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

Abstract

Free fatty acids (FFA) cause apoptosis of pancreatic beta-cells and might contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary beta-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca(2+) stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor beta-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary beta-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced beta-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca(2+) depletion. The IRE1 and resulting JNK activation contribute to beta-cell apoptosis. PERK activation by palmitate also contributes to beta-cell apoptosis via CHOP.

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).

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.

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 eIF2α (P-eIF2α) and anti-ATF3 antibodies after 6–12 hours exposure to FFA are shown. Total eIF2α 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.

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).

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).

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.

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).

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.

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.