MCT1 Expression Research Articles

Effects of Elevated Intraocular Pressure on Retinal Ganglion Cell Density and Expression and Interaction of Retinal Aquaporin 9 and Monocarboxylate Transporters

Introduction: Astrocyte-to-neuron lactate shuttle (ANLS) plays an important role in the energy metabolism of neurons, including retinal ganglion cells (RGCs). Aquaporin 9 (AQP9), which is an aquaglyceroporin that can transport lactate, may be involved in ANLS together with monocarboxylate transporters (MCTs) to maintain RGC function and survival. This study aimed to investigate the impact of elevated intraocular pressure (IOP) on AQP9-MCT interaction and RGC survival. Methods: IOP was elevated in Aqp9 knock-out (KO) mice and wild-type (WT) littermates by anterior chamber microbead injection. RGC density was measured by TUBB3 immunostaining on retinal flat mounts. Immunolabeling, immunoblot, and immunoprecipitation were conducted to identify and quantitate expressions of AQP9, MCT1, MCT2, and MCT4 in whole retinas and ganglion cell layer (GCL). Results: Aqp9 KO and WT mice had similar RGC density at baseline. Microbead injection increased cumulative IOP by approximately 32% up to 4 weeks, resulting in RGC density loss of 42% and 34% in WT and Aqp9 KO mice, respectively, with no statistical difference. In the retina of WT mice, elevated IOP decreased the amount of AQP9, MCT1, and MCT2 protein and changed the AQP9 immunoreactivity and reduced MCT1 and MCT2 immunoreactivities in GCL. Meanwhile, it decreased MCT1 and increased MCT2 that interact with AQP9, without affecting MCT4 expression. Aqp9 gene deletion increased baseline MCT2 expression in the GCL and counteracted IOP elevation regarding MCT1 and MCT2 expressions. Conclusion: The compensatory upregulation of MCT1 and MCT2 with Aqp9 gene deletion and ocular hypertension may reflect the need to maintain lactate transport in the retina for RGC survival.

Dietary potential probiotics and enzymes complex modulates the performance and rumen microbiota in weaned goats.

This trial was performed to investigate the effects of combined feeding of Candida utilis CICC 31170, Bacillus coagulans R11, and Lactobacillus acidophilus NCFM and a multi-enzyme complex on the growth performance, immune parameters, feed digestibility, and rumen microbiota of weaned goats. Thirty weaned goats were randomly divided into CON, PRB, and COB groups and fed different diets. End weight and ADG increased significantly in the PRB and COB groups (P<0.05), and ADFI increased significantly in COB (P<0.05). On day 80, there was a significant increase in IL-10 content in PRB and COB compared to the CON (P<0.05). Highly significant increases in rumen papilla width, epithelial cell thickness, stratum spinosum+basale thickness, and stratum corneum thickness were found in PRB and COB (P<0.05). COB group significantly increased the gene expression of HMGCL and MCT1 in rumen epithelium (P<0.001). The COB group had the tendency to increase the feed digestibility of dry matter and crude fat compared with the CON group (P<0.10). The abundance of Prevotellaceae_unclassified was significantly higher in PRB (P<0.05), and the abundance of Fibrobacteres was significantly higher in COB in comparison to those in CON (P<0.05). The results indicate that the dietary potential probiotics and enzymes complex could modulate the growth performance, immunity, feed digestibility, and rumen microbiota in weaned goats.

Monocarboxylate transporter 4 inhibition potentiates hepatocellular carcinoma immunotherapy through enhancing T cell infiltration and immune attack.

Monocarboxylate transporter (MCT) 4 is a high-affinity lactate transporter that is primarily involved in the maintenance of intracellular pH homeostasis and highly expressed in different tumors. However, the role of MCT4 in modulating immune responses against HCC remains unknown. In this study, we demonstrated that MCT4 was overexpressed in HCC, which was associated with poor prognosis in patients. Genetic or pharmacological inhibition of MCT4 using VB124 (a highly potent MCT4 inhibitor) suppressed HCC tumor growth in immunocompetent mice model by enhancing CD8 + T cell infiltration and cytotoxicity. Such improved immunotherapy response by MCT4 targeting was due to combined consequences characterized by the alleviated acidification of tumor microenvironment and elevated the chemokine (C-X-C motif) ligand (CXCL) 9/CXCL10 secretion induced by reactive oxygen species/NF-κB signaling pathway. Combining MCT4 inhibition improved the therapeutic benefit of anti-programmed cell death 1 immunotherapy in HCC and prolonged mice survival. Moreover, higher MCT4 expression was observed in tumor tissues from nonresponder patients with HCC receiving neoadjuvant therapy with toripalimab. Our results revealed that lactate exportation by MCT4 has a tumor-intrinsic function in generating an immunosuppressive HCC environment and demonstrated the proof of the concept of targeting MCT4 in tailoring HCC immunotherapeutic approaches.

