Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Lactate metabolism is required for inner retinal neuron survival. A , immunofluorescence labeling of retinal transverse cryosections showing MCT2 expression mainly in the inner nuclear layer (INL). Colabeling for PKCα and calretinin illustrates MCT2 expression in RBCs and a subset of ACs. B i – B iv ( top ), ( C ) statistical analysis, and quantification of the cell death assay (TUNEL), performed in organotypic retinal explants, showing occasional TUNEL positive nuclei ( arrows ). When compared to control (CTR, n = 5) treatment with AR-C and Shikonin increased INL cell death, while SR treatment did not. The data were analyzed with the Kruskal-Wallis and Dunn’s multiple comparison post hoc tests. B i -B iv ( bottom ), ( D ) quantification of RBCs per 100 μm retinal length revealed a significant density reduction of this cell type after treatment with SR, AR-C, and Shikonin, supporting a dependence of RBCs on extracellular lactate. The data were analyzed by one-way ANOVA with Tukey’s multiple comparison post hoc test. Each dot reflects a single retinal explant. E , schematic summary, showing the transporters, applied drugs used throughout the investigation, and their respective effects on lactate metabolism. Shikonin and FX-11 inhibit lactate synthesis directly, while AR-C155858 and SR-13800 block lactate transport. Graphs display mean values ± SD; asterisks indicate ∗ p < 0.05, ∗∗ p < 0.01. SR = MCT1 inhibitor; Shikonin = PKM2 inhibitor. ACs, amacrine cells; AR-C = MCT2 inhibitor; INL, inner nuclear layer; LDH-A, lactate dehydrogenase A; MCT1, monocarboxylate transporter one; MCT2, monocarboxylate transporter 2; ONL, outer nuclear layer; PKCα, protein kinase Cα; PKM2, pyruvate kinase M2; RBCs, rod bipolar cells.

Article Snippet: Since there is only a limited number of commercial MCT inhibitors available, we used three potent and specific inhibitors to isolate MCT isoforms: SR-13800 for MCT1 (SR; Tocris, Cat. No. 5431), AR-C155858 (AR-C; Tocris, Cat. No. 4960) for MCT1 and MCT2, and Syrosingopine (Syro; Sigma-Aldrich, Cat. No. SML1908) to inhibit MCT1 and MCT4.

Techniques: Immunofluorescence, Labeling, Expressing, TUNEL Assay, Control, Comparison, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Depolarization of inner retinal cells is reduced and slowed by inhibition of lactate metabolism. A and D , representative images of Fluo4-AM and CoroNa Green-loaded cells in the INL, before and at two time points after bath perfusion with KCl, in controls and retinas incubated with SR, AR-C, and Shikonin. B and E , ( top ), traces of individual experiments in each condition ( red line = mean, gray shadow = SD). B and E , ( bottom ), overview of the kinetic parameters measured in the imaging. C i–iv , statistical analysis of the different parameters in the calcium imaging experiments indicating alterations in the amplitude and time to peak. F i–iv , statistical analysis of the sodium imaging experiments showing specific alterations in all the parameters after MCT2 inhibition. Box plots display the median ± min and max values and the mean in red . Individual values are displayed as open circles ( gray ). The control is represented as a dashed line at 100%, and results are presented as percentage of control. Asterisks indicate ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. INL, inner nuclear layer; MCT, SR= MCT1 inhibitor; AR-C= MCT2 inhibitor; Shikonin = PKM2 inhibitor; FX-11= LDH-A inhibitor. See also Figs. S1 and . LDH-A, lactate dehydrogenase A; MCT, monocarboxylate transporter.

Article Snippet: Since there is only a limited number of commercial MCT inhibitors available, we used three potent and specific inhibitors to isolate MCT isoforms: SR-13800 for MCT1 (SR; Tocris, Cat. No. 5431), AR-C155858 (AR-C; Tocris, Cat. No. 4960) for MCT1 and MCT2, and Syrosingopine (Syro; Sigma-Aldrich, Cat. No. SML1908) to inhibit MCT1 and MCT4.

