Fig 1: Pleiotropic alterations in the MPC1gt/gt E13.5 brain metabolome are rescued by a ketogenic diet.(A) Principal component analysis of brain metabolome data from E13.5 MPC1+/+ and MPC1gt/gt embryos from females maintained on normal or ketogenic (keto) diet and (B) underlying annotated metabolites showing highest variance among principal components. (C) Heat map showing fold changes of annotated metabolites (m/z is indicated) that were found to be significantly different (q-value < 0.01, abs(log2(fold change)) > 0.5) between MPC1gt/gt and MPC1+/+ brain samples from E13.5 embryos maintained on normal or ketogenic diets. (D) The abundance of the indicated metabolites in the brains of E13.5 MPC1+/+ or MPC1gt/gt embryos under normal or ketogenic diet was determined by non-targeted metabolomics.
Fig 2: Neuro-MPC1-KO mice are highly sensitive to pro-convulsant drugs and develop acute epileptic-like seizures.(A) Schematic description of the PTZ kindling protocol. (B) Seizure severity scores reflecting the different clinical symptoms as indicated, obtained for neuro-MPC1-WT or neuro-MPC1-KO. N = 8 independent neuro-MPC1-WT and neuro-MPC1-KO mice. Two way ANOVA (F(7,70)=19, p = 0.0001). (C) Illustration of the recording setups in awake mice indicating the position of surface EEG electrodes and representative example of a seizure recorded in a neuro-MPC1-KO mouse after injection of 35 mg/kg PTZ during surface EEG recordings. The inset shows an example of fast ripples generated during an ictal epileptic discharge. (D–I) GCaMP6S calcium imaging of the CA1 area from hippocampal slices in the presence of Carbachol (50 µM) and PTZ (2 mM). Slices were prepared from WT animals (top, black) or from KO animals with no pre-treatment (bottom, red). (D) Ca2+ sweeps recorded in four representative GCaMP6S-expressing neurons. (E) Raster plots of Ca2+ transient onsets extracted from all recorded neurons in a given slice. (F) Cumulative distribution of the frequency of the calcium events in all the recorded neurons. N = 7, 12 independent experiments. Kolmogorov-Smirnov test (WT vs KO p = 0.0001). (G) Cumulative distribution of the occurrence of neuronal co-activations exceeding chance levels as a function of time N = 7, 12 independent experiments. Kolmogorov-Smirnov test (WT vs KO p = 0.0344). Amplitude (H), and duration (I) of the calcium events recorded in all neurons of the hippocampus. N = 7, 12 independent experiments. Mann-Whitney test (Amplitude: WT vs KO p = 0.5918; Duration: WT vs KO p = 0.9182).
Fig 3: Determination of MPC1 and MPC2 expression and UK5099 blocking effect on pyruvate mitochindrial transportation in squamous esophageal cancer cells(A) MPC1 and MPC2 protein expression in esophageal squamous EC109, KYSE140 and KYSE450 cells by ICC assay. (B) MPC1 and MPC2 protein expression by Western blot. (C) UK5099 treatment reduces the mitochondrial pyruvate concentration in esophageal squamous EC109, KYSE140 and KYSE450 cells in vitro. Data are expressed as mean ± SD, n = 3.*p < 0.05, **p < 0.01, ***p < 0.001. The scale bar is 50 µm.
Fig 4: In vivo spermatocytes have heterogeneous mitochondria and reduced OXPHOS activity.(A) Immunolabeling of NDUFB6, MTCOI, and MPC1 in spermatocytes in testis sections. Line scans are shown as arbitrary units (au) to the right. Scale bars, 20 µm. (B) Quantification showing the percentage of spermatocytes with reduced or heterogeneous staining. L/Z, leptotene/zygotene; P/D, pachytene/diplotene. N = 4. (C) COX/SDH enzyme histochemistry in adult testis sections. Note that mutant sections have much reduced OXPHOS activity. Scale bars, 50 µm. ST, spermatid; SC, spermatocyte; SG, spermatogonium. Data are represented as mean ± SEM. ****p=0.0001; ***p=0.001; **p=0.01. For statistical tests used, see the Materials and methods section.
Fig 5: Induction of Ldh activity by deletion of mitochondrial pyruvate transport does not affect tumor initiation or progression in SCC. a Coupling DMBA/TPA carcinogenesis to transgenic deletion of mitochondrial pyruvate carrier function allowed for an examination of tumorigenesis following stimulation of Ldh activity. Haematoxylin and eosin stain shows similar histology in wild-type and Mpc1-null tumors. Scale bars, 200 µm. b Quantification of time to tumor formation, number of tumors, and tumor volume formed showed that Mpc1 deletion did not affect tumorigenesis. Each bar represents n = 12 mice per genotype. Shown as mean ± SEM. Paired t test was performed, P < 0.05 shown for knockout vs. wild-type tumors. c Western blotting indicated that the genetic deletion of Mpc1 was effective. d Ldh activity on lysate from normal skin and wild-type vs. Mpc1-null tumors. Each bar represents the relative Ldh activity signal for each genotype type where n = 12 mice per genotype. Shown as mean ± SEM. Paired t test was performed, P < 0.05 shown for each tumor genotype vs. control skin. e Mice with just gain of KrasG12D were crossed with floxed-Mpc1 mice to generate one-hit mice with and without Mpc1 expression. Haematoxylin and eosin stain shows similar hyperplasia in wild-type and Mpc1-null tumors. Scale bars, 100 µm. f Ldha activity in lysate generated from wild-type vs. Mpc1-null tumors. Each bar represents the relative Ldh activity signal for each genotype type, where n = 3 mice per genotype. Shown as mean ± SEM. Paired t test was performed, P < 0.05 shown for wild-type vs. knockout hyperplastic skin. g [U-13C6]glucose tracing for glycolytic intermediates shows that while glucose uptake did not change, the production of lactate was increased by loss of Mpc1 in HFSCs. Labeled metabolites were extracted and analyzed by LC–MS from hyperplastic tissue from each genotype. Heatmap depicts percent labeled glycolytic intermediate isotopomers from tissue isolated from six mice per genotype. Student’s paired t test was performed, *P < 0.05; **P < 0.01; ***P < 0.001; NS, P > 0.05; n = 12
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