Intracellular Lactate Concentrations Research Articles

Metabolic regulation of cytoskeleton functions by HDAC6-catalyzed α-tubulin lactylation.

Posttranslational modifications (PTMs) of tubulin, termed the "tubulin code", play important roles in regulating microtubule functions within subcellular compartments for specialized cellular activities. While numerous tubulin PTMs have been identified, a comprehensive understanding of the complete repertoire is still underway. In this study, we report that α-tubulin lactylation is catalyzed by HDAC6 by using lactate to increase microtubule dynamics in neurons. We identify lactylation on lysine 40 of α-tubulin in the soluble tubulin dimers. Notably, lactylated α-tubulin enhances microtubule dynamics and facilitates neurite outgrowth and branching in cultured hippocampal neurons. Moreover, we discover an unexpected function of HDAC6, acting as the primary lactyltransferase to catalyze α-tubulin lactylation. HDAC6-catalyzed lactylation is a reversible process, dependent on lactate concentrations. Intracellular lactate concentration triggers HDAC6 to lactylate α-tubulin, a process dependent on its deacetylase activity. Additionally, the lactyltransferase activity may be conserved in HDAC family proteins. Our study reveals the primary role of HDAC6 in regulating α-tubulin lactylation, establishing a link between cell metabolism and cytoskeleton functions.

Lactate supports Treg function and immune balance via MGAT1 effects on N-glycosylation in the mitochondria.

Current research reports that lactate affects Treg metabolism, although the precise mechanism has only been partially elucidated. In this study, we presented evidence demonstrating that elevated lactate levels enhanced cell proliferation, suppressive capabilities, and oxidative phosphorylation (OXPHOS) in human Tregs. The expression levels of Monocarboxylate Transporters 1/2/4 (MCT1/2/4) regulate intracellular lactate concentration, thereby influencing the varying responses observed in naive Tregs and memory Tregs. Through mitochondrial isolation, sequencing, and analysis of human Tregs, we determined that α-1,3-Mannosyl-Glycoprotein 2-β-N-Acetylglucosaminyltransferase (MGAT1) served as the pivotal driver initiating downstream N-glycosylation events involving progranulin (GRN) and hypoxia-upregulated 1 (HYOU1), consequently enhancing Treg OXPHOS. The mechanism by which MGAT1 was upregulated in mitochondria depended on elevated intracellular lactate that promoted the activation of XBP1s. This, in turn, supported MGAT1 transcription as well as the interaction of lactate with the translocase of the mitochondrial outer membrane 70 (TOM70) import receptor, facilitating MGAT1 translocation into mitochondria. Pretreatment of Tregs with lactate reduced mortality in a xenogeneic graft-versus-host disease (GvHD) model. Together, these findings underscored the active regulatory role of lactate in human Treg metabolism through the upregulation of MGAT1 transcription and its facilitated translocation into the mitochondria.

