Heatmaps of selected pathways that were significantly enriched for hypoglycemia up-regulated genes at 48 hours identified by GSEA.

<p>A) KEGG Lysosome pathway; B) KEGG Glutathione metabolism; C) REACTOME Apoptosis. The genes shown in the heatmaps are the leading edge subsets (the enriched genes) from the GSEA analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150266#pone.0150266.ref020" target="_blank">20</a>]. GSEA score plots are shown alongside the heatmaps: Here a score peak towards the left indicates enrichment of pathway genes at the top of the list of genes ranked by fold-change. All three pathways shown were significantly enriched (corrected p-value ≤ 1e-4) based on 10000 permutations.</p

Increase expression of proteins involved in GSH metabolism and in the establisment of the extracellular matrix.

<p>Immunostaining of GPX3 and GSTO-1 proteins (A), and VERSICAN and FIBULIN-1 (B) in the retina of mice submitted to a hypoglycemic (Hypo) or euglycemic (Eugly) clamp (white arrows showed accumulation of proteins of interest). This experiment is representative of 3 retinas for each group.</p

GSH homeostasis was modulated in the retina of mice suffered single hypoglycemia but not after multiple successive hypoglycemic events.

<p>A) GSH content was measured in protein lysates obtained from retina of single euglycemic (sEugly: white inverted triangle), multiple euglycemic (mEugly: white triangle), single hypoglycemic (sHypo: black square) and multiple hypoglycemic (mHypo: black circle) mice. Results were expressed as percent of sEugly and as mean ± SEM of 2 to 4 samples. *p<0.01 sHypo vs. sEugly. B) We tested the expression of two genes, Gpx3 and Gsto1, in the retina of hypoglycemic and euglycemic animals 48 h after the clamp. We were able to show, by RT-qPCR, the induction of both genes in single hypoglycemic conditions, but not after multiple successive hypoglycemic or euglycemic. <i>RL8</i> (ribosomal protein L8) was used as internal control to normalize RNA expression and results are expressed as percent of respective control (sEugly, mEugly) and as mean ± SEM of 3 retinas, *p<0.04.</p

Multiple successive hypoglycemias did not exacerbate cell death in mouse retina but protect against retinal apoptosis.

<p>A) Flat-mounted retinas were isolated 48 h after the clamp, stained for cell death by TUNEL assay and DAPI counter coloration was performed. White arrows show TUNEL positive cells in single hypoglycemic condition (sHypo) while no TUNEL positive cells were detected when multiple successive hypoglycemia (mHypo) was applied. Control situation with multiple euglycemia (mEugly) or single euglycemia (sEugly) showed, as expected, no TUNEL positive cells. Results were representative of two (sEugly/mEugly) to three (sHypo/mHypo) observed flat-mount retinas for each group. B) Cleaved Caspase 3 antibody showed positive cells (white arrow) in the outer nuclear (ONL) of the hypoglycemic mice retina, while no positive cells were observed in all other conditions. Counterstaining with DAPI was performed to identify the retinal cell layers. Results were representative of four (sEugly), one (mEugly), four (sHypo) or two (mHypo) observed retinas for each group.</p

Identification of differentially expressed genes during hyperinsulinemic/hypoglycemic clamp.

<p>A) Mouse plasma glucose concentration before, during and after the clamp. *p<0.0001 Hypo (n = 18) vs. Eugly (n = 16). B) Heatmap of significantly differentially regulated genes (p≤0.01) at 4 hours and 48 hours, showing mean normalized intensities (n = 3) at each time-point and condition.</p

Differential methylation of the <i>Fxyd3</i> promoter in control and dKO islets.

<p>(A) Upper part: schematic representation of the <i>Fxyd3</i> promoter and the R1 and R2 regions that have been analyzed by sequencing following bisulfite conversion of cytosines. Lower part: schematic representation of the cytosines that were methylated (black dots), not methylated (circles) or not determined (brown dots). (B, C) DNA was prepared from control and dKO adult or neonates islets. Following bisulfite treatment, pyrosequencing was performed to quantitate the methylation of individual CpGs. (B) Global promoter methylation in neonate and adult islets. These data were derived from the calculated means of the percent methylated CpG at each position and for each group of mice. These means were compared for each position between groups using a two-way anova with repeated measurements with post-hoc Tukey test. Data are mean ± sem, n = 6–12 islet preparations; **p<0,01. (C) Quantitative methylation of seven CpGs in islets of control and dKO neonate and adult mice. Data are mean ± sem, n = 6–12 islet preparations; statistics are: #p<0.05 when compared to CTRL neonates (white dashed bars); *p<0.05 when compared to CTRL adults (white bars); £ p<0.05 when compared to dKO neonates (grey dashed bars) and $ p<0.05 when compared to dKO adults (grey bars).</p

Fxyd3 expression is not regulated by exendin-4 or forskolin in adult islets.

