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First published online 9 August 2005
doi: 10.1242/jcs.02513

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ER stress and SREBP-1 activation are implicated in ß-cell glucolipotoxicity

Department of Cell Physiology and Metabolism, University Medical Center, Geneva, CH-1211, Switzerland

View larger version (60K): [in a new window]   Fig. 1. Effects of chronic high glucose treatment on insulin secretion and gene expression in INS-1E cell. (A) INS-1E cells in 24-well dishes were cultured in either standard (11.2 mM) or 30 mM glucose medium for 48 hours. After 5 hours equilibration in 2.5 mM glucose medium, cells were washed twice with KRBH. Insulin release from INS-1E cells stimulated with 20 mM glucose, 20 mM leucine and 20 mM KCl in KRBH buffer containing 2.5 mM (referred to Basal) was determined by radio-immunoassay and expressed as a percentage of cellular insulin content. The equilibration period is necessary to see the glucose-responsiveness of insulin release in INS-1 cells. Cellular insulin content was decreased by 32.4±6.7% after 48 hours treatment with 30 mM glucose. Data represent mean ± s.e.m. of six independent experiments. **P<0.001. (B,C) The gene expression patterns in INS-1E cells cultured in either standard (11.2 mM) or 30 mM glucose medium for 48 hours were quantified by northern blotting. Total RNA samples (20 µg/lane) were analysed by hybridizing with the indicated cDNA probes. Four independent experiments are shown side by side to demonstrate the consistency of the results.

View larger version (51K): [in a new window]   Fig. 2. Comparison of gene expression patterns in INS-1 cells allowing inducible expression, respectively, of ChREBP and the nuclear active form (na) of SREBP-1c. (A) ChREBP*16 cells were cultured in standard medium (11.2 mM glucose) with 0, 75, 150 or 500 ng/ml doxycycline for 24 hours. The culture was continued in 2.5 mmol/l glucose medium with the indicated concentrations of doxycycline for 16 hours, followed by an additional 8 hours in culture medium with 2.5, 6, 12 and 24 mM glucose and with or without doxycycline. The equilibration period is necessary to see the glucose-responsiveness of gene expression. (B) SREBP-1c*233 cells were cultured in 2.5 mM glucose medium with the indicated concentrations of doxycycline for 16 hours, followed by an additional 8 hours in culture medium with 2.5, 6, 12 and 24 mM glucose and with or without doxycycline. SREBP-1c was induced for a total of 24 hours to avoid its apoptotic effects. Gene expression patterns in these cells were evaluated by quantitative northern blotting. 20 µg total RNA samples were analysed by hybridising with indicated cDNA probes. WT, the endogenous wild-type SREBP-1c; NA, the induced nuclear active form of SRENP-1c. The experiments were repeated two times with similar results.

View larger version (14K): [in a new window]   Fig. 3. Overexpression of ChREBP did not affect glucose-stimulated insulin secretion or cell growth. (A) ChREBP*16 cells were cultured with (+Dox) or without (-Dox) 500 ng/ml doxycycline in standard medium (11.2 mM glucose) for 24 hours and then equilibrated in 2.5 mM glucose medium for a further 24 hours. Insulin release from ChREBP*16 cells in KRBH buffer containing the indicated concentrations of glucose was determined by radio-immunoassay and expressed as a percentage of cellular insulin content. Data represent six independent experiments. (B) A WST-1 assay, presented as optical density at 440 nm, showed that ChREBP*16 cell growth/survival was not affected by 48 hours treatment with 500 ng/ml doxycycline.