A57 LONG-TERM EXPOSURE TO ANTICANCER TREATMENTS INCREASES AUTOPHAGY IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background The most common type of esophageal cancer, esophageal squamous cell carcinoma (ESCC), has a 5-year survival rate of only 15%. This low survival rate is to some extent attributed to a high relapse rate, which is, in part, linked to the presence of cancer stem cells (CSC). CSC are a subpopulation of tumor cells that show reduced sensitivity to conventional anticancer therapies, increased potency and self-renewal capacities. With that in mind, ESCC cell lines were exposed in a prolonged manner to anticancer treatments (radiotherapy, 5-FU chemotherapy, and combined therapy) in our laboratory. As expected, an increase in the proportion of CSC (CD44highCD24high) was observed by flow cytometry following the treatments. In pursuit of innovative therapeutic approaches, we conducted an in-depth investigation of the proteomic profiles of these cell lines by mass spectrometry (MS), seeking to unveil novel biological mechanisms. Interestingly, many proteins associated with metabolism and autophagy were modulated. Several alterations were observed by a metabolomic approach as well, namely an increase in intracellular lactate concentration. Recently, a novel role for lactate has been described: lactylation, which is a post-translational modification. Lactylome of those cell lines revealed alteration in proteins related to autophagy and those results led to my research project. Aims Investigate the role of lactylation in autophagy of ESCC. Methods Human ESCC cell lines (including the cell lines exposed to anticancer treatments) will be used as cell culture models. Cell and organoid cultures, mass spectrometry, qPCR, WB, and proliferation assay were used. Results MS analysis of proteins related to autophagy (i.e. RAB1B, CK2, SNX6 et GABARAP) suggests differential regulation of autophagy in cell lines exposed to prolonged treatments. A strong contribution of the 5-FU treatment to the autophagic modification is observable in the double-treated cells. Thus, we confirmed an increase in the LC3BII/I ratio by WB in the cell lines exposed to anticancer treatments. LC3BII/I WB used in combination with Bafilomycin A1 treatment for inhibition of autophagosome and lysosome fusion is a commonly used read-out for autophagic flux. To further study the link between lactylation, autophagy and CSC, we screened 10 human ESCC cell lines for their LC3BII/I ratio. Four cell lines were selected: TE1, TE2, HCE4 and TE5. Using Bafilomycin A1, we confirmed that the autophagic flux in those cell lines was not blocked. Those cell lines will now be used to perform lactylome analysis of sorted CSC cells. Conclusions In brief, our results showed that the autophagic flux is increased in cells exposed to prolonged treatments. This suggests that autophagy could play a role in ESCC response to treatment. Funding Agencies CIHRTRIANGLE, Chaires de recherche du Canada, CRCHUS, Université de Sherbrooke

Evidence for Hypoxia-Induced Shift in ATP Production from Glycolysis to Mitochondrial Respiration in Pulmonary Artery Smooth Muscle Cells in Pulmonary Arterial Hypertension.

The metabolic state of pulmonary artery smooth muscle cells (PASMCs) from patients with pulmonary arterial hypertension (PAH) is not well understood. In this study, we examined the balance between glycolysis and mitochondrial respiration in non-PAH-PASMCs and PAH-PASMCs under normoxia and hypoxia. We investigated the enzymes involved in glycolysis and mitochondrial respiration, and studied the two major energy-yielding pathways (glycolysis and mitochondrial respiration) by measuring extracellular acidification rate (ECAR) and cellular oxygen consumption rate (OCR) using the Seahorse extracellular flux technology. Under both normoxia and hypoxia, the mRNA and protein levels of pyruvate dehydrogenase kinase 1 and pyruvate dehydrogenase were increased in PAH-PASMCs compared with non-PAH-PASMCs. The mRNA and protein levels of lactate dehydrogenase, as well as the intracellular lactate concentration, were also increased in PAH-PASMCs compared with non-PAH-PASMCs under normoxia. However, these were not significantly increased in PAH-PASMCs compared with non-PAH-PASMCs under hypoxia. Under normoxia, ATP production was significantly lower in PAH-PASMCs (59 ± 5 pmol/min) than in non-PAH-PASMCs (70 ± 10 pmol/min). On the other hand, ATP production was significantly higher in PAH-PASMCs (31 ± 5 pmol/min) than in non-PAH-PASMCs (14 ± 3 pmol/min) under hypoxia. There is an underlying change in the metabolic strategy to generate ATP production under the challenge of hypoxia.

Metformin Treatment Induces Different Response in Pheochromocytoma/Paraganglioma Tumour Cells and in Primary Fibroblasts.

