Journal: eLife

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

doi: 10.7554/eLife.64821

Figure Lengend Snippet: ( a ) Left: Representative traces of two different readouts of the Pyronic sensor, expressed in the cytosol of a dentate granule cell (DGC). The lifetime of the donor species mTFP (black line, left Y-axis) is overlapped to the change in the ratio of intensities between the donor and the acceptor species (Δ mTFP/Venus), expressed as the percent change over the initial mTFP/Venus value (gray line, right Y-axis). Both readouts transiently increase in response to a brief exposure to 10 mM pyruvate for 2 min in the bath solution. The application of 2 µM UK5099, an inhibitor of the mitochondrial pyruvate carrier, steadily increased the Pyronic signal to a plateau after 30–35 min. The bars indicate the times of application of both pyruvate and UK5099. Right: Quantification of Pyronic mTFP lifetimes before and after the treatment with UK5099. Data points obtained before and after the application of UK5099 are connected by lines. Box plots represent the 25–75% (Q2—Q3) interquartile range, and the whiskers expand to the lower ( Q1 ) and upper ( Q4 ) quartiles of the distribution (5–95%). The median of the distribution is represented by a horizontal line inside the box, and the mean is represented by a cross symbol (×). The data were compared using a paired Student’s t’s test (N neurons = 82, N slices = 7 and N mice = 4). ( b ) Left: Representative trace of the NAD(P)H autofluorescence signal ( top ), recorded from a population of DGCs in an acute hippocampal slice. These cells expressed the Ca 2+ sensor RCaMP1h, whose fluorescence was simultaneously monitored as a proxy for neuronal activity ( bottom ). A stimulating electrode was placed in the hilus of the dentate gyrus of the hippocampus, and a train of depolarizing pulses was delivered to the DGC axons (antidromic stimulation) before and after the treatment. Treating with UK5099 reduces the baseline and the responses induced by stimulation. Right: Quantification of the normalized amplitudes of the NAD(P)H signal overshoot, with or without UK5099. The baseline before each stimulation (F baseline-i ) was subtracted from the raw trace, and the difference between the baseline and the peak (ΔF=F peak -F baseline-i ) is presented as a percentage change over the baseline (ΔF/F baseline-i ). The data were compared using a paired Student’s t’s test (N slices = 10 and N mice = 6). ( c ) Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded before and after the application of UK5099. Right: The NADH CYT transient was decreased in the presence of UK5099. The Peredox lifetime change from the baseline to the peak of the transient was divided by the magnitude of the RCaMP1h transient (ΔPeredox/ΔRCaMP), as the metabolic responses are correlated with the Ca 2+ spikes . The data were compared using a Wilcoxon matched pairs test (N neurons = 48, N slices = 7 and N mice = 6). ( d ) Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC, with sequential application of the LDH inhibitor GSK-2837808A (LDHi) and UK5099. The data were compared using a Wilcoxon matched pairs test (N neurons = 52, N slices = 6 and N mice = 5). ( e ) Representative trace of the NAD(P)H autofluorescence signal and RCaMP1h fluorescence, simultaneously recorded from a population of DGCs in an acute hippocampal slice. Treating with 2 µM GSK-2837808A (LDH inhibitor) increases the baseline while preserving the waveform of the NAD(P)H responses to stimulation. The subsequent application of 2 µM UK5099, as in panel ( d ), diminished both phases of the NAD(P)H responses, especially the overshoot. The data were compared using a paired t-test (N slices = 4 and N mice = 3).

Article Snippet: Chemical compound, drug , UK5099 , Santa Cruz , Cat#sc-361394; CAS:56396-35-1 , .

