Fig 1.
Insulin and metabolites changes with AICAR incubation.
(A) ZMP levels after AICAR incubation for 1 hour followed by glucose stimulation for 60 minutes (B) zoom view of endogenous ZMP levels after glucose stimulation. (C) Insulin level and stimulation index for INS-1 cells (832/13) after AICAR incubation and after glucose stimulation for 60 minutes. (D) Insulin level and stimulation index for INS-1 cells (832/3) after AICAR incubation and after glucose stimulation for 60 minutes. (E) Heat map showing fold change of metabolites with AICAR treatment. Significantly different values are highlighted with an asterisk. Student’s t-test was performed on each time point comparing the control and AICAR treated samples, with n = 3 or 4 for each replicate.
Fig 2.
AICAR effect on Acetyl-CoA Carboxylase (ACC).
(A) AMPK effect on ACC, (B) phosphoACC and total ACC after AICAR incubation and after glucose treatment for 10 and 20 minutes. Cells were incubated with/without AICAR for 1 h, followed by stimulation with 12C glucose for different time points, resulting in the illustrated levels of (C) malonyl-CoA, (D) ratio of malonyl CoA/acetyl CoA, and (E) palmitoyl-CoA. Student’s t-test was performed on all time points comparing the control and AICAR treated samples, with n = 3 or 4 for each replicate.
Fig 3.
AICAR effect on HMGR and Central Carbon metabolism.
(A) AMPK effect on mevalonate pathway. Cells were incubated with/without AICAR for 1 h, followed by stimulation with 12C glucose for different time points, resulting in the illustrated levels of (B) HMG-CoA, (C) farnesyl pyrophosphate and (D) ratio of farnesyl pyrophophate/ HMG-CoA. Cells were incubated with/without AICAR for 1 h, followed by stimulation with U-13C glucose for different time points, resulting in the illustrated levels of different isotopomers of (E) fructose bisphosphate and (F) Citrate. Student’s t-test was performed on all time points comparing the control and AICAR treated samples, with n = 3 or 4 for each replicate.
Fig 4.
Effect of AICAR on pentose phosphate and purine pathway metabolites.
Cells were incubated with/without AICAR for 1 h, followed by stimulation with 12C glucose for different time points, resulting in the illustrated levels of (A) GAR, (B) PRPP and (C) Ribose and ribulose-5-P. Cells were incubated with/without AICAR for 1 h, followed by stimulation with U-13C glucose for different time points, resulting in the illustrated levels of D) Unlabeled ATP and UTP, (E) +5 labeled ATP and UTP, and (F) +5 labeled ribose+ribulose-5 phosphate. The percentage of maximum was calculated based on the maximum of each species. (G) The purine and pyrimidine pathway (1) PRPP synthase (2) PRPP amidotransferase. Student’s t-test was performed on all time points comparing the control and AICAR treated samples, with n = 3 or 4 for each replicate.
Fig 5.
Effect of AICAR on metabolites in the Kennedy pathway for PE.
(A) CDP-ethanolamine after incubation with/without AICAR for 1 h followed by stimulation with glucose for different time points (B) CDP-ethanolamine levels after starvation for 6 h at low glucose (C) Extracted ion chromatogram and mass spectrum of labeled and unlabeled CDP-ethanolamine-after incubation with +4 ethanolamine for 1 h before glucose treatment for 30 minutes (D) levels of labeled and unlabeled CDP-ethanolamine-after incubation with +4 ethanolamine for 1 h and stimulation with glucose for 30 minutes. (E, F) Levels of PE and LPE after incubation for 1 hour with +4 ethanolamine and stimulation with 12C glucose for 30 minutes. Student’s t-test was performed with n = 3 or 4 for each replicate.
Fig 6.
Effect of AICAR on Ceramides and glycerolipids synthesis pathway.
(A) levels of DAG (34:1) isotopomers after stimulation with U-13C glucose for 65 minutes. (B) ratio of +3 labeled DAG/unlabeled DAG (C) Glycerolipid and Kennedy pathway for PE synthesis: ECT-ethanolamine phosphate cytidylyltransferase, EPT-ethanolamine phosphotransferase, GPAT-glycerol-3-phosphate O-acyltransferase, (D) CDP-ethanolamine levels after incubation of cells at 2 mM glucose for 60 minutes +/- AICAR +/- phospholipase C (PLC) inhibitor (U-73122) 20 μM or +/- phospholipase D inhibitor Cay10593 (60 μM). (E) Ceramide levels after incubation of cells with 50 μM palmitic acid +/- AICAR 250 μM for 1 h before stimulation with U-13C glucose for different time points. Student’s t-test was performed with n = 3 or 4 for each replicate.