Simple SummaryPheochromocytoma/paragangliomas (PPGLs) are neuroendocrine tumours and are often non-metastatic. However, no effective treatment is available for their metastatic form. Recent studies have shown that metformin exhibits antiproliferative activity in many human cancers, including PPGLs. Nevertheless, no data are available concerning whether metformin is also able to inhibit PPGL metastatic spread. A tumour is a very complex system, comprising not only cancer cells, but also other cells that all together form the so-called tumour microenvironment. Cancer-associated fibroblasts are residential or recruited fibroblasts, transformed by cancer cells, to promote tumour growth and spread. Therefore, the interplay between tumour cells and cancer-associated fibroblasts has become an interesting target for cancer therapy. Here, we demonstrate that metformin has different effects on cancer cells and fibroblasts, providing evidence that metformin may hold promise for altering tumour microenvironment homeostasis. Improving our knowledge on malignant tumour microenvironment properties could lead to develop complementary strategies to target tumour spread and progression.Pheochromocytoma/paragangliomas (PPGLs) are neuroendocrine tumours, often non-metastatic, but without available effective treatment for their metastatic form. Recent studies have shown that metformin exhibits antiproliferative activity in many human cancers, including PPGLs. Nevertheless, no data are available on the role of metformin on PPGL cells (two-dimension, 2D) and spheroids (three-dimension, 3D) migration/invasion. In this study, we observed that metformin exerts an antiproliferative effect on 2D and 3D cultures of pheochromocytoma mouse tumour tissue (MTT), either silenced or not for the SDHB subunit. However, metformin did not affect MTT migration. On the other hand, metformin did not have a short-term effect on the proliferation of mouse primary fibroblasts, but significantly decreased their ability to migrate. Although the metabolic changes induced by metformin were similar between MTT and fibroblasts (i.e., an overall decrease of ATP production and an increase in intracellular lactate concentration) the activated signalling pathways were different. Indeed, after metformin administration, MTT showed a reduced phosphorylation of Akt and Erk1/2, while fibroblasts exhibited a downregulation of N-Cadherin and an upregulation of E-Cadherin. Herein, we demonstrated that metformin has different effects on cell growth and spread depending on the cell type nature, underlining the importance of the tumour microenvironment in dictating the drug response.

A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors

Genetically encoded fluorescent biosensors are powerful tools used to track chemical processes in intact biological systems. However, the development and optimization of biosensors remains a challenging and labor-intensive process, primarily due to technical limitations of methods for screening candidate biosensors. Here we describe a screening modality that combines droplet microfluidics and automated fluorescence imaging to provide an order of magnitude increase in screening throughput. Moreover, unlike current techniques that are limited to screening for a single biosensor feature at a time (e.g. brightness), our method enables evaluation of multiple features (e.g. contrast, affinity, specificity) in parallel. Because biosensor features can covary, this capability is essential for rapid optimization. We use this system to generate a high-performance biosensor for lactate that can be used to quantify intracellular lactate concentrations. This biosensor, named LiLac, constitutes a significant advance in metabolite sensing and demonstrates the power of our screening approach.

Woodfordia fruticosa extract supplementation stimulates the growth of Lacticaseibacillus casei and Lacticaseibacillus rhamnosus with adapted intracellular and extracellular metabolite pool.

To investigate the effect of Woodfordia fruticosa extract (WfE) on two probiotic bacteria: Lacticaseibacillus casei and Lacticaseibacillus rhamnosus. WfE supplementation at 0·5 and 1 mg ml-1 stimulated probiotic growth (P < 0·05), enhanced adhesion to CaCO2 cells (P < 0·05) while inhibiting foodborne pathogens Escherichia coli and Staphylococcus aureus (P < 0·05). 1 H-NMR based metabolomic studies indicated higher glucose : lactate and glucose : acetate in the extracellular matrix with significant variation (P < 0·05) in intracellular concentrations of lactate, acetate, glutamate, dimethylamine, phenylalanine, branched-chain amino acids and total cellular lipid composition. Fatty acid methyl ester analysis showed a chemical shift from saturated to unsaturated lipids with WfE supplementation. PCA plots indicated clear discrimination between test groups, highlighting variation in metabolite pool in response to WfE supplementation. Phytonutrient-rich WfE exhibited prebiotic-like attributes, and probiotic bacteria showed altered metabolite pools as an adaptive mechanism. This is the first report providing insights into the prebiotic-like activity of WfE on gut representative probiotics. The extended metabolomic studies shed light on the positive interaction between phytonutrients and beneficial bacteria that possibly help them to adapt to a phytonutrient-rich WfE environment. WfE with potential prebiotic attributes can be used in the development of novel synbiotic functional products targeting gut microbial modulation to improve health.

