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Files in this Data Supplement:
Fig. S1. Overview of the three main branches of the ER stress response. Under basal conditions, the three main ER stress transducers IRE1, ATF6 and PERK remain inactive due to binding to the ER chaperone BiP. When proteins are not properly folded in the ER lumen, BiP dissociates from the luminal side of the ER stress transducers, thereby activating them. Active IRE1 has endoribonuclease activity and causes alternative splicing of XBP1 mRNA. The XBP1s protein is a transcription factor regulating genes involved in protein folding and export from the ER, as well as export of misfolded proteins and ER-associated protein degradation (ERAD). IRE1 also activates JNK. Active ATF6 induces transcription of ER chaperones, such as BiP, and XBP1. PERK phosphorylates eIF2α, thereby inhibiting protein translation and arrival of newly synthesized proteins in the ER. Translation of some proteins such as the transcription factor ATF4 is facilitated, and ATF4 induces expression of CHOP and ATF3. The goal of ER stress signaling is to clear misfolded proteins and restore ER homeostasis. When the ER stress is prolonged and excessive, it will, however, trigger apoptosis through JNK, CHOP and ATF-3. Caspases contribute to the execution of apoptosis. Because protein folding in the ER is Ca2+-dependent, proteins can misfold when the SERCA pump is inhibited and ER Ca2+ stores are depleted.
Fig. S2. Glucolipotoxicity in β-cells. INS-1E (A) and primary rat β-cells (B) were cultured for 12 hours to 6 days without (control, black circles) or with oleate (0.5 mM; gray diamonds); palmitate (0.5 mM, black squares) at 10-11 mM glucose. The results are the means ± s.e.m. of 6-19 independent experiments. **P<0.01; ***P<0.001 vs control. (C) Primary rat β-cells were cultured for 6 days in the presence or absence of oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars) at glucose concentrations of 6.1, 10 and 28 mM. The results are the means ± s.e.m. of seven independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs respective glucose control, lP<0.05 vs low glucose (6.1 mM), mP<0.05 vs medium glucose (10 mM).
Fig. S3. Palmitate induces eIF2-α phosphorylation. INS-1E cells were cultured in the presence or absence of oleate and/or palmitate at glucose concentrations of 11 or 28 mM, or with CPA, used as a positive control. (A) Western blots using a phospho-specific eIF2-α antibody after 6 and 24 h exposure. Total eIF2-α is used as a protein loading control. One representative experiment for three similar experiments is shown. (B) Mean optical density measurements for the 6-hour exposure to oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars); n=3; *P<0.05 vs respective glucose control, mP<0.05 vs glucose 11 mM.
Fig. S4. FFA induce ATF3 expression in INS-1E cells. ATF3 mRNA expression was analyzed by real time PCR and normalized for the expression level of the housekeeping gene GAPDH. INS-1E cells were cultured for 6-48 hours in the presence of oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars) at glucose concentrations of 5.6, 11 or 28 mM. The results represent means ± s.e.m. of six independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs respective glucose control, lP<0.05 vs low glucose (5.6 mM), mP<0.05 vs medium glucose (11 mM).
Fig. S5. FFA induce CHOP expression in INS-1E cells. CHOP mRNA expression was analyzed by real time PCR and normalized for the expression level of the housekeeping gene GAPDH. INS-1E cells were cultured for 6-48 hours in the presence of oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars) at glucose concentrations of 5.6, 11 or 28 mM. The results represent means ± s.e.m. of six independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs respective glucose control, lP<0.05 vs low glucose (5.6 mM), mP<0.05 vs medium glucose (11 mM).
Fig. S6. FFA induce BiP expression in INS-1E cells. BiP mRNA expression was analyzed by real time PCR and normalized for the expression level of the housekeeping gene GAPDH. INS-1E cells were cultured for 6-48 hours in the presence of oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars) at glucose concentrations of 5.6, 11 or 28 mM. The results represent means ± s.e.m. of six independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs respective glucose control, lP<0.05 vs low glucose (5.6 mM), mP<0.05 vs medium glucose (11 mM).