Expression of HIF-1α and Genes Involved in Glucose Metabolism Is Increased in Cervical Cancer and HPV-16-Positive Cell Lines.

Cervical cancer (CC) is the most common cancer in women in the lower genital tract. The main risk factor for developing CC is persistent infection with HPV 16. The E6 and E7 oncoproteins of HPV 16 have been related to metabolic reprogramming in cancer through the regulation of the expression and stability of HIF-1α and consequently of the expression of its target genes, such as HIF1A (HIF-1α), SLC2A1 (GLUT1), LDHA, CA9 (CAIX), SLC16A3 (MCT4), and BSG (Basigin or CD147), which are involved in glucose metabolism. This work aimed to evaluate the expression of HIF-1α, GLUT1, LDHA, CAIX, MCT4, and Basigin in patient samples and CC cell lines. To evaluate the expression level of HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG genes in tissue from patients with CC and normal tissue, the TCGA dataset was used. To evaluate the expression level of these genes by RT-qPCR in CC cell lines, HPV-negative (C-33A) and HPV-16-positive (SiHa and Ca Ski) cell lines were used. Increased expression of HIF1A, SLC2A1, LDHA, SLC16A3, and BSG was found in Ca Ski and CA9 in SiHa compared to C-33A. Similar results were observed in CC tissues compared to normal tissue obtained by bioinformatics analysis. In conclusion, the expression of HIF-1α, GLUT1, LDHA, CAIX, MCT4, and BSG genes is increased in CC and HPV-16-positive cell lines.

Formosanin C inhibits non-small-cell lung cancer progression by blocking MCT4/CD147-mediated lactate export

BackgroundTumor cells reprogram their metabolic network to maintain their uncontrolled proliferation, metastasis, and resistance to cancer therapy. Treatments targeting abnormal cellular metabolism may have promising therapeutic effects. Formosanin C (FC), a diosgenin derived from the rhizoma of Paris polyphylla var. yunnanensis, has shown potent anti-cancer activities against various cancer types. However, the effect of FC on cancer metabolism remains to be elucidated. PurposeIn this research, we aimed to elucidate FC's effect and potential mechanisms on metabolism in lung cancer. MethodsColony formation, transwell cell migration, and apoptosis were detected in multiple NSCLC cell lines to assess the cytotoxicity of FC. 1H NMR metabolomics approach was applied to screen the differential metabolites in H1299 cells and the culture medium. Western blotting, flow cytometry, and other molecular biological techniques were performed to verify the latent mechanism involved in metabolites. An allograft tumor model was employed to investigate the anti-tumor effects of FC in vivo. ResultsFC significantly inhibited monoclonal formation and migration and induced cell cycle arrest and apoptosis in NSCLC cells. FC altered the abundances of 12 metabolites in lung cancer cells and 3 metabolites in the medium. These differential metabolites are primarily involved in glycolysis, citric acid cycle, and glutathione pathways. Notably, there was a remarkable increase in intracellular lactate and a reduction in extracellular lactate after FC treatment. Mechanically, FC downregulated the expression of MCT4 and CD147, blocking the export of lactate. Furthermore, FC also evoked mitochondrial dysfunction coupled with excessive oxidative stress, decreased mitochondrial membrane potential, ATP production reduction, glutathione depletion, and Ca2+ overload. Moreover, FC suppressed tumor progression in vivo with reduced protein levels of the MCT4 and CD147 in tumor tissues. ConclusionFC inhibits lung cancer growth by the novel mechanism in which MCT4/CD147-mediated inhibition of lactate transport and disruption of mitochondrial functions are involved.

Imaging of lactate metabolism in retinal Müller cells with a FRET nanosensor

Müller cells, the glial cells of the retina, provide metabolic support for photoreceptors and inner retinal neurons, and have been proposed as source of the significant lactate production of this tissue. To better understand the role of lactate in retinal metabolism, we expressed a lactate and a glucose nanosensor in organotypic mouse retinal explants cultured for 14 days, and used FRET imaging in acute vibratome sections of the explants to study metabolite flux in real time. Pharmacological manipulation with specific monocarboxylate transporter (MCT) inhibitors and immunohistochemistry revealed the functional expression of MCT1, MCT2 and MCT4 in Müller cells of retinal explants. The introduction of FRET nanosensors to measure key metabolites at the cellular level may contribute to a better understanding of heretofore poorly understood issues in retinal metabolism.