PDK2-enhanced glycolysis promotes fibroblast proliferation in thyroid-associated ophthalmopathy.

The study aimed to investigate the role of pyruvate dehydrogenase kinase (PDK) in regulating glycolysis and proliferation of perimysial orbital fibroblasts (pOFs) during the pathogenesis of thyroid-associated ophthalmopathy (TAO). EdU and BrdU incorporation assays were performed to examine cell proliferation. Lactate production and oxygen consumption assays were conducted to evaluate glycolysis. Real-time PCR was adapted to quantify PDK mRNA levels. Capillary Western immunoassay was adapted to quantify PDK2, Akt, pAkt308 and GAPDH in protein samples. The TAO pOFs exhibited stronger proliferation activity, higher intracellular lactate concentration, and lower oxygen consumption rate than the control pOFs. The PDK inhibitor dichloroacetic acid (DCA) dose-dependently suppressed the proliferation of both TAO and control pOFs. DCA reduced lactate production and promoted oxygen consumption in the TAO pOFs but showed no significant effects on glycolysis in the control pOFs. Among four PDK isotypes, PDK2 was overexpressed in the TAO pOFs. The potential PDK signaling mediator, cytoplasmic Akt, was more abundant in TAO pOFs than control pOFs. Knockdown of PDK2 resulted in lower lactate production, stronger oxygen consumption, weaker proliferation activity, and less cytoplasmic Akt in the TAO pOFs but showed no significant effects in the control pOFs. The Akt inhibitor MK2206 suppressed proliferation in both TAO and control pOFs, and lactate production was inhibited by MK2206 in the TAO OFs but not the control pOFs. To conclude, PDK2 overexpression enhances glycolysis and promotes proliferation via Akt signaling in the TAO pOFs. These findings yield insights that PDK2 is a potential therapeutic target for TAO treatment.

SLC38A2 Overexpression Induces a Cancer-like Metabolic Profile and Cooperates with SLC1A5 in Pan-cancer Prognosis.

Cancer cells have dramatically increased demands for energy as well as biosynthetic precursors to fuel their restless growth. Enhanced glutaminolysis is a hallmark of cancer metabolism which fulfills these needs. Two glutamine transporters, SLC1A5 and SLC38A2, have been previously reported to promote glutaminolysis in cancer with controversial perspectives. In this study, we harnessed the proximity labeling reaction to map the protein interactome using mass spectrometry-based proteomics and discovered a potential protein-protein interaction between SLC1A5 and SLC38A2. The SLC1A5/SLC38A2 interaction was further confirmed by bimolecular fluorescence complementation assay. We further investigated the metabolic influence of SLC1A5 and SLC38A2 overexpression in human cells, respectively, and found that only SLC38A2, but not SLC1A5, resulted in a cancer-like metabolic profile, where the intracellular concentrations of essential amino acids and lactate were significantly increased as quantified by nuclear magnetic resonance spectroscopy. Finally, we analyzed the 5-year survival rates in a large pan-cancer cohort and found that the SLC1A5hi /SLC38A2lo group did not relate to a poor survival rate, whereas the SLC1A5lo /SLC38A2hi group significantly aggravated the lethality. Intriguingly, the SLC1A5hi /SLC38A2hi group resulted in an even worse prognosis, suggesting a cooperative effect between SLC1A5 and SCL38A2. Our data suggest that SLC38A2 plays a dominant role in reprogramming the cancer-like metabolism and promoting the cancer progression, whereas SLC1A5 may augment this effect when co-overexpressed with SLC38A2. We propose a model to explain the relationship between SLC1A5, SLC38A2 and SCL7A5, and discuss their impact on glutaminolysis and mTOR signaling.