Techniques: Inhibition, Incubation, Imaging, Control

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Combined MCT1/MCT2 inhibition decreases intracellular lactate levels in inner retinal neurons. A – C , ( left ), from left to right : fluorescence curves showing lactate influx evoked by stimulation with 10 mM lactate, a diagram showing the transporters affected by the application of each drug and a fluorescence trace showing the effect of each drug on the lactate influx. A – C , ( right ), statistical analysis of the response amplitude. An evident response disruption was noted only when MCT2 was inhibited. However, under MCT1 and MCT4 inhibition, the amplitude was unaltered. D , modulation of intracellular lactate levels by transient depolarization. E , ( top ), effects of the inhibition of different MCTs and retinal depolarization on lactate dynamics. Black dashed lines indicate drug-induced change in slope and red dashed lines indicate the effect of drug + KCl. E , ( bottom ), statistical analysis of the slope of the responses under basal conditions (only drug, left ) and depolarization (drug + K + , right ). The data revealed that MCT2 inhibition led to a reduction in intracellular lactate levels, resulting from lactate consumption. This consumption was exacerbated after depolarization in SR, AR-C, and Syro. Data were analyzed using either a paired Student’s t test or Wilcoxon matched-pair test. The black trace represents the average of one experiment, whereas the light gray shadow represents the SD. Graphs display the mean ± SD. ∗ Indicates p < 0.05. SR = MCT1 inhibitor; AR-C = MCT1 and MCT2 inhibitor; Syro = MCT1 and MCT4 inhibitor. The number of experiments is represented as N = number of explants and n = number of cells recorded. See also Figs. S5 and . MCT, monocarboxylate transporter.

Article Snippet: Since there is only a limited number of commercial MCT inhibitors available, we used three potent and specific inhibitors to isolate MCT isoforms: SR-13800 for MCT1 (SR; Tocris, Cat. No. 5431), AR-C155858 (AR-C; Tocris, Cat. No. 4960) for MCT1 and MCT2, and Syrosingopine (Syro; Sigma-Aldrich, Cat. No. SML1908) to inhibit MCT1 and MCT4.

Techniques: Inhibition, Fluorescence, Disruption

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Inhibition of lactate transport through MCT2 induces alterations in RBC currents. A and C , comparison of the voltage-current relationship of the outward current and calcium current under different conditions. B and D , representative recordings to depolarizing voltage steps. Reciprocal feedback was altered only in the AR-C condition ( p = 0.0042) but was unaffected in the SR condition ( p = 0.1957) compared to controls. E and F , in the presence of 20 mM lactate, we observed a decrease in the outward current ( p = 0.0224) and calcium current ( p = 0.0003) only under MCT2 inhibition, but no differences were noted either in the outward current ( p = 0.3925) or calcium current ( p = 0.8146) when only MCT1 was blocked. G , similar results were obtained when we measured the membrane potential, where AR-C ( p = 0.0321) caused a significant depolarization, while the SR condition was not different from controls ( p = 0.0822). Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison post hoc test. Each dot represents a single recorded cell. Graphs represent the mean ± SD; asterisks indicate ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. SR = MCT1 inhibitor; AR-C= MCT1 and MCT2 inhibitor. See also Fig. S3 and , and Table 1 . MCT, monocarboxylate transporter; RBC, rod bipolar cell.

Article Snippet: Since there is only a limited number of commercial MCT inhibitors available, we used three potent and specific inhibitors to isolate MCT isoforms: SR-13800 for MCT1 (SR; Tocris, Cat. No. 5431), AR-C155858 (AR-C; Tocris, Cat. No. 4960) for MCT1 and MCT2, and Syrosingopine (Syro; Sigma-Aldrich, Cat. No. SML1908) to inhibit MCT1 and MCT4.