Dual Roles of Lactate in EGFR-TKI-Resistant Lung Cancer by Targeting GPR81 and MCT1.

Lactate is critical in modeling tumor microenvironment causing chemotherapy resistance; however, the role of lactate in tyrosine kinase inhibitor (TKI) resistance has not been fully known. The aim of this study was to evaluate whether lactate could mediate TKI resistance through GPR81 and MCT1 in non-small-cell lung cancer (NSCLC). Here, we showed that lactate enhanced the cell viability and restrained erlotinib-induced apoptosis in PC9 and HCC827 cells. GPR81 and AKT expression were significantly increased with the addition of lactate, and siGPR81 reduced AKT expression resulting in a raised apoptosis rate with erlotinib treatment. Furthermore, we found that lactate also promoted MCT1 exposure, and inhibiting MCT1 with AZD3965 markedly impaired the glycolytic capacity. A significant increase of GPR81 and MCT1 expression was observed in insensitive tissues compared with sensitive ones by immunostaining in NSCLC patients. Our results indicate that lactate adopts dual strategies to promote TKI resistance in NSCLC, not only activating AKT signaling by GPR81, but also giving energy supply through MCT1-mediated input. Targeting GPR81 and MCT1 may provide new therapeutic modalities for TKI resistance in NSCLC.

Fibroblast-epithelial metabolic coupling in laryngeal cancer.

To explore the Fibroblast-epithelial metabolic coupling among laryngeal cancers and its prognostic roles METHODS: We reviewed the clinical information of patients with laryngeal cancer in our department. Paraffin-embedded tissues from included patients were immune-stained with antibodies towards MCT4 and TOMM20 and evaluated for stromal and epithelial expression. Survival analysis and Cox regression analysis were applied to investigate the prognostic factor of laryngeal squamous cell carcinoma. TCGA database was used to validate our result. Stromal MCT4 and TOMM20 were both significantly associated with each other among laryngeal cancer tissues. High expression of both Stromal MCT4 and TOMM20 is related to poor prognosis in laryngeal cancer. Stromal MCT4 expression was an independent prognostic indicator for laryngeal cancer. Furthermore, cancer cell MCT4 expression has no relationship with the clinical characteristics of laryngeal cancer. Our results support that the phenomenon of metabolic symbiosis was exist in the laryngeal cancer tissue. In addition, TOMM20 and stromal MCT4 could be used as new therapeutic targets for laryngeal cancer.

Роль экспрессии монокарбоксилатов первого и четвертого типов (MCT1, MCT4) опухолевыми и стромальными клетками рака простаты в определении прогноза заболевания и эффективности радикального лечения

A search for new methods for diagnosing clinically significant prostate cancer is of importance due to the insufficient accuracy of modern methods in detecting aggressive tumors. One of the promising opportunities for the early diagnosis of clinically significant prostate cancer is the assessment of the glycolytic profile of the tumor by determining the expression of monocarboxylates (MCT) types 1 and 4 in tumor cells, as well as in adjacent stromal cells.An analysis of patients of who underwent radical prostatectomy at the Institute of Urology and Reproductive Health of Sechenov University from 2015 to 2017 was carried out. The patients with histologically confirmed prostate adenocarcinoma were included in the study. Among them, the presence or absence of biochemical recurrence during the first year was studied. An immunohistochemical (IHC) study of postoperative specimen was performed to determine the expression of MCT1 and MCT4 by tumor and stromal cells. The correlation between the intensity of their expression and the risk of biochemical recurrence and the tumor characteristics was evaluated.High membrane expression of MCT1 directly correlated with high stromal expression of MCT4 (r=0.314, p<0.003). A significant direct correlation was found between the predominance of stromal expression of MCT4 over membrane expression and biochemical recurrence (r=0.403, p<0.001), as well as a high ISUP group (4 and 5) (r=0.294, p=0.005).Determination of the level of expression of type 1 and 4 monocarboxylate transporters in adenocarcinoma cells and tumor stromal cells can become an effective tool for risk stratification, and may also predict the biological behaviors of the prostate cancer and the efficiency of definitive treatment.

