Journal: Aging Cell

Article Title: Longevity Humans Have Youthful Erythrocyte Function and Metabolic Signatures

doi: 10.1111/acel.14482

Figure Lengend Snippet: The mechanisms of enhanced erythrocyte GSH synthesis and maintained erythrocyte S1P In longevity individuals. (a) Isotopically labeled l ‐glutamine flux experiments were conducted in cultured human RBCs to trace its metabolism in erythrocytes among young, elderly, and longevity individuals. (b) Isotopically labeled l ‐glutamate flux experiments were conducted in cultured human RBCs to trace its metabolism in erythrocytes among young, elderly, and longevity individuals. (c) Protein expression of the alanine‐serine‐cysteine transporter type‐2 (ASCT2), a high‐affinity glutamine transporter, on RBC membrane (left) by western blot in three age groups, ponceau staining (right) as loading control. (d) The quantification of ASCT2 protein bands by densitometry, ** p < 0.01, one‐way ANOVA. (e) Protein expression of the excitatory amino acid transporter 3 (EAAT3), transporter of glutamate and aspartate, on RBC membrane (left) by western blot in three age groups, ponceau staining (right) as loading control. (f) The quantification of EAAT3 protein bands by densitometry, * p < 0.05, one‐way ANOVA. (g) Protein expression of the major facilitator superfamily transporter 2b (MFSD2B), the exclusive S1P transporter, on RBC membrane (left) by western blot in three age groups, ponceau staining (right) as loading control. (h) The quantification of MFSD2B protein bands by densitometry, * p < 0.05, one‐way ANOVA. (i) Erythrocyte SphK1 activity in three age groups. ns, not significant, one‐way ANOVA. (j) Erythrocyte cytosolic GAPDH activity in three age groups, * p < 0.05, ** p < 0.01, ns, not significant, one‐way ANOVA. (k) Protein expression of the GAPDH in RBC total lysate (left) by western blot in three age groups, ponceau staining (right) as loading control. (l) The quantification of GAPDH protein bands by densitometry, * p < 0.05, ** p < 0.01, ns, not significant, one‐way ANOVA. (m) Protein expression of the BPGM in RBC total lysate (left) by western blot in three age groups, ponceau staining (right) as loading control. (n) The quantification of BPGM protein bands by densitometry, * p < 0.05, ns, not significant, one‐way ANOVA. (o) Working model: Longevity individuals exhibit a youth‐like erythrocyte oxygen release function, heightened anti‐ROS capacity, and reduced inflammation levels. This is accompanied by upregulation of the transporter responsible for glutamine and glutamate transport on the erythrocyte membrane in longevity individuals, resulting in elevated levels of glutamate and enhanced synthesis of glutathione. Furthermore, this mechanism contributes to the mitigation of neurotoxicity by decreasing plasma glutamate levels. The decreased membrane MFSD2B in RBC increases the intracellular level of S1P, which facilitates the release of membrane GAPDH, increased cytosolic GAPDH accelerates glycolysis and increases glycolytic RLS shunt (elevated 2,3‐BPG) which in turn leads to enhanced O 2 delivery to counteract aging‐related tissue hypoxia and leading to longevity and healthy aging.

Article Snippet: We used primary antibodies to ASCT2 (Proteintech, # 20350‐1‐AP, 1:8000), EAAT3 (Proteintech, # 12686‐1‐AP, 1:1000), MFSD2B (Invitrogen, # PA5‐21050, 1:1000), GAPDH (Proteintech, # 60004‐1‐Ig, 1:3000), BPGM (Proteintech, # 17173‐1‐AP, 1:4000), and then incubated with secondary antibodies (Abiowell, # AWS0001), Goat anti‐Mouse IgG (H + L), HRP, 1:5000; # AWS0002, Goat anti‐Rabbit IgG (H + L), HRP; 1:5000.

Techniques: Labeling, Cell Culture, Expressing, Membrane, Western Blot, Staining, Control, Activity Assay, Clinical Proteomics