Techniques: Inhibition, Comparison, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Lactate dynamics of MCs under different conditions. A and B , effect of transient depolarization on intracellular glucose ( A ) and lactate ( B ) levels. C , lactate dynamics after inhibition of different MCTs and retinal depolarization. Black dashed lines indicate drug-induced changes in slope and red dashed lines indicate the effect of drugs + KCl. D , statistical analysis of the slope of the responses in the basal condition (drug only) and under depolarization (drug + K+). Accumulation of intracellular lactate was observed after bath application of different MCT inhibitors. However, this increase was less intense after depolarization under all conditions. Data were analyzed either with paired Student’s t test or Wilcoxon matched-pairs test. Graphs display the mean ± SD. ∗ Indicates p < 0.05. SR = MCT1 inhibitor; AR-C = MCT1 and MCT2 inhibitor; Syro = MCT1 and MCT4 inhibitor. The black trace represents the average response, while the light gray shadow represents the SD. The number of experiments is represented as: N = number of explants; n = number of cells recorded. MCs, Müller cells; MCT, monocarboxylate transporter.

Article Snippet: Since there is only a limited number of commercial MCT inhibitors available, we used three potent and specific inhibitors to isolate MCT isoforms: SR-13800 for MCT1 (SR; Tocris, Cat. No. 5431), AR-C155858 (AR-C; Tocris, Cat. No. 4960) for MCT1 and MCT2, and Syrosingopine (Syro; Sigma-Aldrich, Cat. No. SML1908) to inhibit MCT1 and MCT4.

Techniques: Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Extracellular lactate as an alternative energy source for retinal bipolar cells

doi: 10.1016/j.jbc.2024.106794

Figure Lengend Snippet: Model for th e lactate dy namics in inner retinal cells. General model proposing a consumption of lactate by inner retinal cells. Previous work demonstrated the functional expression of MCT1, MCT2, and MCT4 in MCs, where MCT2 (and to a minor degree also MCT1) mainly regulates lactate influx, while MCT4 mediates lactate efflux, contributing to the accumulation of extracellular lactate. Here, we propose that this extracellular lactate produced by MCs and possibly other retinal cell types is consumed by BCs and ACs through MCT2. ACs, amacrine cells; BCs, bipolar cells; MCs, Müller cells; MCT1, monocarboxylate transporter one; MCT2, monocarboxylate transporter 2; MCT4, monocarboxylate transporter 4.

Article Snippet: Since there is only a limited number of commercial MCT inhibitors available, we used three potent and specific inhibitors to isolate MCT isoforms: SR-13800 for MCT1 (SR; Tocris, Cat. No. 5431), AR-C155858 (AR-C; Tocris, Cat. No. 4960) for MCT1 and MCT2, and Syrosingopine (Syro; Sigma-Aldrich, Cat. No. SML1908) to inhibit MCT1 and MCT4.

Techniques: Functional Assay, Expressing, Produced

Journal: iScience

Article Title: The ATP-exporting channel Pannexin 1 promotes CD8 + T cell effector and memory responses