Autophagy induces mTOR-dependent glucose uptake and mTOR-independent lactate utilization in cadmium-treated A549 cells

Cadmium (Cd) is a non-essential heavy metal with many harmful effects, especially tumorigenesis. Previously we established that autophagy-dependent increasing of glycolysis played an important role in Cd-induced cell growth and migration of A549 and HELF cells. In this study, we found Cd could induce autophagy and mTOR in A549 cells, HELF cells and in lung tissues of BALB/c mice. More interestingly, Cd-induced elevation of mTOR was autophagy-dependent and autophagy-induced cell growth and glycolysis was mTOR-dependent. However, in A549 cells, besides the above mTOR-dependent pathway, Cd-induced autophagy could directly induce PKM2 and LDHA independent of mTOR. Further study showed that only in A549 cells could autophagy other than mTOR enable Cd to increase MCT1 expression and MCT1 was involved in autophagy-induced PKM2 and LDHA. Sodium lactate added in the culture medium promoted Cd-induced cell growth of A549 cells, while had no effect on HELF cells. Finally, the effect of autophagy/MCT1/PKM2 pathway on lactate utilization to facilitate Cd-induced A549 cell growth was determined. Above all, we concluded that in HELF cells, autophagy induced mTOR-dependent glycolysis in which GLUT1 and HKII was elevated to promote glucose intake to accelerate cell growth. Whereas, in A549 cells, besides the above pathway to use glucose, autophagy could induce an mTOR-independent glycolysis pathway in which lactate could be used as fuel through autophagy-MCT1-PKM2 to escape glucose deficiency.

355 (PB135) - Cellular transporters as novel metabolic immune checkpoints: NGY-091, a small molecule dual MCT1/4 inhibitor for immuno oncology

Background: Aberrant metabolic reprogramming of malignant cells is known to drive cancer progression and metastasis. The increase in glycolytic flux in cancer cells creates a lactate-rich tumor microenvironment (TME), which tumors exploit by expanding immunosuppressive cell populations such as Tregs and MDSCs that thrive on lactate as a fuel source. Blockade of lactate export from glycolytic cancer cells, while inhibiting lactate uptake by suppressive immune cells, is a novel therapeutic strategy to treat cancer. Lactate transport in cells is predominantly mediated by MCT1 and MCT4 transporters. We have developed a compound, NGY-091, a first-in-class small molecule dual inhibitor of the MCT1 and MCT4 lactate transporters. Materials and Methods: The direct cytotoxic effect of NGY-091 was examined in multiple cancer cell lines. The on-target activity of NGY-091 was validated by measuring intracellular and extracellular lactate levels. in vivo efficacy of NGY-091 was studied using CDX, PDX and syngeneic tumor models. Immune profiling of tumor was performed 2 weeks after treating animals with NGY-091 by flow cytometry. Results: NGY-091 treatment exhibited a potent in vitro cytotoxicity against cancer cells with various levels of MCT1 and MCT4 expressions. NGY-091 strongly blocked lactate import through MCT1 under high lactate conditions and lactate export through MCT4 in high glucose conditions. NGY-091 treatment also inhibited pyruvate entry into the mitochondria through MPC1. Furthermore, direct in vivo tumor cell killing was evident in multiple human CDX and PDX models with the treatment of NGY-091. In syngeneic models of 4T1 and MC38 murine tumors, we observed significant tumor growth inhibition with NGY-091 treatment, and strong synergistic tumor reduction when treatment was combined with various immune checkpoint inhibitors. Profiling of lymphoid and myeloid cells in NGY-091 treated tumors by flow cytometry revealed a significant alteration in immune architecture suggesting activation of antitumor immunity. NGY-091 treated tumors induced a profound increase in effector T cell populations, CD8/Treg ratio, and tumor-suppressive M1 macrophages, while significantly downregulating M2 macrophages. To further investigate if NGY-091 directly alters immune cell activation and functionality in vitro, we treated CD4 T, CD8T, Tregs, and MDSCs in a lactate-rich culture condition, which mimicked the lactate level in the TME. NGY-091 treatment strongly increased effector CD4+ and CD8+ T cells, meanwhile it significantly reduced suppressive function of Treg and MDSCs in vitro. Conclusions: These findings indicate the direct effect of NGY-091 on immune cells and validate the in vivo observations in 4T1 tumors. Therefore, NGY-091 intervenes with two key hallmarks of cancer, metabolism and immunity and provides a novel modality to therapeutically target cancer. Conflict of interest: Ownership: Nirogy Therapeutics

Different Expression Patterns of Metabolic Reprogramming Proteins in Testicular Germ Cell Cancer

