Browsing by Author "Purhonen, Janne"

GRACILE syndrome (Fellman disease) is a neonatal mitochondrial disorder with hepatopathy belonging to Finnish disease heritage. The GRACILE acronym comes from the most important clinical features of the disorder: fetal growth restriction, aminoaciduria, cholestasis (with steatosis and fibrosis), hepatic iron overload, lactic acidosis, and early death during infancy. The syndrome is caused by mutated (homoyzygous c.232A>G) BCS1L, a nuclear gene encoding a respiratory chain complex III assembly factor. BCS1L mutations compromise Rieske iron-sulfur protein incorporation into complex III, which leads to respiratory chain deficiency. Bcs1l c.232A>G knock-in C57BL/6 mouse model mimics the human disorder, and the homozygous mice (GRACILE mice) display growth restriction, progressive liver disease and a short life span. For a vast majority of mitochondrial disorders no effective treatments currently exist. One promising treatment strategy is to increase mitochondrial biogenesis with the idea that respiratory chain deficiency can be compensated by boosting mitochondrial function. In mouse models of mitochondrial myopathies induction of mitochondrial biogenesis with a transgene approach, pharmaceutical intervention or ketogenic diet has alleviated the disease. It has been hypothesized that ketogenic diet activates energy deprivation signals of the cells, which are the most important regulators of mitochondrial biogenesis. So far ketogenic diet has not been studied in animal models of mitochondrial hepatopathies. The objective of this master’s thesis was to study effect of ketogenic diet on hepatopathy of GRACILE mouse model. From weaning on, wild-type and homozygous mutant mice (n = 11 - 14 per group) were fed standard chow or ketogenic diet (90,5 E% fat) for three weeks after which the mice were sacrificed and tissues collected for analysis. The effect of ketogenic diet was evaluated with following methods: liver histology (H&E, Sirius Red, Oil Red O, PAS and semiquantitative scoring), liver triglyceride content, mitochondrial DNA copy number (qPCR), expression of selected genes (qPCR) and respiratory chain proteins (Western blot). The mice tolerated the ketogenic diet, and the diet did not affect weight gain. Livers of wild-type mice on ketogenic diet were considered normal except for slight fat accumulation. In GRACILE mice ketogenic diet ameliorated some aspects of the liver disease (less signs of inflammation and fibrosis) but increased microvesicular steatosis. Mitochondrial DNA copy number was unchanged among groups. Ketogenic diet did not affect gene or protein expression of selected respiratory chain components. The GRACILE mutation increased gene expression of Pgc1a, a gene encoding a master regulator of mitochondrial biogenesis, as well as protein and mRNA levels of respiratory chain complex IV subunits. The ketogenic diet had a clear effect on liver phenotype in the mutant mice, but a longer follow-up time is needed to show the effect on disease progression. The beneficial action of ketogenic diet on liver histology might not be related to increased number of mitochondria or respiratory chain proteins, and the mechanism behind the effect need to be elucidated in further investigations.