Figure 1. Chronic (>8 wk) 25 mM High Glucose Increases Glycolysis in IMS32 Schwann Cells. A, Schematic diagram of glycolysis. Enzymes that promote glycolysis (italicized right of arrow) and those that oppose glycolysis (italicized left of arrow) are regulated by chronic high glucose in Schwann cells (full gene names listed in Table 1). mRNA levels of proglycolytic (B–J) and antiglycolytic (K–M) enzymes during normal (5.6 mM), chronic (>8 wk) high (25 mM), and chronic high returned to normal glucose conditions. N, Glycolysis rate as measured by extracellular acidification rate of 5.6 mM glucose, 25 mM glucose for 24 hours, 4 weeks, and 4 weeks high glucose returned to normal for 24 hours. Data are represented as mean ± SEM. Asterisks indicate significance by Dunnett's post hoc test compared with the 5.6 mM glucose control group. *, P < .05; **, P < .01; ***, P < .001. mRNA levels are expressed relative to normal glucose following normalization with HK genes Gusb, Hprt1, and Hsp90ab1. From: Glucose-Induced Metabolic Memory in Schwann Cells: Prevention by PPAR Agonists Endocrinology. 2013;154(9):3054-3066. doi:10.1210/en.2013-1097 Endocrinology | Copyright © 2013 by The Endocrine Society

Figure 2. Persistence of Glucose-Induced Gene Memory Figure 2. Persistence of Glucose-Induced Gene Memory. A–C, Expression of Fbp1, Fbp2, and Gpd1 at 5.6 mM normal glucose, chronic (>8 wk) 25 mM high glucose, and upon return to 5.6 mM normal glucose for 2 and 4 weeks. Data are represented as mean ± SEM. Asterisks indicate significance by Dunnett's post hoc test compared with the 5.6 mM glucose control group. *, P < .05; **, P < .01; ***, P < .001. mRNA levels are expressed relative to normal glucose after normalization with HK genes Ppib and Hprt1. From: Glucose-Induced Metabolic Memory in Schwann Cells: Prevention by PPAR Agonists Endocrinology. 2013;154(9):3054-3066. doi:10.1210/en.2013-1097 Endocrinology | Copyright © 2013 by The Endocrine Society

Figure 3. The Effect of High Glucose on Oxidative Stress Response, NADH Availability, and ATP Levels. A, Schwann cells maintained at 5.6 mM normal or 25 mM high glucose for more than 8 weeks were measured for ROS formation, and after change of glucose media for 4 hours. B and D, NADH availability and ATP levels at normal and high glucose, and after change to 15 mM glucose for 48 hours. C and E, Effect of the metabolites pyruvate, lactate, and β-hydroxybutyrate ketone body (30 mM) on NADH and ATP for 48 hours. Data are represented as mean ± SEM. Means with different letters are significantly different using Bonferroni post hoc test following one-way ANOVA, P < .05. From: Glucose-Induced Metabolic Memory in Schwann Cells: Prevention by PPAR Agonists Endocrinology. 2013;154(9):3054-3066. doi:10.1210/en.2013-1097 Endocrinology | Copyright © 2013 by The Endocrine Society

Figure 5. PPAR Agonism to Reverse or Prevent Glucose-Induced Gene Memory. A–D, Intervention of metabolic memory: Following 25 mM high glucose (>8 wk), PPARγ agonist pioglitazone (10 μM) was applied for 1 week, indicated by +. Glucose-induced changes in several metabolic genes (Cpt1b, Fbp2, Acaa2, Plin2) were not reversed using this intervention. E–H, Prevention of metabolic memory: PPARγ agonist (pio) and PPARα agonist (feno) (10 μM each) were applied during exposure to 25 mM high glucose (>8 wk). Glucose-induced changes were selectively prevented in rate-limiting metabolic genes (Cpt1b, Fbp1, Fbp2, Pfkl). Data are represented as mean ± SEM. Asterisks indicate significance by Dunnett's post hoc test compared with the 5.6 mM glucose control group. *, P < .05; **, P < .01; ***, P < .001. mRNA levels are expressed relative to normal glucose after normalization with HK genes Ppib and/or Hprt1. From: Glucose-Induced Metabolic Memory in Schwann Cells: Prevention by PPAR Agonists Endocrinology. 2013;154(9):3054-3066. doi:10.1210/en.2013-1097 Endocrinology | Copyright © 2013 by The Endocrine Society

Figure 6. DNA Methylation of Fbp1 after Exposure to 25 mM High Glucose (>8 wk) and after Normalization (5.6 mM) for 1 Week. Location of (A) Fbp1 relative to (B) amplicon regions assayed, (C) CpG sites, and (D) CpG island of Fbp1 using UCSC Genome Browser (http:/genome.ucsc.edu/). Black bars indicate individual CpG sites, red bars indicate decreased methylation following high glucose, and green bars indicate increased methylation. F–N, Percent methylation was obtained for each CpG assayed, location corresponding to CpG ID (E), (n = 8), mean ± SEM. one-way ANOVA followed by Dunnett's Multiple Comparison Test compared with 5.6 mM control; *, P < .05; **, P < .01; ***, P < .001. Data for CpG sites within the same fragment after cleavage reactions are shown together (yellow bracket in C) because data for these sites fails to be uniquely informative. From: Glucose-Induced Metabolic Memory in Schwann Cells: Prevention by PPAR Agonists Endocrinology. 2013;154(9):3054-3066. doi:10.1210/en.2013-1097 Endocrinology | Copyright © 2013 by The Endocrine Society

Figure 4. PPARγ ChIP and Protein Expression Figure 4. PPARγ ChIP and Protein Expression. A, Schwann cells maintained at 25 mM high glucose (>8 wk) exhibit decreased PPARγ binding to the promoter of Cpt1b, Acaa2, Gpd1, and Fbp2 using chromatin immunoprecipitation. Data are shown as percent input, which represents the DNA sequence enriched by immunoprecipitation with PPARγ antibody normalized to input DNA (n = 8). Student's t test compares 5.6 vs 25 mM glucose (*, P < .05). B, Representative Western blot of PPARγ in the cytoplasmic and nuclear fraction at 5.6 mM glucose, chronic (>8 wk) 25 mM glucose, and after return to 5.6 mM glucose for 1 week. C, Quantification of PPARγ protein levels normalized to actin levels. Data represented as mean ± SEM (n = 3). From: Glucose-Induced Metabolic Memory in Schwann Cells: Prevention by PPAR Agonists Endocrinology. 2013;154(9):3054-3066. doi:10.1210/en.2013-1097 Endocrinology | Copyright © 2013 by The Endocrine Society

Figure 7. DNA Methylation of Fbp2 after Exposure to 25 mM High Glucose (>8 wk) and after Normalization (5.6 mM) for 1 Week. Location of (A) Fbp2 relative to (B) amplicon regions assayed and (C) CpG sites using UCSC Genome Browser (http:/genome.ucsc.edu/). Black bars indicate individual CpG sites, red bars indicate decreased methylation following high glucose, and green bars indicate increased methylation. E–J, Percent methylation was obtained for each CpG assayed, location corresponding to CpG ID (D), (n = 8), mean ± SEM. one-way ANOVA followed by Dunnett's Multiple Comparison Test compared with 5.6 mM control; *, P < .05; **, P < .01; ***, P < .001. From: Glucose-Induced Metabolic Memory in Schwann Cells: Prevention by PPAR Agonists Endocrinology. 2013;154(9):3054-3066. doi:10.1210/en.2013-1097 Endocrinology | Copyright © 2013 by The Endocrine Society