Metabolic reprogramming is an emerging hallmark of cancer, involving the overexpression of metabolism-related proteins, such as glucose and monocarboxylate transporters and intracellular glycolytic enzymes. The biology of testicular germ cell tumors (TGCTs) is very complex, and although their metabolic profile has been scantily explored, some authors have recently reported that the metabolic rewiring of cancer cells resulted in an association with aggressive clinicopathological characteristics. In this study we have investigated, by immunohistochemical analysis, the expression of key proteins sustaining the hyperglycolytic phenotype in pure seminoma (SE, nr. 35), pure embryonal carcinoma (EC, nr. 17) tissues samples, and normal testes (nr. 5). GLUT1, CD44, PFK-1, MCT1, MCT4, LDH-A, and PDH resulted in more expression in EC cells compared to SE cells. TOM20 was more expressed in SE than in EC. GLUT1, MCT1, and MCT4 expression showed a statistically significant association with SE histology, while for EC, the association resulted in being significant only for GLUT1 and MCT4. Finally, we observed that EC resulted as negative for p53, suggesting that the GLUT1 and MTC overexpression observed in EC could be also attributed to p53 downregulation. In conclusion, our findings evidenced that EC exhibits a higher expression of markers of active aerobic glycolysis compared to SE, suggesting that the aggressive phenotype is associated with a higher glycolytic rate. These data corroborate the emerging evidence on the involvement of metabolic reprogramming in testicular malignancies as well, highlighting that the metabolic players should be explored in the future as promising therapeutic targets.

Shoutai Wan Improves Embryo Survival by Regulating Aerobic Glycolysis of Trophoblast Cells in a Mouse Model of Recurrent Spontaneous Abortion.

Background During embryo implantation, the blastocyst exhibits a high capacity for aerobic glycolysis, which results in a unique microenvironment of high lactate/low pH at the maternal-fetal interface. Shoutai Wan (STW) is an effective Chinese herbal formula widely used in the clinical treatment of recurrent spontaneous abortion (RSA). However, the specific molecular mechanism by which STW prevents abortion is yet to be elucidated. Methods Female CBA/J mice were allocated into six groups randomly and then mated with BALB/c mice as the control group, DBA/2 mice as the RSA model, CBA/J×DBA/2 mice treated with dydrogesterone as the DQYT group, or CBA/J×DBA/2 mice treated with low, medium, and high-dose STW as the STW-L, STW-M, and STW-H groups, respectively. Drug administration started 14 days before mating and ended on the 14th day of pregnancy. The embryo loss rate of each group was calculated on day 14 of gestation, and the pregnancy outcomes of the mice in each group were observed. The mouse serum was collected to determine the levels of progesterone (P) and chorionic gonadotropin (CG). The activities of HK2, PKM2, and LDHA, the key glycolytic enzymes in each group, were detected. The expressions of lactate, ATP, HK2, PKM2, LDHA, MCT4, GLUT1, and GPR81 as well as the morphology of trophoblast cells were examined. Results The embryo loss rate and adverse pregnancy outcomes were significantly increased (P < 0.05) in the RSA model group. After dydrogesterone or different doses of STW treatment, the embryo loss rate and adverse pregnancy outcomes were rescued to varying degrees (P < 0.05). Interestingly, there was no significant difference among the groups in terms of serum P and CG (P < 0.05). Moreover, the activities of key glycolytic enzymes, lactate, ATP, HK2, PKM2, LDHA, MCT4, GLUT1, GPR81 protein or mRNA expression, and morphological abnormalities of trophoblast cells improved significantly in the RSA mice after dydrogesterone or different doses of STW treatment (P < 0.05). Conclusion STW can promote aerobic glycolysis in trophoblast cells of RSA mouse embryos, thereby improving the microenvironment of the maternal-fetal interface and enhancing embryo implantation.

Involvement of SLC16A1/MCT1 and SLC16A3/MCT4 in l-lactate transport in the hepatocellular carcinoma cell line.

Fourteen isoforms of the monocarboxylate transporter (MCT) have been reported. Among the MCT isoforms, MCT1, MCT2, and MCT4 play a role in l-lactate/proton cotransport and are involved in the balance of intracellular energy and pH. Therefore, MCT1, MCT2, and MCT4 are associated with energy metabolism processes in normal and pathological cells. In the present study, we evaluated the expression of MCT1, MCT2, and MCT4 and the contribution of these three MCT isoforms to l-lactate uptake in hepatocellular carcinoma (HCC) cells. In HepG2 and Huh-7 cells, l-lactate transport was pH-dependent, which is characteristic of MCT1, MCT2, and MCT4. Furthermore, l-lactate uptake was selectively inhibited by MCT1 and MCT4 inhibitors in HepG2 and Huh-7 cells. Kinetic analysis of HepG2 cells demonstrated that l-lactate uptake was biphasic. Although the knockdown of MCT1 and MCT4 in the HepG2 cells decreased the uptake of l-lactate, the knockdown of MCT2 had no effect on the uptake of l-lactate. Consequently, we concluded that both MCT1 and MCT4 were involved in the transport of l-lactate in HepG2 and Huh-7 cells at pH 6.0. In contrast, PXB-cells, freshly isolated hepatocytes from humanized mouse livers, showed lower MCT4 expression and l-lactate uptake at pH 6.0 compared to that in HCC cell lines. In conclusion, MCT4, which contributes to l-lactate transport in HCC cells, is significantly different in HCC compared to normal hepatocytes, and has potential as a target for HCC treatment.