Differential Gene Set Enrichment Analysis: a statistical approach to quantify the relative enrichment of two gene sets.

Gene Set Enrichment Analysis (GSEA) is an algorithm widely used to identify statistically enriched gene sets in transcriptomic data. However, GSEA cannot examine the enrichment of two gene sets or pathways relative to one another. Here we present Differential Gene Set Enrichment Analysis (DGSEA), an adaptation of GSEA that quantifies the relative enrichment of two gene sets. After validating the method using synthetic data, we demonstrate that DGSEA accurately captures the hypoxia-induced coordinated upregulation of glycolysis and downregulation of oxidative phosphorylation. We also show that DGSEA is more predictive than GSEA of the metabolic state of cancer cell lines, including lactate secretion and intracellular concentrations of lactate and AMP. Finally, we demonstrate the application of DGSEA to generate hypotheses about differential metabolic pathway activity in cellular senescence. Together, these data demonstrate that DGSEA is a novel tool to examine the relative enrichment of gene sets in transcriptomic data. DGSEA software and tutorials are available at https://jamesjoly.github.io/DGSEA/. Supplementary data are available at Bioinformatics online.

Monocarboxylate Transporter 4 Is a Therapeutic Target in Non-small Cell Lung Cancer with Aerobic Glycolysis Preference

Targeting metabolic reprogramming is an emerging strategy in cancer therapy. However, clinical attempts to target metabolic reprogramming have been proved to be challenging, with metabolic heterogeneity of cancer being one of many reasons that causes treatment failure. Here, we stratified non-small cell lung cancer (NSCLC) cells, mainly lung adenocarcinoma, based on their metabolic phenotypes and demonstrated that the aerobic glycolysis-preference NSCLC cell subtype was resistant to the OXPHOS-targeting inhibitors. We identified that monocarboxylate transporter 4 (MCT4), a lactate transporter, was highly expressed in the aerobic glycolysis-preference subtype with function supporting the proliferation of these cells. Glucose could induce the expression of MCT4 in these cells through a ΔNp63α and Sp1-dependent pathway. Next, we showed that knockdown of MCT4 increased intracellular lactate concentration and induced a reactive oxygen species (ROS)-dependent cellular apoptosis in the aerobic glycolysis-preference NSCLC cell subtype. By scanning a panel of monoclonal antibodies with MCT4 neutralizing activity, we further identified a MCT4 immunoglobulin M (IgM) monoclonal antibody showing capable anti-proliferation efficacy on the aerobic glycolysis-preference NSCLC cell subtype. Our findings indicate that the metabolic heterogeneity is a critical factor for NSCLC therapy and manipulating the expression or function of MCT4 can be an effective strategy in targeting the aerobic glycolysis-preference NSCLC cell subtype.

Effect of URAT1 inhibition with verinurad on proximal tubule intracellular lactate: A mathematical modeling analysis and hypothesis for antiproteinuric effect

Verinurad (RDEA3170) lowers serum uric acid by inhibiting URAT1, a urate/carboxylate co‐transporter in the proximal tubule (PT) of the kidney. In a recent clinical trial, verinurad also reduced proteinuria, but the mechanism remains unknown. One proposed hypothesis is that the inhibition of URAT1 augments protein reabsorption by increasing intracellular lactate in proximal tubule cells. The increased availability of lactate may lead to a restoration of the reabsorption of protein by increasing ATP levels and/ or improving endocytosis of protein by lowering endosomal pH. The goal of this study was to mathematically evaluate the effect of URAT1 inhibition with verinurad on intracellular lactate concentration.A mathematical model of urate and lactate transport in the rat proximal tubule was adapted to simulate transport in humans. The transporter model was further adjusted to represent states of hyperuricemia and altered expression of uric acid transporters that likely occur in hyperuricemia. The transporter model was linked to a systems description of plasma uric acid and the pharmacokinetics and pharmacodynamics of verinurad were incorporated into the model.The model was able to reproduce observed verinurad pharmacokinetics, as well as the observed changes in plasma and urinary uric acid. The model predicted that URAT1 inhibition with verinurad increases intracellular lactate concentration in the human PT by 26% in normouricemic and 20% in hyperuricemic conditions.Model simulations indicate that verinurad may substantially increase the availability of lactate inside PT epithelial cells. Since lactate is used as a fuel by the PT, increased availability of lactate may potentially help restore protein reabsorption capacity in fuel‐deficient PT cells by increasing ATP levels. In addition, increased intracellular lactate could help maintain the low endosomal pH necessary for protein reabsorption. This hypothesis remains to be experimentally proven; further non‐clinical/clinical data would be valuable to support model predictions.Urate and lactate proximal tubule transport model.Figure 1

