Peroxisome-generated hydrogen peroxide as important mediator of lipotoxicity in insulin-producing cells

Abstract

Objective: Type 2 diabetes is a complex disease that is accompanied by elevated levels of nonesterified fatty acids (NEFAs), which contribute to β-cell dysfunction and β-cell loss, referred to as lipotoxicity. Experimental evidence suggests that oxidative stress is involved in lipotoxicity. In this study, we analyzed the molecular mechanisms of reactive oxygen species-mediated lipotoxicity in insulin-producing RINm5F cells and INS-1E cells as well as in primary rat islet cells.

Research design and methods: The toxicity of saturated NEFAs with different chain lengths upon insulin-producing cells was determined by MTT and propidium iodide (PI) viability assays. Catalase or superoxide dismutase overexpressing cells were used to analyze the nature and the cellular compartment of reactive oxygen species formation. With the new H₂O₂-sensitive fluorescent protein HyPer H₂O₂ formation induced by exposure to palmitic acid was determined.

Results: Only long-chain (>C14) saturated NEFAs were toxic to insulin-producing cells. Overexpression of catalase in the peroxisomes and in the cytosol, but not in the mitochondria, significantly reduced H₂O₂ formation and protected the cells against palmitic acid-induced toxicity. With the HyPer protein, H₂O₂ generation was directly detectable in the peroxisomes of RINm5F and INS-1E insulin-producing cells as well as in primary rat islet cells.

Conclusions: The results demonstrate that H₂O₂ formation in the peroxisomes rather than in the mitochondria are responsible for NEFA-induced toxicity. Therefore, we propose a new concept of fatty acid-induced β-cell lipotoxicity mediated via reactive oxygen species formation through peroxisomal β- oxidation.

FIG. 1.

Toxicity of saturated NEFAs according to chain length (C10:0-C18:0) in RINm5F insulin-producing cells. Cells were incubated for 24 h with saturated NEFAs of different chain lengths and viability was determined by MTT assay (A) or propidium iodide staining (B). EC₅₀ values were calculated by nonlinear regression analysis. Data are means ± SEM from 4–6 individual experiments. *P < 0.01 as compared with C10:0, C11:0, C12:0, or C13:0. Other comparisons were not significant (ANOVA/Tukey test for multiple comparisons).

FIG. 2.

Immunocytochemical staining for catalase, peroxisomes, and mitochondria in catalase or mitocatalase overexpressing RINm5F insulin-producing cells. RINm5F insulin-producing cells that overexpressed catalase in the cytosol (Catalase, A and B) or in the mitochondria (MitoCatalase, C and D) were seeded overnight on collagen-coated coverslips. After fixation with 4% paraformaldehyde, the cells were stained for catalase (red) and for the peroxisomal membrane protein 70 (PMP-70 green) or the mitochondrial respiratory chain enzyme cytochrome c-oxidase IV (COX-4 green) followed by nuclear counterstaining with DAPI (blue). To quantify the colocalization between catalase and the peroxisomes or mitochondria 20 images of two independent preparations were analyzed with the colocalization add-in of the CellR software (Olympus, Hamburg, Germany). The analyses showed that 56.2 ± 3.3% (n = 58) of catalase were localized in the peroxisomes (A) and 5 ± 0.4% (n = 74) in the mitochondria (B). For the MitoCatalase expressing cells, a proportion of 86.3 ± 2.6% (n = 90) of catalase was detected in the mitochondria (C) and 5.1 ± 1.6% (n = 62) in the peroxisomes (D). Data are means ± SEM of (n) individual cells. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Palmitic acid toxicity in RINm5F insulin-producing cells that overexpress different antioxidative enzymes. RINm5F cells that stably overexpressed the cytosolic antioxidative enzymes copper zinc superoxide dismutase (CuZnSOD) and catalase (Cat) or the mitochondrial antioxidative enzymes manganese superoxide dismutase (MnSOD) and catalase with a mitochondrial leader sequence (Mito-Cat) were incubated with palmitic acid (100 μmol/l) for 24 h; viability was determined by MTT assay. Mock-transfected RINm5F cells served as controls; untreated cells were set as 100% viability. Data are means ± SEM from five individual experiments. **P < 0.01 as compared with control cells (ANOVA/Dunnett test for multiple comparisons).

FIG. 4.