Black Ginger (Kaempferia parviflora) Extract Enhances Endurance Capacity by Improving Energy Metabolism and Substrate Utilization in Mice.

Black ginger (Kaempferia parviflora) extract (KPE), extracted from KP, a member of the ginger family that grows in Thailand, has a good promotion effect on cellular energy metabolism and therefore has been used to enhance exercise performance and treatment of obesity in previous studies. However, the effect of single-dose administration of KPE on endurance capacity has not been thoroughly studied, and whether the positive effect of KPE on cellular energy metabolism can have a positive effect on exercise capacity in a single dose is unknown. In the present study, we used a mouse model to study the effects of acute KPE administration 1 h before exercise on endurance capacity and the underlying mechanisms. The purpose of our study was to determine whether a single administration of KPE could affect endurance performance in mice and whether the effect was produced through a pro-cellular energy metabolic pathway. We found that a single administration of KPE (62.5 mg/kg·bodyweight) can significantly prolong the exercise time to exhaustion. By measuring the mRNA expression of Hk2, Slc2a4 (Glut4), Mct1, Ldh, Cd36, Cpt1β, Cpt2, Lpl, Pnpla2 (Atgl), Aco, Acadm (Mcad), Hadh, Acacb (Acc2), Mlycd (Mcd), Pparg, Ppargc1a (Pgc-1α), Tfam, Gp, Gs, Pfkm, Pck1 (Pepck), G6pc (G6pase), Cs, and Pfkl in skeletal muscle and liver, we found that acute high-concentration KPE administration significantly changed the soleus muscle gene expression levels (p < 0.05) related to lipid, lactate, and glycogen metabolism and mitochondrial function. In gastrocnemius muscle and liver, glycogen metabolism-related gene expression is significantly changed by a single-dose administration of KPE. These results suggest that KPE has the potential to improve endurance capacity by enhancing energy metabolism and substrate utilization in muscles and liver.

The Blastocyst Creates a Specialized Microenvironment to Promote Endometrial Receptivity and Implantation

Background: At the time of implantation, the blastocyst produces a significant amount of lactic acid (LA), creating a microenvironment characterized by high lactate and low pH. Historically considered a ‘by-product’ of metabolism, studies have revealed lactate to be a key regulator of cell function, with a lactate-specific receptor, GPR81, recently identified. Further, lactate is an important signalling molecule in cancers promoting ECM breakdown, angiogenesis and immunosuppression, processes vital for successful implantation. Hence, LA could be an early embryonic signal to facilitate endometrial remodelling for receptivity and implantation. Aim: To determine the role of blastocyst-derived lactate on cellular functions/remodelling and transcriptional changes essential for endometrial receptivity and successful implantation. Methods: To assess functional changes to the endometrium, hormonally primed ECC-1 and Ishikawa cells were exposed to LA, LA + neutralized pH (nLA) or acidic pH (pHL), before analysis of tight junction integrity (TER), cellular proliferation and changes to gene expression. LA’s effects on endometrial stromal cell decidualization and migratory capacity, as well as HUVEC endothelial tube formation/angiogenesis, were also determined. Results: Cellular TER and proliferation were significantly downregulated in ECC-1 cells exposed to 2.5, 5 and 7.5mM LA ( P&lt;0.01), while exposure to nLA or pHL alone had no effect. Upregulation of GLUT4, GPR81, VEGF, SNAI1, and RELA mRNA expression was observed following exposure of Ishikawa cells to combined LA + pHL(P&lt;0.05), while MCT-1 expression decreased. Additionally, 5mM LA increased the migratory capacity of decidualized stromal cells (P&lt;0.05) without changing the extent of decidualization. HUVEC tube formation was significantly increased by 5mM LA exposure ( P&lt;0.01). Conclusion: The specialized microenvironment created by the blastocyst, of both high lactate and low pH, enhances endometrial remodelling for receptivity and implantation through functional changes to epithelial, stromal and endothelial cells in the endometrium. Therefore, LA appears to be an important signalling molecule in the maternal-fetal dialogue underpinning implantation.