Techniques: Fluorescence, Activity Assay, Preserving

Journal: eLife

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

doi: 10.7554/eLife.64821

Figure Lengend Snippet: ( a ) Representative images of DGCs expressing the Pyronic sensor, pseudocolored according to the lifetime of the donor species (mTFP). Cells perfused in ACSF only (Control), or in the presence of the MPC inhibitor UK5099, are shown before and after the application of 10 mM pyruvate as in . Images in the bottom were taken at the peak value of the response to pyruvate. The color scale bar to the right shows the range of lifetime for Pyronic mTFP. ( b ) The change in the Pyronic mTFP lifetime (LT) in response to external pyruvate application was diminished in DGCs after incubating with UK5099. Data points obtained before and after the application of UK5099 are connected by lines. The data were compared using a paired Student’s t’s test (N neurons = 82, N slices = 7 and N mice = 4).

Article Snippet: Chemical compound, drug , UK5099 , Santa Cruz , Cat#sc-361394; CAS:56396-35-1 , .

Techniques: Expressing, Control

Journal: eLife

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

doi: 10.7554/eLife.64821

Figure Lengend Snippet: ( a ) Top: Low-magnification image of an acute brain slice of the hippocampus, showing the placement of the stimulating electrode in the hilus (H) and the O 2 electrode in the layer of dentate granule cells (DGCs). The red square represents the area used for imaging the NAD(P)H autofluorescence signals. Bottom: The identification of the area of interest (dashed-line polygon drawn in the DGC layer), was performed in infrared-illuminated/differential interference contrast images, obtained at high magnification (×60 objective) and a pixel binning of 8. ( b ) Left: Representative traces of NAD(P)H and FAD + autofluorescence signals, extracellular oxygen concentration and RCaMP fluorescence, simultaneously recorded from a population of DGCs in a hippocampal slice. The shaded area corresponds to the application of 2 µM of the MPC inhibitor UK5099. Right: Effect of UK5099 treatment on the baseline level of each signal. Data points obtained before and after the application of UK5099 are connected by lines (N slices = 10 and N mice = 6 for NAD(P)H and FAD + autofluorescence signals, along with RCaMP fluorescence). As shown in the representative trace, there were some experiments with oxygen and autofluorescence signals recorded simultaneously, but the full dataset for O 2 measurements contained stand-alone O 2 recordings as well (N slices = 10 and N mice = 5).

Article Snippet: Chemical compound, drug , UK5099 , Santa Cruz , Cat#sc-361394; CAS:56396-35-1 , .

Techniques: Slice Preparation, Imaging, Concentration Assay, Fluorescence

Journal: eLife

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

doi: 10.7554/eLife.64821

Figure Lengend Snippet: ( a ) The traces represent the average normalized NAD(P)H signals (mean ± SD) before and after 2 µM UK5099 (as in ), elicited by antidromic stimulation. The baseline before each stimulation (F baseline-i ) was subtracted from the raw trace. The difference between the baseline and the dip (ΔF=F dip -F baseline-i ), presented as percent change over the baseline (ΔF/F baseline-i ), was compared between the two conditions. Other panels show the comparisons for the time from the beginning of the stimulation to the minimum value of the initial dip and the recovery time from that minimum value to 50% of the amplitude of the dip. Additionally, we compared the overshoot/dip ratio, which reflects the relative contributions of the reductive and the oxidative phases to the NAD(P)H signal (re/ox), independently of changes in the baseline autofluorescence . This comparison also shows a decreased mitochondrial NADH production in stimulated neurons, following MPC inhibition. ( b ) Average normalized FAD + traces, expressed as percent change from the baseline before each stimulation (F baseline-i ). The difference between the baseline and the initial peak, or the undershoot (ΔF=F peak or undershoot -F baseline-i ), were calculated as a percent change over the baseline (ΔF/F baseline-i ). The time from the beginning of the stimulation to the peak, and the recovery time from the peak to 50% of its amplitude, were also compared. ( c ) Average normalized O 2 recordings, expressed as percent change from the baseline before each stimulation. The graphs show the difference between the baseline and the dip in the O 2 signal elicited by stimulation (Δ[O 2 ]=[O 2 ] dip -[O 2 ] baseline-i ), either as the absolute change (expressed in µM) or as percent change over the baseline (Δ[O 2 ]/[O 2 ] baseline-i ). The time from the beginning of the stimulation to the minimum value of the [O 2 ] upon stimulation, and the recovery time from this value to 50% of its amplitude, were also compared. ( d ) Average normalized RCaMP traces, expressed as percent change from the baseline before each stimulation (F baseline-i ). The difference between the intensities at baseline and at the peak (ΔF=F peak -F baseline-i ), expressed as A.U./px or as the percent change over the baseline (ΔF/F baseline-i ), were compared between the recordings before and after MPC inhibition. The time from the beginning of the stimulation to the peak, and the recovery time from the peak to 50% of its amplitude, were also compared. Surprisingly, neither the dip of the NAD(P)H signal nor the transient increase in O 2 consumption in the tissue were as attenuated as the NAD(P)H overshoot, although both were mildly decreased. A likely explanation for these changes is that MPC inhibition partially depolarizes mitochondria, slowing down events that require an energized mitochondria. These events include Ca 2+ influx, the adenine nucleotide exchanger and Complex V ( ; reviewed by ; ), all of which further dissipate the mitochondrial membrane potential and/or the proton gradient, contributing to NADH oxidation. Data points obtained before and after the application of UK5099 are connected by lines (N slices = 10 and N mice = 6 for NAD(P)H and FAD + autofluorescence signals, along with RCaMP fluorescence). Oxygen recordings were performed simultaneously to the autofluorescence signals in most of the experiments, except one (N slices = 10 and N mice = 5). A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise.

