The present data clearly show that NEFA toxicity increases with increasing chain length. NEFAs are oxidized not only in the mitochondria, but also in the peroxisomes. Fatty acid β-oxidation is a property of peroxisomes in most, if not all, organisms (27
). Moreover, in yeast and plants, peroxisomes are the sole sites of fatty acid β-oxidation (44
). One product of peroxisomal β-oxidation is H2
, whereas mitochondrial β-oxidation generates reducing equivalents (27
). Mitochondrial β-oxidation is tightly coupled to the respiratory chain and oxidative phosphorylation. It provides acetyl-CoA for further oxidation in the TCA cycle and ensures the production of ATP, which is not an apparent function of peroxisomal β-oxidation. In peroxisomal β-oxidation, the initial step generates H2
and energy is lost as heat. Peroxisomal β-oxidation results in chain shortening of long- and very long-chain fatty acids, which are poor substrates for mitochondrial β-oxidation. The shortened fatty acids are subsequently transported to the mitochondria in a carnitine-dependent manner for further degradation (27
The observation of the chain length-dependent increase in the toxicity of saturated fatty acids to insulin-producing cells prompted us to study the role of oxygen free radicals generated by peroxisomal β-oxidation as mediators of lipotoxicity. In the first step of peroxisomal β-oxidation, FAD-containing acyl-CoA oxidase introduces a double bond at the β-position of the fatty-acyl-CoA ester and the hydrogen atoms are transferred to molecular oxygen to yield H2
). Remarkably, in rat liver, for example, about 20% of total oxygen consumption is accounted for peroxisomal oxidase activity (45
). For the detoxification of H2
, the oxidoreductase catalase, which has a high turnover rate, is expressed in the peroxisomes of most tissues (28
), but not in those of pancreatic β-cells (29
). This low catalase enzyme activity could also be found in RINm5F cells, which makes these cells well suited as model cells for H2
Experiments with insulin-producing cells overexpressing antioxidative enzymes showed that only cytosolic catalase provided protection against palmitic acid-induced toxicity; mitochondrial catalase was not protective. The superoxide radical detoxifying isoenzymes MnSOD and CuZnSOD were also not protective, indicating that the formation of superoxide radicals does not play as crucial a role in lipotoxicity as does the formation of H2O2. Indeed, determination of ROS production by DCF fluorescence measurements provided support for this hypothesis by showing a reduction in ROS generation in insulin-producing cells that overexpressed catalase in the peroxisomes and the cytosol, but not in the mitochondria. Using the H2O2-sensitive HyPer protein as a novel, specific method for detecting H2O2, we clearly identified H2O2 as the main reactive oxygen species formed during palmitic acid treatment. To determine the subcellular site of H2O2 formation, the HyPer protein was fused to a peroxisome- or a mitochondrion-targeting sequence to allow organelle-specific expression.
These experiments show that peroxisomes were a major site of H2O2 formation in insulin-producing cells, whereas the mitochondria were a minor site. Primary rat islet cells are well equipped with peroxisomes, and also showed an increased peroxisomal H2O2 formation in response to palmitic acid, as demonstrated in this study.
An additional argument for this hypothesis is the fact that overexpression of catalase in the peroxisomes and the cytosol (RINm5F-Cat) significantly reduced H2
production not only in the peroxisomes, but also in the mitochondria, though catalase was not overexpressed in the latter organelle. H2
as a membrane-permeable ROS (23
) can diffuse from its site of generation in the peroxisome into the mitochondria where it is detected through the HyPer-Mito protein. Thus, the source of the elevated H2
concentration detected in the mitochondria is at least in part H2
-generated in the peroxisomes.
However, in contrast to the peroxisomes in other cell types, insulin-producing cells do not appear to express catalase mRNA or protein (29
). This lack of catalase expression leaves insulin-producing cells badly protected against potentially hazardous effects of H2
generated through peroxisomal β-oxidation. Mitochondrial β-oxidation may not be able to cope with the elevated levels of NEFAs that are associated with obesity and type 2 diabetes, resulting in a higher proportion of fatty acids being metabolized through peroxisomal β-oxidation, leading to increased H2
formation. The membrane-permeable H2
could leave the peroxisomes and harm insulin biosynthesis and secretion (11
), as has been shown for insulin-secreting cells after exposure to high NEFA concentrations for extended periods (15
). The results of the present study indicate that ROS generated in the peroxisomes are the major cause of lipotoxicity-mediated β-cell dysfunction. This does not detract from the fact that mitochondrial ROS formation may contribute to this phenomenon, in particular with respect to direct negative effects on mitochondrial function.
NADPH oxidase activation could also be a source of long-chain fatty acid-mediated superoxide radical generation (22
) that has been postulated to cause lipotoxicity in insulin-producing cells (15
). Palmitic acid has been shown to activate this plasma membrane enzyme in aortic smooth muscle and endothelial cells in a protein kinase C-dependent manner, leading to superoxide radical formation (49
). In insulin-producing cells, palmitic acid can induce superoxide radical formation along with increased expression of the NADPH oxidase p47phox
). However, data from the present study indicated that cytosolic CuZnSOD did not protect insulin-producing cells against palmitic acid-induced toxicity, suggesting that an increased rate of superoxide radical formation per se is insufficient to explain NEFA-induced lipotoxicity.
Thus, we hypothesize that NEFA-induced lipotoxicity is mediated by H2
generated during peroxisomal β-oxidation of palmitic acid as the physiologically most abundant long-chain saturated fatty acid. This provides an interesting new concept for lipid-induced glucose intolerance in obesity and diabetic hyperglycemia as a result of β-cell dysfunction in patients with type 2 diabetes. The lack of catalase expression in pancreatic β-cell peroxisomes (29
) explains the exceptional susceptibility of pancreatic β-cells to lipotoxicity (14