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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

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).
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Figure 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).

Mentions: ROS generation in RINm5F insulin-producing control cells as measured by the DCF fluorescence method was increased by about 40% in response to exposure to 100 μmol/l palmitic acid (Fig. 4). Overexpression of catalase in the peroxisomes and the cytosol, but not in the mitochondria, significantly reduced palmitic acid-induced (100 μmol/l) ROS generation.


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

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

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3012172&req=5

Figure 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).
Mentions: ROS generation in RINm5F insulin-producing control cells as measured by the DCF fluorescence method was increased by about 40% in response to exposure to 100 μmol/l palmitic acid (Fig. 4). Overexpression of catalase in the peroxisomes and the cytosol, but not in the mitochondria, significantly reduced palmitic acid-induced (100 μmol/l) ROS generation.

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