doi: 10.1016/j.isci.2024.110290

Figure Lengend Snippet: Panx1 controls the activation of effector CD8 + T cells through export of eATP and late extracellular lactate accumulation (A) WT Nur77-GFP CD8 + T cells were activated in the presence of PBS or Panx1i, with the addition of vehicle or eATP. Nur77-GFP representative histograms (left) and average Nur77-GFP + percentages (right) are shown. (B and C) CD4-Cre or CD4-Cre Panx1 fl/fl CD8 + T cells were activated in vitro (anti-CD3/CD28 + IL-2) for up to 48 h, with the addition of vehicle or eATP at either the beginning of cultures (B) or at 20 h after activation (C). (B) Average percentages of CD69 + cells at 3 h after activation. (C) Average percentages of CD44 + and CD69 + cells at 24 h after activation (left); average percentages of CellTracer Violet − cells (CTV % divided) at 48 h after activation (right). (D) CD4-Cre or CD4-Cre Panx1 fl/fl effector-like CD8 + T cells (with prolonged exposure to IL-2) were incubated in the presence or absence of eATP; average percentages of CD69 + cells and of ECAR values are shown. (E) WT (CD4-Cre) or Panx1-KO (CD4-Cre Panx1 fl/fl ) effector-like and memory-like CD8 + T cell cultures were harvested, and intracellular lysates and supernatants were submitted for untargeted metabolomics (GC-MS) analysis. Enrichment analysis showing pathways preferentially represented in the metabolites from the supernatants of WT effector-like CD8 + T cells (WT > Panx1-KO). Levels (arbitrary units [AU]) of l-lactate in the supernatants (left) and intracellular lysates (right) of effector-like WT and Panx1-KO CD8 + T cells are shown below. (F–G) CD4-Cre or CD4-Cre Panx1 fl/fl CD8 + T cells were activated in vitro (anti-CD3/CD28 + IL-2) for 72 h, and l-lactate measurements (mM) were done. (F) Intracellular lactate levels. (G) Extracellular lactate levels, in the presence or absence of inhibitors for MCT1 (SR13800) and MCT4 (VB124). (H) Average percentages of IFNγ + CD8 + T cells at 72 h after activation, with addition of sodium lactate +/− MCT1i. (I) Representative histograms (left) and average percentages of CTV % divided and CD69 + cells at 48 h after activation, with addition of vehicle, MCT1i, MCT4i, or MCT1/MCT4i. (G–K) CD4-Cre or CD4-Cre Panx1 fl/fl CD8 + T cells were activated in vitro (anti-CD3/CD28 + IL-2) for up to 72 h, in the presence of the indicated metabolites or inhibitors. (J) Average percentages of CD44 + and CD69 + cells after 24 h of activation (left) and of CTV % divided cells at 48 h after activation (right), with the addition of vehicle or sodium lactate. (K) ECAR and OCR kinetics (left) and average baseline values (right) at 72 h after activation, with the addition of vehicle or sodium lactate. (L) Average percentages of CD69 + cells at 48 h after activation, with addition or sodium lactate +/− oligomycin. (M) CD4-Cre or CD4-Cre Panx1 fl/fl effector-like CD8 + T cells were incubated in the presence or absence of sodium lactate (right); average percentages of CD69 + cells and of ECAR values are shown. (N) WT Nur77-GFP CD8 + T cells were activated in the presence of PBS or Panx1i, with the addition of vehicle or sodium lactate; average percentages of Nur77-GFP + cells (left), CD69 + cells (center), or CD44 + cells (right) are shown. (O) WT (CD4-Cre) or Panx1-KO (CD4-Cre Panx1 fl/fl ) P14 cells (CD45.2 + ) were transferred into LCMV-infected WT CD45.1 + mice. Some mice were treated with sodium lactate between days 1 and 3 post-infection, and spleen P14 cells were analyzed at day 7 post-infection. Flow cytometry plots showing expression of CD127 and KLRG1 (left) and the average numbers of TE, MP, and DN P14 cells per spleen (right) are shown. (A–D, F–O) Data from two to three independent experiments; n = 3–14 per experimental group. (E) Data from three biological replicates per experimental group (from n = 3 mice per group). ns: not significant ( p > 0.05); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA with Tukey’s post-test (A–D, F–O) unpaired t test (E), two-way ANOVA with Bonferroni’s post-test (M). See also Figures S5 and .

Article Snippet: SR13800 , Tocris , Cat# 5431.

Techniques: Activation Assay, In Vitro, Incubation, Gas Chromatography-Mass Spectrometry, Infection, Flow Cytometry, Expressing

Journal: iScience

Article Title: The ATP-exporting channel Pannexin 1 promotes CD8 + T cell effector and memory responses

doi: 10.1016/j.isci.2024.110290

Figure Lengend Snippet:

Article Snippet: SR13800 , Tocris , Cat# 5431.

Techniques: Purification, Virus, Recombinant, Blocking Assay, Saline, Cell Stimulation, Cell Isolation, Isolation, Infection, In Vitro, Software