Palmitic acid-induces production of reactive oxygen species in RINm5F insulin-producing cells that overexpress catalase in the cytosol (Cat) or in the mitochondria (Mito-Cat). To determine ROS generation, cells were loaded with 10 μmol/l of DCF-DA dye for 30 min and then cultured with 100 μmol/l palmitic acid for 24 h. DCF fluorescence was measured after 24 h and normalized to that of untreated cells. Data are means ± SEM from seven individual experiments. ##P < 0.01 as compared with untreated cells (t test, unpaired, two-tailed); **P < 0.01 as compared with control cells (ANOVA/Dunnett test for multiple comparisons).

FIG. 5.

Live cell fluorescence microscopy for detection of H₂O₂ in RINm5F insulin-producing cells using the H₂O₂ sensor proteins HyPer-Mito and HyPer-Peroxi. Cells that stably expressed the H₂O₂ sensor protein HyPer in the mitochondria (HyPer-Mito A and B) or peroxisomes (HyPer-Peroxi C and D) were incubated with 100 μmol/l palmitic acid for 24 h. Shown are representative images at 0 h (A and C) and 24 h (B and D). Fluorescence at 504/520 nm is depicted in red and fluorescence at 427/520 nm is shown in green. Increased H₂O₂ generation is indicated by a color change from green to yellow to red. The group specified as “PA 0 h” comprises cells that were analyzed immediately after NEFA treatment (0 h). E: To quantify the hydrogen peroxide production images of RINm5F cells were analyzed with the CellR software (Olympus, Hamburg, Germany). The fluorescence intensities of individual cells were measured at 504/520 nm and 427/520 nm and the ratio of both wavelength pairs indicates the H₂O₂ production. Data are means ± SEM from four individual experiments. *P < 0.05 as compared with H₂O₂ production at 0 h (t test, unpaired, two-tailed). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

Live cell fluorescence microscopy for detection of H₂O₂ in primary rat islet cells using the H₂O₂ sensor protein HyPer-Peroxi. Primary rat islet cells were infected with HyPer-Peroxi lentivirus at a MOI of 10. Islet cells which expressed the H₂O₂ sensor protein HyPer in peroxisomes (HyPer-Peroxi) were incubated with 500 μmol/l palmitic acid for 24 h (C and D). Shown are representative images at 0 h (A and C) and 24 h (B and D). Fluorescence at 504/520 nm is depicted in red and fluorescence at 427/520 nm is shown in green. Increased H₂O₂ generation is indicated by a color change from green to yellow to red. The group specified as “PA 0 h” comprises cells that were analyzed immediately after NEFA treatment (0 h). Cells specified as “untreated” were cultivated under control conditions in medium with 1% ethanol and the appropriate BSA concentration (BSA:NEFA ratio of 2%: 1 mmol/l) in the absence of NEFAs. E: To quantify the hydrogen peroxide production images of primary rat islet cells were analyzed with the CellR software (Olympus, Hamburg, Germany). The fluorescence intensities of individual cells was measured at 504/520 nm and 427/520 nm and the ratio of both wavelength pairs indicates the H₂O₂ production. Data are means ± SEM from four individual experiments. *P < 0.05 as compared with H₂O₂ production at 0 h (t test, unpaired, two-tailed). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

Localization of H₂O₂ production in RINm5F insulin-producing cells after palmitic acid treatment. Cells that stably expressed the H₂O₂ sensor protein HyPer in peroxisomes (A) or mitochondria (B) and catalase in the cytosol (Cat) or mitochondria (Mito-Cat) were treated with 50 or 100 μmol/l palmitic acid for 24 h. The fluorescence ratio (504/520 nm to 427/520 nm), which is an indicator of H₂O₂ production, was measured spectrofluorometrically. Shown are the changes in the fluorescence ratios after 24 h normalized to the fluorescence ratios of untreated cells. Data are means ± SEM from 10 individual experiments. #P < 0.05 vs. untreated cells (0 μmol/l PA), *P < 0.05, **P < 0.01 vs. control cells (ANOVA/Dunnett test for multiple comparisons).

FIG. 8.

Localization of H₂O₂ production in INS-1E insulin-producing cells after palmitic acid treatment. Cells that stably expressed the H₂O₂ sensor protein HyPer in the peroxisomes or the mitochondria were treated with 500 μmol/l palmitic acid for 24 h. The fluorescence ratio (504/520 nm to 427/520 nm), which is an indicator of H₂O₂ production, was measured spectrofluorometrically. Shown are the changes in the fluorescence ratios after 24 h normalized to the fluorescence ratios of untreated cells. Data are means ± SEM from 14 individual experiments. *P < 0.05 vs. HyPer-Peroxi; ##< 0.01 vs. untreated cells (t test, unpaired, two-tailed).