Effect of AICAR on Ceramides and glycerolipids synthesis pathway.

<p>(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.</p

Effect of AICAR on metabolites in the Kennedy pathway for PE.

<p>(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.</p

AICAR effect on HMGR and Central Carbon metabolism.

<p>(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.</p

Purification of NR from PAB076-Conditioned Media.

<p>A) HPLC trace of media collected from strain PAB076 grown to OD 60 in 2x YPD and supplemented with 5 mM NA. B) Preparative SP-Sephadex chromatography with fractions analyzed by HPLC. NR eluted at 20 to 50 mM NaCl in fractions 27 to 36. C) Intracellular NAD⁺ determination of strain BY4742 grown in NA-free SDC media and NA-free SDC media supplemented with 10 µM NR from fractions produced in panel B.</p

Insulin and metabolites changes with AICAR incubation.

<p>(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.</p

Effect of AICAR on pentose phosphate and purine pathway metabolites.

<p>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.</p

<i>S. cerevisiae</i> strains used in this study.

<p><i>S. cerevisiae</i> strains used in this study.</p

Heatmap illustrating alterations in metabolite levels in tissues collected using different methods of anesthesia.

<p>Data are expressed as fold change versus cervical dislocation. n = 8 mice per method of anesthesia or euthanasia. • indicates p < 0.05 versus cervical dislocation after false discovery rate correction.</p

Principal component analysis of untargeted metabolomics data from tissues collected using different methods of anesthesia.

<p>Two-dimensional PCA score plots reveal separation in metabolite profiles induced by different methods of anesthesia and euthanasia in C57BL/6J mice. Tissues analyzed were a) skeletal muscle, b) heart, c) liver, d) white adipose and e) serum. Methods of anesthesia and euthanasia were: CD, cervical dislocation euthanasia (red); CO2, Carbon dioxide euthanasia (green); Iso-Cont, continuous isoflurane anesthesia (dark blue); Iso-OD, isoflurane overdose euthanasia (light blue); Ket, ketamine anesthesia (pink); Pent, pentobarbital anesthesia (orange). Ellipses represent the 95% confidence interval.</p

Annotated features most responsible for differentiating methods of anesthesia and euthanasia in each tissue as determined by PLS-DA analysis.

<p>Annotated features most responsible for differentiating methods of anesthesia and euthanasia in each tissue as determined by PLS-DA analysis.</p