S99 dephosphorylation), PKM_K137Q (PKM K137 acetylation), and PKM_K137R (PKM K137 deacetylation), were evaluated. The results showed that the secondary structure, tertiary structure, and polymer formation were affected among different PKM variants. In addition, the glycolytic activity of PKM_K137Q was decreased because of its weakened binding with phosphoenolpyruvate. In the PKM_K137R variant, the actin phosphorylation level exhibited a decrease, suggesting a low kinase activity of PKM_K137R. The results of molecular simulation showed a 42% reduction in the interface area between PKM_K137R and actin, in contrast to wtPKM and actin. These findings are significant for revealing the mechanism of how PTMs regulate PKM function and provide a theoretical foundation for the development of precise meat quality preservation technology.

Better maintenance of enzymatic capacity and higher levels of substrate transporter proteins in skeletal muscle of aging female mice.

This study investigated sex-specific differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and also metabolite transporter protein levels in the skeletal muscles of adult (5 months old), middle-aged (12 months old), and advanced-aged (24 months old) mice. While gastrocnemius glycogen content increased with age regardless of sex, gastrocnemius triglyceride levels increased only in advanced-aged female mice. Aging decreased creatine kinase and adenylate kinase activities in the plantaris muscle of both sexes and in the soleus muscle of male mice but not in female mice. Irrespective of sex, phosphofructokinase and lactate dehydrogenase (LDH) activities decreased in the plantaris and soleus muscles. Additionally, hexokinase activity in the plantaris muscle and LDH activity in the soleus muscle decreased to a greater extent in aged male mice compared with those in aged female mice. Mitochondrial enzyme activities increased in the plantaris muscle of aged female mice but did not change in male mice. The protein content of the glucose transporter 4 in the aged plantaris muscle and fatty acid translocase/cluster of differentiation 36 increased in the aged plantaris and soleus muscles of both sexes, with a significantly higher content in female mice. These findings suggest that females possess a better ability to maintain metabolic enzyme activity and higher levels of metabolite transport proteins in skeletal muscle during aging, despite alterations in lipid metabolism. Our data provide a basis for studying muscle metabolism in the context of age-dependent metabolic perturbations and diseases that affect females and males differently.

Positive feedback regulation between glycolysis and histone lactylation drives oncogenesis in pancreatic ductal adenocarcinoma

BackgroundMetabolic reprogramming and epigenetic alterations contribute to the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). Lactate-dependent histone modification is a new type of histone mark, which links glycolysis metabolite to the epigenetic process of lactylation. However, the role of histone lactylation in PDAC remains unclear.MethodsThe level of histone lactylation in PDAC was identified by western blot and immunohistochemistry, and its relationship with the overall survival was evaluated using a Kaplan-Meier survival plot. The participation of histone lactylation in the growth and progression of PDAC was confirmed through inhibition of histone lactylation by glycolysis inhibitors or lactate dehydrogenase A (LDHA) knockdown both in vitro and in vivo. The potential writers and erasers of histone lactylation in PDAC were identified by western blot and functional experiments. The potential target genes of H3K18 lactylation (H3K18la) were screened by CUT&Tag and RNA-seq analyses. The candidate target genes TTK protein kinase (TTK) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) were validated through ChIP-qPCR, RT-qPCR and western blot analyses. Next, the effects of these two genes in PDAC were confirmed by knockdown or overexpression. The interaction between TTK and LDHA was identified by Co-IP assay.ResultsHistone lactylation, especially H3K18la level was elevated in PDAC, and the high level of H3K18la was associated with poor prognosis. The suppression of glycolytic activity by different kinds of inhibitors or LDHA knockdown contributed to the anti-tumor effects of PDAC in vitro and in vivo. E1A binding protein p300 (P300) and histone deacetylase 2 were the potential writer and eraser of histone lactylation in PDAC cells, respectively. H3K18la was enriched at the promoters and activated the transcription of mitotic checkpoint regulators TTK and BUB1B. Interestingly, TTK and BUB1B could elevate the expression of P300 which in turn increased glycolysis. Moreover, TTK phosphorylated LDHA at tyrosine 239 (Y239) and activated LDHA, and subsequently upregulated lactate and H3K18la levels.ConclusionsThe glycolysis-H3K18la-TTK/BUB1B positive feedback loop exacerbates dysfunction in PDAC. These findings delivered a new exploration and significant inter-relationship between lactate metabolic reprogramming and epigenetic regulation, which might pave the way toward novel lactylation treatment strategies in PDAC therapy.

Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis.

Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms. We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays. We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20. Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases.

Lack of Sestrins Is Associated with Increased Glycolytic Flux

Introduction: Sestrin1, 2, and 3 are a family of stress-response proteins implicated in many diseases. How Sestrins affect cell survival through AMP-activated kinase (AMPK) and protein synthesis through mechanistic target of rapamycin in complex 1 (mTORC1) is most studied; however, how Sestrins affect metabolism is poorly understood. Hypothesis: Independent of AMPK/mTORC1 activation, Sestrins negatively regulate glycolytic flux by inhibiting glycolytic enzyme activity through binding inhibition. Results: Wildtype (WT) HEK293T cells were transfected with plasmids expressing individual FLAG-tagged Sestrins and then deprived of glucose for 180 min or deprived of glucose for 150 min followed by glucose addback for 30 min. FLAG-tagged Sestrins were immunoprecipitated and found to directly interact with Hexokinase2 and Phosphofructokinase such that binding inversely correlates with glucose concentration. Additionally, WT and Sestrin-knockout (TKO) HEK293T cells were subjected to the glycolytic and mitochondrial stress tests and analyzed for metabolic flux via Seahorse analysis. TKO cells demonstrated higher glycolytic flux, but lower mitochondrial flux, compared to WT cells. Cells were then treated with inhibitors against AMPK and mTORC1 and subjected to the glycolytic stress test. Surprisingly, glycolytic flux in TKO cells was unaffected by the inhibitors while WT cells reduced glycolytic flux suggesting Sestrins can regulate glycolytic flux independent of AMPK/mTORC1 activation. Thereafter, WT and TKO cells were glucose-deprived for 120 min or glucose-deprived for 110 min followed by glucose addback for 10 min and then characterized by steady-state metabolomics. As expected, TKO cells possessed elevations in glycolytic metabolites with glucose addback compared to WT cells. Similarly, WT and TKO cells were deprived of glucose for 110 min followed by stable isotope tracing with [13C6]glucose for 10- and 30-minutes and metabolites were profiled by mass spectrometry. TKO cells possessed higher abundances of glycolytic metabolites as well as elevated labeled extracellular lactate. Conclusions: These data support a model wherein Sestrins directly act as negative regulators for glycolytic enzyme activity, thus regulating glycolytic flux. This work is funded by F32DK126312 (P.A.R.), P30DK048520 Pilot and Feasibility Award (P.A.R.), T32DK120521 (A.E.L.), P30CA046934 Cancer Center Support Grant, R01AR080051 (A.B.), R01DK015658 (S.R.K.), P30DK048520 (P.S.M.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antigen-Independent Activation of CD8 T Cells Promotes Salt Sensitive Hypertension