MOG analogues to explore the MCT2 pharmacophore, α-ketoglutarate biology and cellular effects of N-oxalylglycine

α-ketoglutarate (αKG) is a central metabolic node with a broad influence on cellular physiology. The αKG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other αKG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving compound stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that, under our experimental conditions, glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG.

P5. Monocarboxylate transporter expression directly affects degeneration of the intervertebral disc in mice

<h3>BACKGROUND CONTEXT</h3> Disc degeneration is the central component involved in numerous common spinal pathologies. Low back pain remains a leading cause of disability in the world, yet treatment remains nonspecific and of poor efficacy. Degenerative disc disease (DDD) affects millions of people every year, yet the mechanisms driving degeneration remain poorly understood. Recent work has shown that high levels of intracellular lactate are involved in driving disc degeneration. Whether this loss of ability to export lactate is specific to a few models or common across modes of degeneration is yet to be determined. <h3>PURPOSE</h3> We utilized different mouse models of intervertebral disc (IVD) degeneration in order to better understand the role of lactate and lactate transport in DDD. <h3>STUDY DESIGN/SETTING</h3> Randomized, controlled animal trial. <h3>PATIENT SAMPLE</h3> Female mice (C57BL/6J, n=30). <h3>OUTCOME MEASURES</h3> Mice were euthanized at 2, 4 and 8 weeks. The IVD was evaluated by histological and immunofluorescence (IF) analysis. RNA extraction from disc tissue was analyzed with QPCR. Data are presented as means +/-SD. <h3>METHODS</h3> Female mice (C57BL/6J, n=30) were randomly assigned to one of four models of disc degeneration: 1) lumbar disc poke, 2) tail disc poke, 3) spinal instability and 4) sham. In group 1, a retroperitoneal approach exposed the IVD of the lumbar spine, and a 27G needle was used to injure the disc. In group 2, the needle was inserted in the tail IVD. In group 3, lumbar instability is induced by resection of bilateral facet joints and supra/interspinous ligaments. A sham group was used for each. <h3>RESULTS</h3> In group 1 and 2, the degenerative process at 2, 4 and 8 weeks was characterized by loss of nucleus pulposus (NP) cells and the gradual increase in matrix components. The distinction between NP and annulus fibrosus (AF) or endplate cartilage (ECP) was lost. Sham mice had no significant disc degeneration. There was increased expression of COL10 and MMP13 in the NP and decreased expression of GLUT1 (NP marker) in the disc-poke groups. The expression of a central lactate transporter (MCT4) was also reduced in injured mice, and there was an increase in catabolic chondrocyte markers. In group 2 there was also decreasedhexokinase-2 (HK2) expression and an increase in MCT3 expression. In group 3, disordered AF was seen 2 weeks postoperatively, with decreased disc space and fewer NP cells. The proteoglycan inner layer of AF and periphery of NP were decreased at 8 weeks. If confirmed an increase in COL10 and MMP13 in the inner AF and NP. Both NP and CEP of sham mice maintained strong expression of MCT4 while the NP and CEP in injured mice had reduced expression of MCT4. <h3>CONCLUSIONS</h3> Our results demonstrate a common molecular pathway whereby discs degenerate after direct injury or after becoming unstable. In our model there is rapid degeneration of the IVD in mice who exhibit up/down-regulation of several key markers. Importantly, MCT4 is down-regulated, while MCT3 is upregulated. Decreased MCT4 expression is a key and common step in the degeneration of the IVD. While MCT4 is associated with lactate exportation, and its loss results in elevated intracellular lactate and disc degradation, MCT3 is rarely expressed and may be acting as a "rescue" lactate transporter here. Decreased HK2 gene expression led to reduced glycolysis and mitochondrial dysfunction, leading to increased formation of ROS and oxidative stress; HK2 may be important to disc degeneration. We demonstrated a regulatory role for lactate transport in DDD, with decreased lactate exportation common to different degenerative mechanisms. Promoting MCT4 or MCT3 gene expression may represent a novel therapeutic option in DDD. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

