Fig. 5.

Investigating potential mechanisms of folate cycle activation during chronic stress in INS1E β cells.

(A) Diagram showing sources of NAD⁺ and serine for folate cycling activation. ADP, adenosine 5′-diphosphate; ATP, adenosine 5′-triphosphate; TCA, tricarboxylic acid; SHMT1, serine hydroxymethyltransferase 1; CH₂-THF, 5,10-methylenetetrahydrofolate; P-pyruvate, 3-phosphohydroxypyruvate; P-serine, 3-phosphoserine. (B) Glucose stimulation of INS1E β cells transfected with Apollo-NADP⁺. Cells were treated with 100 μM NMN for 24 hours to elevate NAD⁺ levels. UK (50 μM) was added 1 hour before imaging to block NADP⁺ reduction by pyruvate cycling. (C) Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 μM) stimulation of INS1E β cells transfected with Apollo-NADP⁺ at 1 or 15 mM glucose. UK (50 μM) was added 1 hour before imaging to block pyruvate cycling. (D) Serine stimulation (0.3 mM) of unstressed (0 μM TBM) and stressed (8 hours of 100 μM TBM) INS1E β cells transfected with Apollo-NADP⁺. Cells were pretreated with 1 mM asparagine for 1 hour before imaging to promote extracellular serine uptake. Imaging was done at low glucose (1 mM) to avoid activation of pyruvate cycling. (E) Serine stimulation (0.3 mM) of INS1E β cells transfected with Apollo-NADP⁺. Cells were treated with 100 μM NMN for 24 hours to elevate NAD⁺ levels or treated with both 100 μM NMN and 5 μM MTX for 24 hours to block NAD⁺ conversion to NADP⁺. One hour before imaging, cells were pretreated with 1 mM asparagine to promote extracellular serine uptake. Imaging was done at low glucose (1 mM) to avoid activation of pyruvate cycling. (F) Simplified diagram of potential factors required to trigger NADP⁺ reduction through folate cycling. n = 3 to 6 replicates. *P < 0.05, **P < 0.01, and ***P < 0.001.