While CD8+ T cells (CD8Ts) have recently emerged as key players in hypertension, the precise mechanisms underlying their involvement remain incompletely understood. Our previous research uncovered the critical role of CD8T activation during the initial phase of blood pressure elevation, facilitating their direct interaction with the distal convoluted tubule (DCT) via the interferon-gamma (IFNγ)-PDL1 pathway. Importantly, we have now discovered that CD8T activation during this early phase occurs in an antigen-independent manner. In this study, we delve into the contribution of the P2X7 receptor to CD8T activation and its subsequent promotion of sodium retention in the kidney. We hypothesize that blocking this receptor on CD8Ts will disrupt CD8T-tubular interaction and lead to a reduction in blood pressure. To investigate whether CD8T activation in hypertension necessitates T cell receptor (TCR) stimulation, we employed Rag2−/−/OT1mice, which exclusively harbor transgenic OT1 CD8Ts, recognizing only ovalbumin peptides but not other antigens. These mice, without any exposure to ovalbumin, were induced to develop hypertension through the DOCA + salt (DOCA) model. Blood pressure was monitored using radio-biotelemetry, and CD8T activation was confirmed using flow cytometry. In our in-vitro studies, CD8Ts were obtained from both WT and P2X7 KO mice and stimulated with BZATP or ATP. The endpoints include calcium influx (measured via the intracellular calcium indicator Fluo-4), IFNγ transcription, and glycolytic activity (assessed by Seahorse metabolic flux analyzer) as indicators of activation. To validate our findings in an in-vivo context, we measured the activation profiles of CD8Ts obtained from WT and P2X7 KO mice subjected to sham or DOCA treatment, employing RT-PCR and flow cytometry. Additionally, we conducted an adoptive transfer experiment, transferring naïve WT CD8Ts to P2X7 KO mice undergoing DOCA treatment. Blood pressure was consistently monitored via radio-biotelemetry. Our observations revealed that CD8Ts from Rag2−/−/OT1 mice become activated in response to the DOCA pressor, leading to increased IFNγ production and contributing to salt-sensitive hypertension, even in the absence of TCR-mediated antigen stimulation. Agonists of the P2X7 receptor, such as BZATP and ATP, elicited calcium influx in CD8Ts, upregulated IFNγ transcription, and enhanced glycolytic activity, all indicative of T cell activation. Furthermore, CD8Ts pre-stimulated with the P2X7 agonist BZATP displayed increased tubular PDL1 expression and sodium retention in our co-culture model when compared to naïve CD8Ts. Notably, DOCA-treated P2X7 KO CD8Ts were unable to induce an increase in blood pressure in our adoptive transfer model. However, transferring sham WT CD8Ts to DOCA-treated P2X7 KO mice successfully restored hypertension to WT levels. Our findings suggest a novel mechanism in which P2X7 receptor stimulation contributes to CD8T activation in the pathogenesis of hypertension. The deficiency of this receptor on CD8Ts prevents their activation by ATP, resulting in attenuated blood pressure elevation. As such, the CD8T P2X7 receptor presents itself as a promising target for the improvement of blood pressure management. Funding: NIH R01-HL146713, AHA 23TPA1076467. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1131: Delivery Of Allogeneic Mitochondria Reverses Signatures Of Endothelial Aging &amp; Senescence

Mitochondrial dysfunction is a key factor in vascular aging, including endothelial cell (EC) dysfunction. This research investigates whether introducing mitochondria from allogeneic fibroblasts into human microvascular ECs can mitigate the cellular abnormalities associated with aging. Mitochondria were transferred into ECs of different passages (p6-14) using centrifugation. Post mito-transfer, the metabolic activity, mitochondrial network (density), and the proliferative capacity of ECs were evaluated through extracellular flux analysis, confocal microscopy, and 5-ethynyl-2'-deoxyuridine incorporation (proliferation assay), respectively. Results show that compared to age matched control ECs, mito-transferred ECs exhibited no change in their oxygen consumption rates linked to mitochondrial respiration. Additionally, while early passage ECs (p6-7) had no change in glycolytic activity (compared to controls), middle (p10-11) and late passage ECs (p10-14) had considerable increases in their glycolytic activity post mito-transfer. Mitochondrial network analysis additionally revealed that the mitochondrial density of early passage ECs were unaffected by mito-transfer, while late passage ECs had enhanced mitochondrial networks, including total volume, and branching post mito-transfer. Lastly, late passage ECs demonstrated a 25% increase in proliferation after mito-transfer. These findings indicate that aging in ECs is linked to increased oxidative phosphorylation metabolism, and that mito-transfer can reduce senescence-associated oxidative glucose metabolism and enhance EC proliferation. This positions mito-transfer as a promising strategy for improve EC function in cardiovascular aging.

Characteristic features of in vitro differentiation of human naïve CD4+ T cells to induced regulatory T cells (iTreg) and T helper (Th) 17 cells: Sharing of lineage-specific markers