Article Snippet: Chemical compound, drug , UK5099 , Santa Cruz , Cat#sc-361394; CAS:56396-35-1 , .

Techniques: Comparison, Inhibition, Membrane, Fluorescence

Journal: eLife

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

doi: 10.7554/eLife.64821

Figure Lengend Snippet: ( a ) Filmstrip of the metabolic response in the cytosol of stimulated DGCs, before and after the application of 2 µM UK5099 (as in ). The images were pseudocolored according to the lifetime of Peredox and RCaMP. The time stamp at −10 s corresponds to the baselines, ten seconds prior to the stimulation. A train of depolarizing pulses was delivered at time zero, which is revealed by an increase in the RCaMP lifetime because of the intracellular Ca 2+ spike. The bottom row of images corresponds to the peak NADH response in the cytosol (+40 s), in control conditions. The color scale bar to the right shows the range of lifetimes for both sensors. The UK5099-treated DGCs exhibit a lower Peredox lifetime at baseline and an attenuated NADH CYT response to stimulation. ( b–c ) Effects of UK5099 on the parameters derived from the Peredox and RCaMP transients, respectively. The full dataset of was used (N neurons = 48, N slices = 7 and N mice = 6), except for comparisons of the time to peak and decay of the NADH CYT response, when only neurons that presented a ΔPeredox Lifetime ≥ 0.05 ns after UK5099 were analyzed (N neurons = 13, N slices = 3 and N mice = 3). A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise.

Article Snippet: Chemical compound, drug , UK5099 , Santa Cruz , Cat#sc-361394; CAS:56396-35-1 , .

Techniques: Control, Derivative Assay

Journal: eLife

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

doi: 10.7554/eLife.64821

Figure Lengend Snippet: ( a–b ) Effects of the sequential application of 2 µM GSK-2837808A and 2 µM UK5099 (as in ) on the parameters derived from the Peredox and RCaMP transients, respectively. The full dataset of was used (N neurons = 52, N slices = 6 and N mice = 5), except for comparisons of the time to peak and decay of the NADH CYT response, when only neurons that presented a ΔPeredox Lifetime ≥ 0.05 ns during all the experiment were included (N neurons = 49, N slices = 6 and N mice = 5). For multiple comparisons of normally distributed data, a repeated measures ANOVA was used, with a Student-Newman-Keuls post-test. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test otherwise. The ratio (after UK5099)/(before UK5099) was included for each parameter, to compare the conditions UK5099/Control and ( UK5099+LDHi )/ LDHi alone (as in ). For UK5099/Control , the full dataset of was used (N neurons = 48, N slices = 7 and N mice = 6), except for comparisons of the time to peak and decay of the NADH CYT response, when only neurons that presented a ΔPeredox Lifetime ≥ 0.05 ns after UK5099 were analyzed (N neurons = 13, N slices = 3 and N mice = 3). A Student’s t test or a Welch’s t-test were used for comparisons between normally distributed data, or a non-parametric Mann-Whitney test was used otherwise. ( c ) Effects of the sequential application of GSK-2837808A and UK5099 (as in ) on the ratio of lifetimes ΔPeredox/ΔRCaMP. ( d ) Comparison of the size effect of LDH inhibition on the NADH CYT transients elicited with antidromic stimulation (data from experiments in , N neurons = 52, N slices = 6 and N mice = 5) or synaptic stimulation (data from 3F in , N neurons = 54, N slices = 5 and N mice = 5), using a Mann-Whitney test.

