Roduct creatinine along with the phosphorylated kind of creatine interfere with creatine transport by MCT12. None on the tested compounds appeared to influence creatine uptake drastically (P , 0.5358, ANOVA) (Experiment 1: creatine only: 225 + 25 pmol/h/oocyte, arginine: 165 + 28 pmol/h/oocyte, glycine: 168 + 27 pmol/h/oocyte, ornithine: 164 + 34 pmol/h/oocyte and phosphocreatine: 169 + 35 pmol/h/oocyte; (n ?five ?7); Experiment two: creatine only: 89 + 9 pmol/h/oocyte), creatinine 95 + ten pmol/h/oocyte). To enable comparison of all compounds, the results are displayed as percentage in Figure 3D. SLC16A12 mutation alters transport properties In an SLC16A12 mutation screen of sufferers with age-related cataracts (ARCs) we identified a novel heterozygous DNA sequence alteration (Fig. 4A) that maps to position c.1219G.A; p.G407S, affecting an evolutionary hugely conserved amino acid that localizes towards the final extracellular loop.3-Fluoro-4-iodo-2-methoxypyridine Purity This alteration was not located in 400 alleles from men and women representing the general population. Potential development of ARCs within this group is unlikely as a result of this sequence alteration. The patient with all the c.1219G.A alteration was diagnosedStatistical significance (two-way ANOVA) and fold change determined by the observed intensity (log2 ratio).SLC16A12 cRNA. The log2 ratio of 22.6485 corresponds to a six.3-fold reduction in creatine levels (P , 0.0001, unpaired t-test) (Fig. 2A), suggesting efflux of creatine. Efflux and uptake of creatine by MCT12 To test whether creatine was indeed specifically transported by MCT12, efflux experiments had been performed, as recommended by the metabolomics study.2-Fluoroacrylic acid structure The time course (0, 15 and 60 min) showed a considerable enhance in radioactivity (disintegrations per minute, DPM) (P , 0.0001, ANOVA) in the medium when oocytes had been coinjected together with the chaperone and SLC16A12 cRNA (32 + three, 1805 + 262, 6869 + 636 DPM for the three time points, n ?9), verifying creatine efflux. Only minimal alterations (ns, ANOVA) inside the amount of radioactivity were observed in noninjected (17 + 1, 68 + 34, 198 + 100 DPM, n ?9) oocytes and those injected with the chaperone CD147 cRNA alone (40 + 17, 73 + 21, 157 + 57 DPM, n ?ten). At time points 15 and 60, radioactivity within the medium of oocytes injected using the transporter was considerably unique from the noninjected and chaperone only injected oocytes (P , 0.0001, ANOVA) (Fig. 2B). Remaining radioactivity in oocytes right after the last time point was drastically decreased (P , 0.0001) in coinjected (12 165 + 991 DPM) compared with noninjected (34 418 + 951 DPM) and CD147 only injected oocytes (31 958 + 968 DPM, nNI ?ten, nCD147 ?9, nCD147+ hMCT12 ?eight) (Supplementary Material, Fig.PMID:23829314 S2). These benefits confirmed the information obtained in the metabolomics study and demonstrated that the efflux of creatine will depend on MCT12. In an uptake experiment, we tested whether creatine transport is bidirectional. In oocytes expressing MCT12, creatine uptakeHuman Molecular Genetics, 2013, Vol. 22, No.Figure 2. Creatine is transported by MCT12. (A) Substantially reduce creatine levels (6.3-fold) were detected in oocytes coinjected with SLC16A12 and its chaperone CD147 compared with oocytes injected only with CD147. (B) Creatine efflux in noninjected oocytes (NI), and oocytes expressing CD147 or CD147 + hMCT12. Content of 14C creatine within the medium was recorded as disintegrations per minute at distinct time points (0, 15 and 60 min). (C) Creatine uptake. Measurements were taken ten min just after addition of 1.