In vitro induced regulatory T cells (iTreg) and IL-17 producing T cells (Th17-like cells) can be generated in culture from native CD4+ T cells in peripheral blood by different sets of cytokines. In the presence of transforming growth factor (TGF)-β plus interleukin (IL)-2, cells differentiate into Treg cells with increased expression of the forkhead box P3 (FOXP3). In the presence of TGF-β, IL-6, IL-1β and IL-23, cells differentiate into Th17 cells that produce IL-17A. However, protocols for the generation of human iTreg and Th17 are still controversial. In this study, we characterized the biological features of iTreg and Th17 cells differentiated from peripheral blood naïve CD4+ T cells in vitro using the established protocols. We showed that cells obtained from Treg or Th17 culture conditions shared some phenotypic markers. Cells under Treg conditions had an up-regulated FOXP3 gene and a down-regulated RAR-related orphan receptor C (RORC) gene. Cells derived from the Th17 condition exhibited a down-regulated FOXP3 gene and had significantly higher RORC gene expression than Treg cells. Both resulting cells showed intracellular production of IL-17A and IL-10. Th17 condition-cultured cells exhibited more glycolytic activity and glucose uptake compared to the Treg cells. The findings suggest that cells obtained from established protocols for the differentiation of iTreg and Th17 cells in vitro are possibly in the intermediate stage of differentiation or may be two different types of cells that share a lineage-specific differentiation program.

Single-cell transcriptome analysis reveals heterogeneity of neutrophils in non-small cell lung cancer.

Lung cancer stands out as a highly perilous malignant tumor with severe implications for human health. There has been a growing interest in neutrophils as a result of their role in promoting cancer in recent years. Thus, the present study aimed to investigate the heterogeneity of neutrophils in non-small cell lung cancer (NSCLC). Single-cell RNA sequencing of tumor-associated neutrophils (TANs) and polymorphonuclear neutrophils sourced from the Gene Expression Omnibus database was analyzed. Moreover, cell-cell communication, differentiation trajectories and transcription factor analyses were performed. Neutrophils were found to be closely associated with macrophages. Four major types of TANs were identified: a transitional subcluster that migrated from blood to tumor microenvironment (TAN-0), an inflammatory subcluster (TAN-1), a subpopulation that displayed a distinctive transcriptional signature (TAN-2) and a final differentiation state that promoted tumor formation (TAN-3). Meanwhile, TAN-3 displayed a marked increase in glycolytic activity. Finally, transcription factors were analyzed to uncover distinct TAN cluster-specific regulons. The discovery of the dynamic characteristics of TANs in the present study is anticipated to contribute to yielding a better understanding of the tumor microenvironment and advancing the treatment of NSCLC.

Novel Inositol 1,4,5-Trisphosphate Receptor Inhibitor Antagonizes Hepatic Stellate Cell Activation: A Potential Drug to Treat Liver Fibrosis.

Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.

Computational Modeling to Identify Drugs Targeting Metastatic Castration-Resistant Prostate Cancer Characterized by Heightened Glycolysis.

Metastatic castration-resistant prostate cancer (mCRPC) remains a deadly disease due to a lack of efficacious treatments. The reprogramming of cancer metabolism toward elevated glycolysis is a hallmark of mCRPC. Our goal is to identify therapeutics specifically associated with high glycolysis. Here, we established a computational framework to identify new pharmacological agents for mCRPC with heightened glycolysis activity under a tumor microenvironment, followed by in vitro validation. First, using our established computational tool, OncoPredict, we imputed the likelihood of drug responses to approximately 1900 agents in each mCRPC tumor from two large clinical patient cohorts. We selected drugs with predicted sensitivity highly correlated with glycolysis scores. In total, 77 drugs predicted to be more sensitive in high glycolysis mCRPC tumors were identified. These drugs represent diverse mechanisms of action. Three of the candidates, ivermectin, CNF2024, and P276-00, were selected for subsequent vitro validation based on the highest measured drug responses associated with glycolysis/OXPHOS in pan-cancer cell lines. By decreasing the input glucose level in culture media to mimic the mCRPC tumor microenvironments, we induced a high-glycolysis condition in PC3 cells and validated the projected higher sensitivity of all three drugs under this condition (p < 0.0001 for all drugs). For biomarker discovery, ivermectin and P276-00 were predicted to be more sensitive to mCRPC tumors with low androgen receptor activities and high glycolysis activities (AR(low)Gly(high)). In addition, we integrated a protein-protein interaction network and topological methods to identify biomarkers for these drug candidates. EEF1B2 and CCNA2 were identified as key biomarkers for ivermectin and CNF2024, respectively, through multiple independent biomarker nomination pipelines. In conclusion, this study offers new efficacious therapeutics beyond traditional androgen-deprivation therapies by precisely targeting mCRPC with high glycolysis.