Article Snippet: Chemical compound, drug , UK5099 , Santa Cruz , Cat#sc-361394; CAS:56396-35-1 , .

Techniques: Derivative Assay, Control, MANN-WHITNEY, Comparison, Inhibition

Journal: eLife

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

doi: 10.7554/eLife.64821

Figure Lengend Snippet: ( a ) Representative traces of simultaneously recorded NAD(P)H and FAD+ autofluorescence signals, and RCaMP fluorescence, in a population of DGCs in an acute brain slice. ( b ) Effects of the sequential application of 2 µM GSK-2837808A and 2 µM UK5099 on the parameters derived from the NAD(P)H and RCaMP transients. The treatment with GSK-2837808A alone significantly elevated the baseline of the NAD(P)H autofluorescence by 31 ± 12%, likely because of the combination of two factors: an increased pyruvate availability and a higher NADH transfer from the cytosol to the mitochondria via the MAS. Since the responses to stimulation seems to occur independently from the changes in the NAD(P)H baseline. The FAD + signal at baseline was unaltered by LDH inhibition and showed the expected trends after MPC inhibition with UK5099. Baselines were compared using a repeated measures ANOVA, with a Student-Newman-Keuls post-test (N slices = 4 and N mice = 3). Comparisons for other parameters were restricted to before and after the application of UK5099, in the continuous presence of an LDH inhibitor. These comparisons were performed using a paired t-test.

Article Snippet: Chemical compound, drug , UK5099 , Santa Cruz , Cat#sc-361394; CAS:56396-35-1 , .

Techniques: Fluorescence, Slice Preparation, Derivative Assay, Inhibition

Journal: eLife

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

doi: 10.7554/eLife.64821

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , UK5099 , Santa Cruz , Cat#sc-361394; CAS:56396-35-1 , .

Techniques: Plasmid Preparation, Recombinant, Derivative Assay, Produced, Software

Journal: STAR Protocols

Article Title: Protocol to assess bioenergetics and mitochondrial fuel usage in murine autoreactive immunocytes using the Seahorse Extracellular Flux Analyzer

doi: 10.1016/j.xpro.2024.102971

Figure Lengend Snippet: Metabolic pathways influenced by fuel inhibitors utilized in this protocol Metabolism incorporates the products from several intracellular pathways for the production of cellular fuel. These networks, including glycolysis (beginning with glucose), fatty acid oxidation (beginning with long-chain fatty acid), and glutaminolysis (beginning with glutamine) terminate in the production of metabolites that can feed directly into the tricarboxylic acid (TCA) cycle. Selective inhibition of these individual pathways can provide crucial insight into the metabolic reliance and flexibility of differing cell types. 2DG is a glucose mimic and selective inhibitor of hexokinase activity, playing a key role in regulating glucose oxidation. Hexokinase catalyzes the phosphorylation of glucose into glucose-6-phosphate, thus ultimately preventing the conversion of glucose into pyruvate. UK5099 is a selective inhibitor of pyruvate import into the mitochondria for utilization in the TCA cycle. BPTES is a specific inhibitor of glutaminase activity, inhibiting the breakdown of glutamine into α-ketoglutarate (α-KG). Thioridazine inhibits peroxisomal β-oxidation, impeding peroxisomal fatty acid oxidation (FAO) and halting the breakdown of long-chain fatty acids into acetyl-CoA for the TCA cycle. Etomoxir, as a mitochondrial FAO inhibitor, specifically disrupts the action of CPT1, preventing the conversion of acyl coenzyme A (CoA) to acetyl-carnitine, thereby inhibiting the mitochondrial-derived utilization of long-chain fatty acids to fuel the TCA cycle. Image created with BioRender.com .

