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PIMT and RBC metabolism
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Highlights of metabolites in this group are presented in Figure 4D including several tryptophan metabolites (kynurenine, nicotinamide, hydroxyindoleacetate), PPP and glutathione homeostasis metabolites (ribose phosphate, glutathionyl-cysteine, ergothioneine). The increase of these metabolites in WT but not PCMT1 KO mice demonstrate a defect in the ability of PCMT1 KO RBC to further activate the PPP following additional exogenous insults. Thus, in addition to impairing protein repair, lack of PCMT1 pre- vents compensatory metabolic shifts to environmental insults.
Increased in vivo hemolysis in response to systemic oxi- dant stress
In order to allow the study of PCMT1 KO RBC in adult mice in vivo, bone marrow transplant (BMT) was carried out with donors being PCMT1 KO or WT controls (5 weeks of age prior to seizure onset in PCMT1 KO) and recipients being Ubi-GFP recipient mice that express GFP in all blood lineages. This allowed monitoring of engraftment by evalu- ating the progressive decline in GFP positivity in circulating blood cells via flow cytometry (Figure 5A). No significant difference was seen in the rate of RBC engraftment from PCMT1 KO versus WT donors ( approximately 56 days) (Figure 5B). Consistent with what was observed for RBC from 5-week-old PCMT1 KO mice compared to WT con-
trols, the metabolic phenotypes of RBC from PCMT1 KO→WT mice showed significant accumulation of SAM compared to WT→WT RBC, as well as decreases in glu- tathione (total pool – including reduced and oxidized) and increases in dopamine and tryptophan metabolites (indole, indole acetate, 3-methyldioxyindol) (Figure 5C). Conversely, BMT PCMT1 KO→WT RBC showed higher levels of glycolytic intermediates (glucose 6-phosphate, glyceraldehyde 3-phosphate, 2,3-diphosphoglycerate and isobaric isomer, phosphoenolpyruvate) and lower steady state levels of PPP metabolites (6-phosphogluconate and ribose phosphate) or other pathways involved in NADPH homeostasis (folate, pyruvoyl-THF – Figure 5C). Together, these results suggested a lower antioxidant capacity of RBC from PCMT1 KO donors compared to WT donors; howev- er, this difference did not affect the ability of hematopoietic stem cells to generate RBC or RBC to circulate in an other- wise healthy state.
In order to test the effects of oxidant stress that was increased from normal healthy conditions, mice were treat- ed intravenously with a biotinylating reagent such that 100% of RBC were biotinylated. Mice were then injected with PHZ, which induces oxidant stress and is known to be toxic to RBC (Figure 6A). Clearance of RBC was then mon- itored by assessing the percentage of Biotin + RBC over time in peripheral blood. Significantly higher clearance of
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Figure 3. Organ metabolomics in wild-type and PCMT1-/- knockout mice. (A) Organ metabolomics in wild-type (WT) and PCMT1-/- knockout (PCMT1 KO) mice; (B) metabolomics of brain; (C) heart; (D) kidney; (E) liver and (F) and spleen in WT and PCMT1 KO mice. Each panel illustrates the results from Partial Least Square- Discriminant Analyses and the top 25 significant metabolites by t-test for each organ.