Fig. S7. FFA induce XBP1 expression in INS-1E cells. Total XBP1 mRNA expression was analyzed by real time PCR and normalized for the expression level of the housekeeping gene GAPDH. INS-1E cells were cultured for 6-48 hours in the presence of oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars) at glucose concentrations of 5.6, 11 or 28 mM. The results represent means ± s.e.m. of six independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs respective glucose control, lP<0.05 vs low glucose (5.6 mM), mP<0.05 vs medium glucose (11 mM).
Fig. S8. FFA activate the UPR reporter in INS-1E cells. INS-1E cells were co-transfected with the UPR luciferase reporter, responsive to ATF6 and XBP1s, and the internal control pRL-CMV, encoding Renilla luciferase. After overnight transfection, the cells were exposed for 24 hours to oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars) at glucose concentrations of 5.6, 11 or 28 mM and assayed for firefly and Renilla luciferase activities. The results were normalized for Renilla luciferase activity. The results are means ± s.e.m. of ten independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs respective glucose control, lP<0.05 vs low glucose (5.6 mM), mP<0.05 vs medium glucose (11 mM).
Fig. S9. Exposure of INS-1E cells to FFA leads to XBP1 splicing. Spliced XBP1 mRNA expression was analyzed by real time PCR and normalized for the expression level of the housekeeping gene GAPDH. INS-1E cells were cultured for 6-48 hours in the presence of oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars) at glucose concentrations of 5.6, 11 or 28 mM. The results represent means ± s.e.m. of six independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs respective glucose control, lP<0.05 vs low glucose (5.6 mM), mP<0.05 vs medium glucose (11 mM).
Fig. S10. Role of ATF3 in FFA-mediated β-cell apoptosis. (A) Apoptosis in dispersed islet cells after a 72-hour exposure to 0.5 mM palmitate at 10 mM glucose. Islets were isolated from wild type (wt, open bars) or ATF3−/− mice (black bars). Results are means ± s.e.m. of 4-6 independent experiments. *P<0.05, **P<0.001 vs control. (B and C) INS-1E cells were transfected with two siRNAs against ATF3 mRNA (#1 and #2, gray bars), negative siRNA (N, black bars) or control medium (C, open bars) and exposed to CPA, 0.5 mM palmitate or vehicle (control) for 14 hours. (B) One representative ATF3 Western blot and optical densitometry of ATF3 protein expression levels of four independent experiments are shown. Results are expressed as ATF3 per β-actin protein and are means ± s.e.m. *P<0.05 vs vehicle-treated control cells, #P<0.05 vs non-transfected cells (C, open bars). (C) Percentage apoptosis after exposure to CPA or palmitate for 14 hours. Results are means ± s.e.m. of five independent experiments. *P<0.05, **P<0.001 vs vehicle-treated control cells, #P<0.05 vs non-transfected cells (C, open bars). (D) Primary rat β-cells were transfected with an siRNA against ATF3 (si, gray bars), negative siRNA (N, black bars) or control medium (C, open bars) and exposed to CPA, 0.5 mM palmitate or vehicle (control) for 24 hours. Primary β-cell ATF3 and β-actin Western blots (n=1) and apoptosis (n=3) are shown. *P<0.05 vs vehicle-treated control cells; #P<0.05 vs non-transfected cells (C, open bars).
Fig. S11. High glucose sensitizes INS-1E cells to ER stress-induced apoptosis. Following a culture for 36 hours at glucose concentrations of 10-11 (gray bars) or 28 mM (black bars), INS-1E (A) or primary rat β-cells (B) were exposed to CPA (3 to 50 µM) for 16 hours at these different glucose concentrations. The results are the means ± s.e.m. of four to five independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs respective glucose control, mP<0.05 vs 10-11 mM glucose.
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