Article Snippet: UK5099 , GlpBio , Cat#GC11865.

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Derivative Assay

Journal: STAR Protocols

Article Title: Protocol to assess bioenergetics and mitochondrial fuel usage in murine autoreactive immunocytes using the Seahorse Extracellular Flux Analyzer

doi: 10.1016/j.xpro.2024.102971

Figure Lengend Snippet: Concentrations of metabolic modulators and reagents utilized in this assay

Article Snippet: UK5099 , GlpBio , Cat#GC11865.

Techniques: Injection, XF Assay

Journal: STAR Protocols

Article Title: Protocol to assess bioenergetics and mitochondrial fuel usage in murine autoreactive immunocytes using the Seahorse Extracellular Flux Analyzer

doi: 10.1016/j.xpro.2024.102971

Figure Lengend Snippet: Lupus GCB cells are dependent on glycolysis and glutaminolysis for mitochondrial fueling, whereas lupus Tfh cells are metabolically flexible Metabolic analysis of splenocytes from symptomatic Yaa DKO mice (A–D). Effects of catabolic pathway inhibitors (A) UK5099, (B) BPTES, (C) etomoxir (Eto.), and (D) thioridazine (Thio.), on the maximal respiratory capacity (MRC) (Percentage (%) of untreated) of GCB and Tfh cells. Results are representative of at least 3 independent experiments. Error bars represent mean ± SEM; ns, not significant, ∗, p < 0.05, by unpaired two-tailed Student’s t -test. Figure reprinted, with permission, from Wilson et al. (2023).

Article Snippet: UK5099 , GlpBio , Cat#GC11865.

Techniques: Metabolic Labelling, Two Tailed Test

Journal: STAR Protocols

Article Title: Protocol to assess bioenergetics and mitochondrial fuel usage in murine autoreactive immunocytes using the Seahorse Extracellular Flux Analyzer

doi: 10.1016/j.xpro.2024.102971

Figure Lengend Snippet:

Article Snippet: UK5099 , GlpBio , Cat#GC11865.

Techniques: Recombinant, Software

Journal: British Journal of Pharmacology

Article Title: Glucagon up‐regulates hepatic mitochondrial pyruvate carrier 1 through cAMP‐responsive element‐binding protein; inhibition of hepatic gluconeogenesis by ginsenoside Rb1

doi: 10.1111/bph.14758

Figure Lengend Snippet: MPC1 is needed for glucagon to promote HGP. (a) Lactate accumulation in the liver of mice injected with 2 mg·kg−1 glucagon (n = 8 each group); (b) lactate contents in primary mouse hepatocytes treated with glucagon (100 nM, 6 hr) in the presence or absence of UK5099 (300 nM; n = 6 each group); (c, d) lactate and glucose production in primary mouse hepatocytes when pyruvate (10 mM) was used as the only substrate (n = 6 each group); (e, f) glucose production in primary mouse hepatocytes when methyl pyruvate (10 mM), alanine (10 mM), or glutamine (10 mM) were used as the only substrate, individually (n = 6 each group). The glucose production was expressed as the percentage of the basal level in PYR, MP, or Ala group. Data shown are individual values with means ± SD. *P < .05, significantly different as indicated. Ala, alanine; Ctrl, control; Gln, glucagon; Glu, glutamine; MP, methyl pyruvate; ns, not significant; PYR, pyruvate

Article Snippet: UK5099 and CPI‐613 were from Apexbio Technology LLC (Houston, USA).

Techniques: Injection