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First published online 17 June 2008
doi: 10.1242/jcs.026062

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Initiation and execution of lipotoxic ER stress in pancreatic β-cells

Daniel A. Cunha^1,*, Paul Hekerman^1,*, Laurence Ladrière^1,*, Angie Bazarra-Castro¹, Fernanda Ortis¹, Marion C. Wakeham¹, Fabrice Moore¹, Joanne Rasschaert¹, Alessandra K. Cardozo¹, Elisa Bellomo², Lutgart Overbergh³, Chantal Mathieu³, Roberto Lupi⁴, Tsonwin Hai⁵, Andre Herchuelz⁶, Piero Marchetti⁴, Guy A. Rutter², Décio L. Eizirik¹ and Miriam Cnop^1,7,

¹ Laboratory of Experimental Medicine, Université Libre de Bruxelles CP-618, Route de Lennik 808, 1070 Brussels, Belgium
² Department of Cell Biology, Division of Medicine, Faculty of Medicine, Imperial College London, London, UK
³ Laboratory for Experimental Medicine and Endocrinology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium
⁴ Department of Endocrinology and Metabolism, Metabolic Unit, University of Pisa, Pisa, Italy
⁵ Department of Molecular and Cellular Biochemistry and Center for Molecular Neurobiology, Ohio State University, Columbus, OH, USA
⁶ Laboratory of Pharmacology, Université Libre de Bruxelles, Brussels, Belgium
⁷ Division of Endocrinology, Erasmus Hospital, Brussels, Belgium

View larger version (21K): [in this window] [in a new window]   Fig. 1. Glucolipotoxicity in rat β-cells. INS-1E cells (A) and rat primary β-cells (B) were cultured for 72 hours in the presence or absence of oleate (0.5 mM, gray bars), palmitate (0.5 mM, black bars) or an equimolar combination of oleate and palmitate (0.25 mM each, hatched bars) at glucose concentrations (G, shown in mM) as indicated. The results are the means ± s.e.m. of 7-15 independent experiments. **P<0.01, ***P<0.001 vs respective glucose control (i.e. not exposed to FFA and cultured at the same glucose concentration); l, P<0.05 vs low glucose (5.6 or 6.1 mM); m, P<0.05 vs medium glucose (10 or 11 mM).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Glucolipotoxicity in human islets. Human islets were cultured for 72 hours or 6 days in the presence or absence of oleate (gray bars) or palmitate (black bars) at a glucose concentration of 6.1 or 28 mM, as indicated. The results are the means ± s.e.m. of four to seven independent experiments. *P<0.05, **P<0.01 vs respective control.

View larger version (31K): [in this window] [in a new window]   Fig. 3. FFA activate PERK signaling. INS-1E cells were cultured in the presence or absence of oleate (O, gray diamonds), palmitate (P, black squares) or oleate plus palmitate (OP, white circles) at 11 mM glucose. Western blots using phospho-specific PERK (P-PERK), phospho-specific eIF2 (P-eIF2) and anti-ATF3 antibodies after 6-12 hours exposure to FFA are shown. Total eIF2 or β-actin was used as control for protein loading. One representative experiment for three to six similar experiments is shown, as well as mean optical density measurements of the western blots following 6-, 12- and 24-hour FFA exposure; n=2-6. ATF3 and CHOP mRNA expression following a 6- to 48-hour FFA exposure was analyzed by real-time PCR, normalized for the expression level of the housekeeping gene GAPDH and expressed as fold induction of the control. The results represent means ± s.e.m. of 6-11 independent experiments. *P<0.05, **P<0.01 vs control.

View larger version (32K): [in this window] [in a new window]   Fig. 4. FFA activate IRE1 and ATF6 signaling. INS-1E cells were cultured in the presence or absence of oleate (O, gray diamonds), palmitate (P, black squares) or oleate plus palmitate (OP, white circles) at a glucose concentration of 11 mM. The mRNA expression of BiP, XBP1s and XBP1t following a 6- to 48-hour FFA exposure was analyzed by real-time PCR, normalized for the expression level of the housekeeping gene GAPDH and expressed as fold induction. The results represent means ± s.e.m. of 6-11 independent experiments. For the measurement of UPR luciferase reporter activity, INS-1E cells were co-transfected with the UPR reporter, which is responsive to ATF6 and XBP1s, and with the internal control pRL-CMV, encoding Renilla luciferase. After overnight transfection, the cells were exposed for 24 hours to FFA. The firefly results were normalized for Renilla luciferase activity and are means ± s.e.m. of ten independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs control (C).