Lowering the increased intracellular pH of human-induced pluripotent stem cell-derived endothelial cells induces formation of mature Weibel-Palade bodies.

Differentiation of human‐induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial‐like cells (hiPSC‐derived endothelial cells [hiPSC‐ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC‐ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel‐Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC‐ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC‐ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC‐ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC‐ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC‐EC. Mechanistically, pro‐VWF dimers require the lower pH environment of the trans‐Golgi network for maturation and tubulation. These data show that while hiPSC‐ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function.

Diverse Roads Taken by 13C-Glucose-Derived Metabolites in Breast Cancer Cells Exposed to Limiting Glucose and Glutamine Conditions.

In cancers, tumor cells are exposed to fluctuating nutrient microenvironments with limiting supplies of glucose and glutamine. While the metabolic program has been related to the expression of oncogenes, only fractional information is available on how variable precarious nutrient concentrations modulate the cellular levels of metabolites and their metabolic pathways. We thus sought to obtain an overview of the metabolic routes taken by 13C-glucose-derived metabolites in breast cancer MCF-7 cells growing in combinations of limiting glucose and glutamine concentrations. Isotopologue profiles of key metabolites were obtained by gas chromatography/mass spectrometry (GC/MS). They revealed that in limiting and standard saturating medium conditions, the same metabolic routes were engaged, including glycolysis, gluconeogenesis, as well as the TCA cycle with glutamine and pyruvate anaplerosis. However, the cellular levels of 13C-metabolites, for example, serine, alanine, glutamate, malate, and aspartate, were highly sensitive to the available concentrations and the ratios of glucose and glutamine. Notably, intracellular lactate concentrations did not reflect the Warburg effect. Also, isotopologue profiles of 13C-serine as well as 13C-alanine show that the same glucose-derived metabolites are involved in gluconeogenesis and pyruvate replenishment. Thus, anaplerosis and the bidirectional flow of central metabolic pathways ensure metabolic plasticity for adjusting to precarious nutrient conditions.

Bi-allelic mutations ofLONP1encoding the mitochondrial LonP1 protease cause pyruvate dehydrogenase deficiency and profound neurodegeneration with progressive cerebellar atrophy

LonP1 is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. We identified a novel homozygous missense LONP1 variant, c.2282 C>T, (p.Pro761Leu), by whole-exome and Sanger sequencing in two siblings born to healthy consanguineous parents. Both siblings presented with stepwise regression during infancy, profound hypotonia and muscle weakness, severe intellectual disability and progressive cerebellar atrophy on brain imaging. Muscle biopsy revealed the absence of ragged-red fibers, however, scattered cytochrome c oxidase-negative staining and electron dense mitochondrial inclusions were observed. Primary cultured fibroblasts from the siblings showed normal levels of mtDNA and mitochondrial transcripts, and normal activities of oxidative phosphorylation complexes I through V. Interestingly, fibroblasts of both siblings showed glucose-repressed oxygen consumption compared to their mother, whereas galactose and palmitic acid utilization were similar. Notably, the siblings' fibroblasts had reduced pyruvate dehydrogenase (PDH) activity and elevated intracellular lactate:pyruvate ratios, whereas plasma ratios were normal. We demonstrated that in the siblings' fibroblasts, PDH dysfunction was caused by increased levels of the phosphorylated E1α subunit of PDH, which inhibits enzyme activity. Blocking E1α phosphorylation activated PDH and reduced intracellular lactate concentrations. In addition, overexpressing wild-type LonP1 in the siblings' fibroblasts down-regulated phosphoE1α. Furthermore, in vitro studies demonstrated that purified LonP1-P761L failed to degrade phosphorylated E1α, in contrast to wild-type LonP1. We propose a novel mechanism whereby homozygous expression of the LonP1-P761L variant leads to PDH deficiency and energy metabolism dysfunction, which promotes severe neurologic impairment and neurodegeneration.