View larger version (63K): [in a new window]   Fig. 4. Characteristics of the INS-1 stable cell line expressing DN-SREBP-1c. (A) Phase-contrast images (top) and immunofluorescence staining (bottom) with a rabbit polyclonal antibody against SREBP-1 showed that DN-SREBP-1c protein was induced by doxycycline in an all-or-none manner. DN-SREBP-1c*23 cells were cultured with (+Dox) or without (-Dox) 500 ng/ml doxycycline for 48 hours. (B) Gel-shift assay of nuclear extracts from DN-SREBP-1c*23 cells using the SRE sequence of the IRS2 promoter. DN-SREBP-1c*23 cells were cultured in either standard (11.2 mM) or 30 mM glucose medium for 48 hours, in the presence (+Dox) or absence (-Dox) of 500 ng/ml doxycycline. Monoclonal SREBP-1-specific antibody was used for the supershift experiment. Experiments were repeated two to three times with similar results. (C) EMSA of nuclear extracts from non-induced DN-SREBP-1c*23 cells using the SRE sequence of the IRS2 promoter. DN-SREBP-1c*23 cells were cultured in either standard (11.2 mM) or 30 mM glucose medium for 48 hours. Monoclonal SREBP-2-specific antibody was used for the supershift experiment. Experiments were repeated twice with similar results.

View larger version (48K): [in a new window]   Fig. 5. Induction of DN-SREBP-1c largely prevented chronic high-glucose-induced glucolipotoxicity in INS-1 cells. (A) DN-SREBP-1c diminished lipid droplets accumulation, as seen by staining with Oil Red-O. DN-SREBP-1c*23 cells were cultured in either standard (11.2 mM; Control) or 30 mM glucose (High glucose) medium for 48 hours, in the presence (+Dox) or absence (-Dox) of 500 ng/ml doxycycline. (B) DN-SREBP-1c prevented apoptosis. DNA fragmentation was assessed in DN-SREBP-1c*23 cells cultured in 30 mM glucose medium for 0, 24, 48, and 72 hours, in the presence (+Dox) or absence (-Dox) of 500 ng/ml doxycycline. (C) DN-SREBP-1c partially restored glucose-stimulated insulin secretion. DN-SREBP-1c*23 cells in 24-well dishes were cultured in either standard (11.2 mM; Control) or 30 mM glucose (High Glucose) medium for 48 hours. After 5 hours equilibration in 2.5 mM glucose medium, cells were washed twice with KRBH. Insulin release from DN-SREBP-1c*23 cells stimulated with either 2.5 (Basal) or 20 mM glucose was determined by radio-immunoassay and expressed as a percentage of cellular insulin content. Data represent mean ± s.e.m. of six independent experiments. *P<0.01; **P<0.001.

View larger version (66K): [in a new window]   Fig. 6. Effects of DN-SREBP-1c on gene expression patterns induced by chronic high-glucose in INS-1 cells. DN-SREBP-1c*23 cells were cultured in either standard (11.2 mM; -) or 30 mM (+) glucose medium for 48 hours, in the presence (+Dox) or absence (-Dox) of 500 ng/ml doxycycline. The gene expression pattern in these cells was quantitatively evaluated by northern blotting. 20 µg total RNA samples were analysed by hybridising with the indicated cDNA probes. The experiments were repeated twice with similar results.

View larger version (27K): [in a new window]   Fig. 7. ER stress and SREBP-1 activation are implicated in ß-cell glucolipotoxicity. (A) The gene expression patterns in INS-1E cells cultured in either standard (11.2 mM; -) or 30 mM (+) glucose medium for 48 hours were quantified by northern blotting. Total RNA samples (20 µg/lane) were analysed by hybridizing with the indicated cDNA probes. Four independent experiments are shown side by side to demonstrate the consistency of the results. (B) Gel-shift assay of nuclear extracts from isolated rat islets using the SRE sequence of the IRS2 promoter. SREBP-1 protein binding activity was assessed in rat islets treated with 30 mM glucose for 0 (freshly isolated), 1, 2, 3, 4, and 5 days or alternatively with two ER stress inducers, thapsigargin (1 µM) and tunicamycin (10 µg/ml) for 1 day. The mouse monoconal antibody against SREBP-1 was used for supershift experiments.

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