View larger version (28K): [in this window] [in a new window]   Fig. 5. FFA activate ER stress signaling in rat primary β-cells and human islets. (A) FACS-purified rat β-cells were cultured for 24 hours in the presence of oleate (gray bars), palmitate (black bars) or oleate plus palmitate (hatched bars) at glucose concentrations of 10 and 28 mM. (B) Human islets were cultured for 48 hours in the absence (white bars) or presence of oleate (gray bars) or palmitate (black bars) at glucose concentrations of 6.1 and 28 mM. ATF3, CHOP, XBP1s and BiP mRNA expression was analyzed by real-time PCR, normalized for the expression level of the housekeeping genes GAPDH (for rat β-cells) or β-actin (for human islets) and expressed as fold induction of the control. The results represent means ± s.e.m. of three (human) or four to seven (rat) independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs control (white bars).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Non-metabolized non-toxic methyl FFA do not elicit ER stress. INS-1E cells were exposed to control (C), palmitate (0.5 mM, P) or methyl-palmitate (0.5 mM, MeP) at 11 mM glucose. After 72 hours, the percentage of apoptotic cells was determined; n=4-5 (left). mRNA expression of ER stress markers was analyzed after 24 hours by real-time PCR and normalized for the housekeeping gene GAPDH; n=4. *P<0.05, **P<0.01 vs control.

View larger version (32K): [in this window] [in a new window]   Fig. 7. FFA deplete ER calcium stores in β-cells. (A) INS-1E cells were cultured for 24 hours in the presence of oleate (O, red line), palmitate (P, green line) or control (C, black line) at a glucose concentration of 11 mM. The intracellular Ca2+ concentration was measured at baseline and following acute thapsigargin stimulation (arrow). The traces shown are the means of 5-6 independent experiments (278-368 cells/experiment). (B) Following infection with an ER-targeted aequorin-encoding adenovirus, INS-1E cells were cultured for 3-24 hours in the presence of oleate (O, red line), palmitate (P, green line), an equimolar mixture of oleate and palmitate (OP, blue line) or control (C, black line) at a glucose concentration of 11 mM. Prior to the measurements, cells were depleted of Ca2+ and the aequorin was reconstituted with coelenterazine n in Ca2+-free KRBB. External CaCl2 (1.5 mM) was then reintroduced at t=20 seconds to allow the reestablishment of ER Ca2+ levels. The results are the means ± s.e.m. of four independent experiments. Bold diamond symbols represent Ca2+ values significantly different from control condition (P<0.05).

View larger version (33K): [in this window] [in a new window]   Fig. 8. CHOP knockdown delays CPA- and FFA-mediated β-cell apoptosis. INS-1E cells were transfected with siRNA against CHOP mRNA (si, gray bars), negative (inactive) siRNA (N, black bars) or empty liposomes (C, white bars) and exposed to CPA (positive control), 0.5 mM palmitate or vehicle (control) for 6-24 hours. (A,B) One representative CHOP western blot after 24-hour exposure and optical densitometry of CHOP protein expression from four independent experiments are shown. Results are expressed as CHOP protein normalized to β-actin protein (used as a loading control) and are means ± s.e.m. (C,D) Percentage of cells undergoing apoptosis after exposure to CPA for 6-24 hours (C) or palmitate for 14-24 hours (D). Results are means ± s.e.m. of four to seven independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs control; #P<0.05 vs non-transfected cells.

View larger version (19K): [in this window] [in a new window]   Fig. 9. Involvement of JNK in FFA-induced β-cell apoptosis. (A) INS-1E cells were co-transfected with the AP-1 reporter and the internal control pRL-CMV, encoding Renilla luciferase. After transfection and overnight recovery, the cells were exposed for 24 hours to control or palmitate (0.5 mM) at 11 mM glucose in the presence or absence of the JNK inhibitors SP600125 (25 µM, gray bars) or L-TAT-JNKi (1 µM, black bars), and assayed for firefly and Renilla luciferase activities. The results were normalized for Renilla luciferase activity and are means ± s.e.m. of four to seven independent experiments. (B) After 72 hours, the percentage of apoptotic cells was determined. n=8-14. *P<0.001 vs control; #P<0.01 vs similar condition without JNK inhibitor.

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