The role of peroxisomes in hepatocytes and pancreatic Beta-cells in mouse. Relationship of peroxisomes with mitochondrie

Peroxisomes are plastic organelles, present in almost all eukaryotic cells, which play an essential role in intermediary lipid metabolism. Inherited diseases with defects in peroxisomal function give rise to multiple organ defects which have been mimicked in mouse models for these diseases. However, their particular function in different cell types and tissues remains largely obscure. In this thesis we investigated the consequences of peroxisome inactivity in hepatocytes and in pancreatic β-cells. Whereas, historically, liver was the tissue in which peroxisomal function was first and most extensively studied, only scarce information is hitherto available on peroxisomes in β-cells of the pancreas.

In early reports on Zellweger syndrome patients, lacking functional peroxisomes, hepatic mitochondrial abnormalities were documented which were later recapitulated in mouse models in which the peroxisome biogenesis factor PEX5 was deleted from hepatocytes. In the latter, the mitochondrial ultrastructural changes were accompanied by a reduced mitochondrial membrane potential and reduced activities of the respiratory complexes I, III and V. Our aim was to search for the mechanisms linking peroxisome dysfunction to mitochondrial disruption. We showed that the mitochondrial anomalies are associated with increased reactive oxygen species. Furthermore, as the ultrastructure of mitochondria and activity of complex I are unchanged in brain, muscle and heart lacking functional peroxisomes these mitochondrial abnormalities appeared to be hepatocyte selective. We further investigated whether peroxisomal metabolites that are specifically enriched in hepatocytes could be a causative factor by either manipulating their levels or comparing their levels in different mouse models of peroxisomal β-oxidation deficiency and correlating them with complex I activity. The severe reduction in the levels of docosahexaenoic acid (DHA) in phospholipids of mitochondria from Pex5-/- hepatocytes could be restored by administering DHA orally to L-Pex5-/- mice. However, no improvements were seen in the complex I activity after the treatment. Furthermore, wild type mice treated with phytol diet accumulated significant amounts of branched chain fatty acids (BCFAs) – phytanic and pristanic acid but did not show any impairment in complex I activity in liver. Moreover, dicarboxylic acids (DCAs) were not found to accumulate in the livers of L-Pex5-/- mice. Also complex I activity in the livers of Mfp1-/- mice fed coconut diet which were previously shown to accumulate DCAs was not decreased. Lastly, we measured the levels of the presumed mito-toxic bile acid intermediates, DHCA and THCA in the livers of adult and prenatal mice lacking either PEX5 or the peroxisomal β-oxidation enzyme MFP2. However, the levels did not correlate with the observed mitochondrial dysfunction. In summary, although we could not find the exact link between absence of peroxisomes and mitochondrial problems, we could exclude the role of depletion of DHA and accumulation of BCFAs, DCAs or bile acid intermediates in mediating the mitochondrial abnormalities.

Previous literature supported contrasting ideas about peroxisomal metabolism being either harmful or beneficial for the functioning of β-cells. Therefore, the second aim of this thesis was to investigate the role of peroxisomes in pancreatic β-cells by generating and phenotyping β-cell specific Pex5 knockout mice (Rip-Pex5-/-). We found that glucose homeostasis in these mutant mice is disturbed which was characterized by increased fed as well as fasted blood glucose levels and glucose intolerance. The circulating insulin levels were reduced after a bolus of glucose in Rip-Pex5-/- mice which can be attributed to reduction in total pancreatic insulin content as well as β-cell mass. No changes were found in the glucose stimulated insulin secretion ex vivo, insulin content per islet and cytoplasmic as well as mitochondrial ROS production in cultured islet cells. However, the mitochondrial membrane potential was increased in cultured islet cells of mutant mice. Taken together, these results suggest that peroxisomes contribute to the normal functioning of healthy β-cells, however at this moment we were unable to unravel the mechanisms behind this relationship.

Overall, this work pointed towards an essential role of peroxisomes in the normal functioning of hepatocytes and pancreatic β-cells, two cell types of utmost importance in metabolic processes.