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

Elsner M, Gehrmann W, Lenzen S - Diabetes (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Germany.

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.

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Related in: MedlinePlus

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.)
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Figure 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.)

Mentions: Although primary rat islet cells as well as untransfected RINm5F insulin-producing cells showed virtually no immunostaining for catalase (data not shown), RINm5F insulin-producing cells transfected to overexpress catalase in the cytosol (Cat) showed a homogenous distribution of catalase in the cytoplasm and a dot-like pattern of catalase colocalization with peroxisomes (Fig. 2A); on the other hand, catalase did not colocalize with mitochondria (Fig. 2B). Catalase was not detectable in the cytoplasm or in the peroxisomes of insulin-producing cells that overexpressed catalase in the mitochondria (Mito-Cat; Fig. 2C); in these cells, catalase colocalized specifically with the mitochondria (Fig. 2D).


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

Elsner M, Gehrmann W, Lenzen S - Diabetes (2010)

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.)
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Related In: Results  -  Collection

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Figure 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.)
Mentions: Although primary rat islet cells as well as untransfected RINm5F insulin-producing cells showed virtually no immunostaining for catalase (data not shown), RINm5F insulin-producing cells transfected to overexpress catalase in the cytosol (Cat) showed a homogenous distribution of catalase in the cytoplasm and a dot-like pattern of catalase colocalization with peroxisomes (Fig. 2A); on the other hand, catalase did not colocalize with mitochondria (Fig. 2B). Catalase was not detectable in the cytoplasm or in the peroxisomes of insulin-producing cells that overexpressed catalase in the mitochondria (Mito-Cat; Fig. 2C); in these cells, catalase colocalized specifically with the mitochondria (Fig. 2D).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Germany.

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.

Show MeSH
Related in: MedlinePlus