<p>Primary islets from adult control (A, B) and dKO (C, D) mice were incubated with DMSO (vehicle) or 10 µM forskolin (fsk) for the indicated periods of time after O/N rest. Then, RNA (A, C) or proteins (B, D) were extracted for quantitative analysis of Fxyd3 expression. Data are mean ± sem, n = 3 to 4. (E) Induction of IGF1-R expression in control islets after 18 hours of forskolin treatment. (F) <i>Fxyd3</i> is down-regulated during post-natal development in control but not in dKO islets. Neonates were 3 to 4 days-old. Data are mean ± sem, n = 10 to 11 samples per group, ***p<0,001.</p

<i>Fxyd3</i> promoter methylation in neonates and in diabetic mouse and human islets.

<p>(A) Ctrl neonates received daily injections for 7 days of Ex9–39 (2,5 nmol/kg) or Ex4 (2,5 nmol/kg) and GIP (80,4 nmol/kg). Methylation of CpG sites was determined by pyrosequencing. Data are mean ± sem, n = 3 experiments, *p<0,05, **p<0,01 as compared to control. (B) <i>Fxyd3</i> mRNA expression in islets prepared as in (A). Data are mean ± sem, n = 4 experiments. (C) <i>Fxyd3</i> mRNA expression in islets from <i>db/+</i> and <i>db/db</i> mice. These data were derived from the calculated means of the percent methylated CpG at each position and for each group of mice. These means were compared for each position between groups using a paired t-test. Data are mean ± sem, n = 4, *p<0,05. (D) Pyrosequencing analysis of global <i>Fxyd3</i> promoter methylation in islets from <i>db/+</i> and <i>db/db</i> mice. Data are mean ± sem, n = 4, **p<0,01. (E) Pyrosequencing analysis of the methylation of individual CpGs. Data are mean ± sem, n = 4, *p<0,05, **p<0,01, ***p<0,001. (E, F) <i>FXYD3</i> expression in human islets assessed by Affymetrix chip analysis (F), probeset g13528881_3p_a_at; (G), probeset g11612675_3p_a_at. Data are mean ± sem, n = 10 per group, **p<0,01 and ***p<0,001 compared to controls. (H) <i>FXYD3</i> mRNA expression measured by real-time PCR using a separate set of control (n = 8 donors) and T2D (n = 8 donors) humans islets. Data are mean ± sem, *p<0,05. (I) Inverse correlation of CpG -43 methylation and Fxyd3 expression in islets from Ctrl and T2DM patients.</p

Fxyd3 is overexpressed in dKO islets.

<p>(A) <i>Fxyd3</i> mRNA level in primary islets from control (CTRL), dKO, <i>Glp1r^−/−</i> or <i>Gipr^−/−</i> mice. Data are mean ± sem, n = 4–5; **p<0,01, ***p<0,001. (B) Western blot analysis and quantification of Fxyd3 expression in primary islets from CTRL, dKO, <i>Glp1r^−/−</i> and <i>Gipr^−/−</i> mice. Data are mean ± sem, *p<0,05, n = 4–5. (C) Confocal immunofluorescence microscopy detection of insulin and Fxyd3 in CTRL and dKO islets cultured on ECM plates. (D) Quantitative RT-PCR analysis of <i>Fxyd</i> family members expression in control and dKO islets. Data are mean ± sem n = 4–5, ***p<0,001 compared to CTRL.</p

Hypermethylation of the <i>Fxyd3</i> promoter reduces transcriptional activity.

<p>(A) ChIP analysis using H3K4me3 antibody reveals enrichment in this histone mark at the transcriptional start site of the <i>Fxyd3</i> gene in dKO as compared to control islets. Results are expressed as percent of total input. Data are mean ± sem, n = 3 experiments, *p<0,05. (B) Luciferase activity measured in MIN6 cells transfected with a basic pGL3 or a <i>Fxyd3</i>promoter-PGL3 reporter construct. Data are mean ± sem, n = 7 experiments realized in triplicates, ***p<0,001. (C) The same sequence of the mouse <i>Fxyd3</i> promoter was sub-cloned into the pCpGL vector (free of CpG). Following in vitro methylation (grey bar) or mock treatment (white bar), basic or <i>Fxyd3</i>promoter-pCpGL plasmids were transfected into MIN6 cells and luciferase activity was measured 48 h later. Methylation significantly reduces luciferase activity. Data are mean ± sem, n = 3 experiments realized in triplicates, **p<0,01. (B, C) Plasmids were co-transfected each time with Renilla reporter vector for normalization.</p