Intra-cellular lactate concentration in T lymphocytes from septic shock patients \u2014 a pilot study

BackgroundSepsis-associated hyperlactatemia is a widely used biomarker, associated with initial severity and poor outcomes. This increased circulating lactate concentration has been proposed to result in part from a mismatch between oxygen delivery and demand in organs. However, other mechanisms may participate. In particular, a metabolic reprogramming similar to the Warburg effect initially described in cancer cells could lead to increased lactate production by immune cells such as T lymphocytes after sepsis. The objective of this study was to set up a protocol for lactate measurement in T lymphocytes, and to evaluate whether lactate production by T lymphocytes was increased in septic shock patients.MethodsWe first optimized protocols for lactate and pyruvate measurements in T lymphocytes purified from healthy volunteers’ blood, either stimulated with phytohaemagglutinine (PHA) or left untreated. We then conducted a pilot study to confirm the feasibility of this protocol in samples from septic shock patients.ResultsPHA stimulation induced aerobic glycolysis in human lymphocytes ex vivo, with increased lactate and pyruvate productions. To correctly measure this phenomenon, minimal cell number was 250,000 and optimal culture duration was 40 h. We also observed a significant correlation between lactate concentration in T lymphocytes and in their culture supernatants. We were able to measure lactate concentration in T lymphocytes from septic shock patients. Our preliminary results showed that intra-lymphocyte lactate concentration was not different between patients and healthy volunteers.ConclusionThis protocol should now be tested in a larger cohort of patients. The association between immune cell metabolic reprogramming as measured by lactate concentration in T cells and functionality represents an exciting field for research.

Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells.

BackgroundThe chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma.Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways.In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells.MethodsConfocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay.ResultsOur studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity.ConclusionsOur data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression.

Intracellular lactate-mediated induction of estrogen receptor beta (ERβ) in biphasic malignant pleural mesothelioma cells.

Biphasic malignant pleural mesothelioma (MPM) is the second most common histotype of MPM. It is histologically characterized by the concomitant presence of epithelioid and sarcomatoid features, the latter associated with worse prognosis. In this report we describe that silencing of AKT1 in spindle-shaped biphasic MPM cells promotes the shift toward an epithelioid phenotype. Furthermore, AKT1 silencing resulted in decreased expression of the lactate/H+ symporter MCT4 and its chaperone CD147/Basigin, and in the induction of estrogen receptor β (ERβ) expression. We provide evidence that ERβ expression is induced by increased intracellular lactate concentration. Spheroid culturing and tumor growth of ERβ negative biphasic MPM in nude mice resulted in the induction of ERβ expression and response to the selective agonist KB9520. In both models, the treatment with the ERβ agonist results in reduced cell proliferation, decreased expression of MCT4 and CD147/Basigin and increased acetylation and inactivation of AKT1. Collectively, in response to metabolic changes, ERβ expression is induced and exerts an anti-tumor effect through selective agonist activation. The possibility to reverse the more aggressive biphasic mesothelioma histotype by targeting ERβ with a selective agonist could represent a new effective treatment strategy.