198. Lactate shuttle: a key component in osteosarcoma

<h3>BACKGROUND CONTEXT</h3> Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents and prognosis remains poor, particularly when in the spine. However, the unique metabolism of OS may allow for novel, targeted therapies. Increased glucose uptake and upregulated glycolysis in aerobic conditions are hallmarks of cancer (the"Warburg effect"). Lactate, once considered a mere byproduct of this increased glycolysis, has now proven to be a critical metabolite in most tumors, including OS. Recent work has highlighted a role for lactate transport in osteosarcoma, although whether OS cells produce or consume lactate is currently debated. Here we present data exploring the role of lactate transport in osteosarcoma by examining up and down regulation of the major lactate transporters, the monocarboxylate transporters (MCTs). <h3>PURPOSE</h3> Due to the poor prognosis of spinal osteosarcoma, identifying novel targets for cancer therapy is crucial. Here, we studied the importance of lactate as a metabolite for OS progression. Drugs specific to non-invasive biomarkers of metabolism such as lactate transporters in OS cells could help design future clinical trials. <h3>METHODS</h3> Gene expression profiles of various OS cell lines (SaOs, MNNG, HOS) in varying metabolic conditions (varying lactate and glucose concentrations) were analyzed. qPCR and immunoblotting were used to analyze expression patterns of overall MCT1 and MCT4, and surface expression of MCTs were determined using immunofluorescence microscopy. For each trial, cell proliferation was analyzed via CyQuant direct cell proliferation kit (Fischer Sci). Experiments were performed in triplicate. Conditions included combinations of low, normal, and high glucose (1, 6, and 20 mM respectively) with low, and high lactate (0.1, 1, and 5 mM respectively). All data are presented as the means ± SD where appropriate. We performed statistical analysis using Student'st-test; p less than 0.05 was considered statistically significant. <h3>RESULTS</h3> MCT1 and MCT4 were expressed in OS cell lines. MCT4 expression was upregulated in high glucose (20mM) conditions, but reduced drastically in the absence of glucose. On the other hand, MCT1 expression was not perturbed even in the absence of glucose. Conversely, MCT1, which is a bidirectional transporter of lactate, is expressed irrespective of presence of glucose. Finally, use of commercial MCT inhibitors namely AZD396, BAY8002, BAY87-2243 and Syrosingopine resulted in decreased cell survival, with simultaneous inhibition of MCT1 and MCT4 leading to the most profound effects. <h3>CONCLUSIONS</h3> The main lactate exporter, MCT4, was upregulated in high glucose conditions, indicating an increase in glycolysis to lactate with increased lactate export. Further, cells were not viable when cultured with lactate alone, unlike numerous other nontumorous tissues that uptake and use lactate as a fuel. OS cells appear to preferentially produce and export lactate, particularly if glucose is abundant, regardless of the local oxygen tension (the "Warburg effect"). Together these data suggest that lactate exportation via MCT4s is critical to osteosarcomagenesis and may serve as a unique target for spinal osteosarcomas. <h3>FDA DEVICE/DRUG STATUS</h3> AZD 3695 (Investigational/Not Approved), Syrosingopine (Not approved for this indication), BAY-8002 (Not approved for this indication)

Low oxygen tension suppresses the death of chondrocyte-like ATDC5 cells induced by interleukin-1ß.

The articular cartilage is an avascular tissue, and oxygen tensions in its superficial and deeper zones are estimated to be 6% and 1%. Degeneration of the articular cartilage begins from the surface zone in osteoarthritis. We previously reported that monocarboxylate transporter-1, a transmembrane transporter for monocarboxylates, played an essential role in the interleukin-1β-induced expression of NADPH oxidase-2, a reactive oxygen species-producing enzyme, and reactive oxygen species-dependent death of mouse chondrogenic ATDC5 cells cultured in a normal condition (20% oxygen). Here, we investigated the effect of oxygen tension on interleukin-1β-induced events described above in ATDC5 cells. Interleukin-1β induced the death of ATDC5 cells under 20% and 6% oxygen but did not under 2% and 1% oxygen. Interleukin-1β induced Mct1 (monocarboxylate transporter-1 gene) and Nox2 (NADPH oxidase-2 gene) mRNAs' expression under 20% oxygen in 24h, respectively, but not under 2% oxygen. On the other hand, a 24-h incubation with interleukin-1β upregulated the expression of Nos2 (inducible nitric oxide synthase gene) mRNA irrespective of oxygen tension. Furthermore, inhibition of I-κB kinase suppressed the interleukin-1β-induced expression of Mct1 mRNA in the cells cultured under 20% and 2% oxygen, indicating NF-κB plays an essential role in the induction of the Mct1 gene expression. The results suggest that interleukin-1β induces monocarboxylate transporter-1 in an oxygen tension-dependent manner required for cell death in ATDC5 cells. These results might explain some part of the degenerative process of the articular cartilage, which begins from its superficial zone in the pathogenesis of osteoarthritis.