Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC3012172&req=5

Figure 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.)

Mentions: The key finding of H2O2 generation in the peroxisomes of RINm5F cells after exposure to palmitic acid was confirmed in primary rat islet cells. Single rat islet cells were lentivirally transduced with Hyper-Peroxi cDNA and incubated with palmitic acid for 24 h. In the palmitic acid treated islet cells a clear shift toward red fluorescence was detectable, which corresponds to a higher H2O2 concentration in comparison with untreated cells (Fig. 6). The fluorescence shift of single cells was quantified with the CellR software (Olympus, Hamburg, Germany). Palmitic acid treatment induced a significant 125% increase of H2O2 production in comparison with the control condition (Fig. 6E).


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

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

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

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

Figure 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.)
Mentions: The key finding of H2O2 generation in the peroxisomes of RINm5F cells after exposure to palmitic acid was confirmed in primary rat islet cells. Single rat islet cells were lentivirally transduced with Hyper-Peroxi cDNA and incubated with palmitic acid for 24 h. In the palmitic acid treated islet cells a clear shift toward red fluorescence was detectable, which corresponds to a higher H2O2 concentration in comparison with untreated cells (Fig. 6). The fluorescence shift of single cells was quantified with the CellR software (Olympus, Hamburg, Germany). Palmitic acid treatment induced a significant 125% increase of H2O2 production in comparison with the control condition (Fig. 6E).

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