Abstract P1-08-03: Targeting lactate metabolism as a novel therapeutic target in platinum-resistant triple negative breast cancer (TNBC)

Abstract Introduction TNBC is a heterogeneous disease and is often associated with expression of a stem cell signature (CD44+, CD24-). Recently platinum agents have been shown to improve the pathological response rates in the neo-adjuvant setting; however a significant proportion of patients with TNBC will relapse. The study objective was to evaluate the potential of targeting glycolysis, mitochondrial function and lactate metabolism in platinum resistant TNBC cell lines. Methods Stem cell signature and glycolytic phenotyping: CD44 and CD24 antibodies were used to measure the percentage of stem cells in each of the cell lines using flow cytometry(FACS). Protein expression of hexokinase, Glut1 transporter, lactate dehydrogenase A/B(LDHA and LDHB), and monocarboxylic transporter 1 and 4 (MCT1 and MCT4) was determined in MCF7(control), MDA-MB231, MDA-MB468, and HCC38 cells. Proliferation and apoptosis assays: IC50s for platinum, metformin, and MCT1 inhibitor AR-C155858, Tocris (MCT1I) were calculated by the sulforhodamine B (SRB) assay. Propidium iodide and AnnexinV staining intensity used to measure apoptosis by FACS. Metabolic analysis: The change in lactate and glucose levels in culture media post-treatment with metformin and MCT1I was quantified by 1H NMR. Intracellular lactate and ATP concentrations were determined by lactate(BioVision) and ATP determination assays(Invitrogen). Mitochondrial membrane potential and Reactive oxygen species (ROS) were measured by MitoTracker Red and MitoSOX Red. 2NBDG was used to assess cell glucose uptake. Combination index SRBs: Cells were treated with increasing concentrations of MCT1I and metformin for 72h. The combination index values were calculated using CalcuSyn® software. Results MDA-MB231 and HCC38 cells were resistant to cisplatin and associated with a stem cell signature (CD44+, CD24-) which was not detected in the other cell lines. MCT1 was highly expressed in HCC38 cells but not at all in MDA-MB231. In contrast, MCT4 was abundantly expressed MDA-MB231 but minimally in HCC38 cells. In these two cell lines, only MDA-MB231 was sensitive to metformin which reduced ATP production, induced ROS generation, and increased the percentage of apoptotic cells. HCC38 also produced ROS, but ATP production and apoptosis were unaffected. HCC38 cells were more sensitive to MCT1I than other cells, which was associated with increased intracellular lactate. The NMR data showed significant increases in lactate in culture media following treatment with metformin in MDA-MB231 and HCC38. Only HCC38 had increased 2NBDG uptake and reduced of glucose levels in culture medium by metformin. Metformin significantly enhanced the anticancer efficacy of MCT1I in HCC38 cells, as a synergistic effect of the two agents was observed across the range of different concentrations; whilst an antagonistic effect was shown in MDA-MB231 cells with almost all concentrations. Conclusions Targeting lactate metabolism selectively inhibits a platinum-resistant stem cell population of TNBC, this effect is enhanced when combined with metformin. The inhibition of glycolysis and lactate metabolism may represent a new therapeutic strategy for this population and warrants further study. Citation Format: Narumi Harada, Vidhya Varghese, Lingmin Xie, Laura M Kenny. Targeting lactate metabolism as a novel therapeutic target in platinum-resistant triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-03.

Hypoxia‐induced carbonic anhydrase IX facilitates lactate transport in human breast cancer cells by non‐catalytic interaction

The most aggressive and invasive tumor cells, which often reside in hypoxic environments, rely on extensive glycolysis to meet their large demand for energy and biosynthetic precursors. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms that regulate lactate transport in MCF‐7 human breast cancer cells under normoxia and hypoxia. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Measurements of intracellular pH and lactate concentration with the FRET‐based lactate nanosensor laconic show that CAIX augments lactate flux via MCT1 by a non‐catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+‐shuttle His200, functions as a “proton‐collecting/distributing antenna” to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of MCF‐7 cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.This work was supported by the Research Initiative Membrane Biology, the Stiftung Rheinland‐Pfalz für Innovation, the Graduiertenfördungskommission Rheinland‐Pfalz, and the DFG.