Journal: Scientific Reports
Article Title: Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis
Figure Lengend Snippet: MTO1-defective cells exhibit altered expression of metabolism genes and differ from GTPBP3-defective cells in the bioenergetics profile. ( A ) qRT-PCR analysis of mRNA expression of genes related to glycolysis ( GLUT1: glucose transporter 1, PKF1: phosphofructokinase, LDHA and LDHB: lactate dehydrogenase A and B, respectively, PDK4: pyruvate dehydrogenase kinase 4 and MPC1: mitochondrial pyruvate carrier 1 ); glutaminolysis ( ASCT2: glutamine/amino acid transporter 2, SN2: glutamine/amino acid transporter system N , and GLS: glutaminase ); cellular fatty acid (FA) uptake ( FAT/CD36: fatty acid translocase , and FABP3: fatty acid binding protein 3 ); mitochondrial fatty acid (FA) uptake ( CPT1a and CPT1b: carnitine palmitoyltransferase I a and b ); fatty acid oxidation ( LCAD: long-chain acyl-CoA dehydrogenase, MCAD:medium-chain acyl-CoA dehydrogenase , and HADH: hydroxyacyl-CoA dehydrogenase ); fatty acid synthesis ( ACC: Acetyl-CoA carboxylase and FAS: fatty acid synthase ), in MTO1 (MTO1 HF) human fibroblasts. Data are expressed as fold change respect to WT HF and shown in a heatmap. The colour and the corresponding value in log2 scale are plotted on the right. ( B ) Oxygen consumption rates of digitonin-permeabilized cells in the presence of 5 mM ADP, 0.2 mM octanoyl-carnitine (Oct) and malate ( M ), which was added at three different concentrations (0.05, 0.1 and 2 mM). ( C ) Oxygen consumption rates of digitonin-permeabilized cells in the presence of 5 mM ADP, 0.2 mM octanoyl-carnitine (Oct), 2 mM malate ( M ) and after sequential addition of substrates for Complex I (5 mM pyruvate ( P ) and 10 mM glutamate ( G ); PGMOctp) and Complex II (succinate ( S ); PGMSOctp), the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (CCCP, stepwise titration in 0.05 μM increments; PGMSOcte), and the Complex I inhibitor (0.5 μM rotenone; Se). Oxygen consumption rates (OCRs), expressed as picomoles (pmol) per second (s) per million of cells (Mill), were normalized to the mitochondrial copy number (mtDNA/nDNA ratio) in each sample. p denotes phosphorylation of ADP + Pi to ATP. e denotes electron transfer system (ETS) capacity at optimum CCCP concentration (noncoupled respiration). Data represent the means ± SD from at least 3 independent determinations. Differences from wild-type (WT) or Negative Control (NC) values were found to be statistically significant at *p
Article Snippet: For mRNA quantification, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.
Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Titration, Concentration Assay, Negative Control