Introduction A characteristic histological feature of alcoholic liver disease (ALD), known since the 1970s, is the presence of megamitochondria (MM) in hepatocytes. In a recent study conducted in more than 100 patients, the presence of MM was associated with a lower risk of death within 90 days.1Mitochondria are dynamic organelles and their shape is intimately involved with their function, under physiological conditions the changes in mitochondrial morphology are regulated by mitochondrial-shaping proteins (MSP). However, the mechanisms driving MM formation, their role, function and involvement in the pathogenesis of ALD is currently unknown, which is the aim of this study.
Method Human precision cut liver slices (PCLS) and hepatoma cells VL-17A (positive for ADH/CYP2E1) were cultured with EtOH alone or in combination with FFA, to induce hepatotoxicity. Cell viability was evaluated by FACS (AnnexinV/PI positive cells) or ATP quantification. Post-treatment, changes in the mitochondrial shape were assessed by confocal and electron microscopy; MSP expression and activation was also analysed by Western blot on isolated mitochondria. Additionally, mitochondrial functionality was evaluated in intact cells using the Seahorse Bioscience analyser. Changes in inflammatory cytokine production were assessed by Cytokine Bead Array.
Results The toxic effect of EtOH (+/-FFA) was confirmed by an increase in the percentage of dead cells or a reduction in ATP levels. Cells that survived the toxic insult showed a dramatically increased proportion of MM; however they also had a compromised ability to grow. Mitochondrial functionality, in terms of coupling efficiency and spare respiratory capacity, was also impaired. Pro inflammatory cytokine profile was found in the supernatants of these cultures. This was associated with a reduced activity in the MSP related to mitochondrial fragmentation.
Conclusion Cellular survival strategies, in the face of a chronic toxic stimulus, are a necessary requirement to ensure continuing functionality of the organ. We show here that growth retarded hepatocytes, which survived the EtOH insult, are characterised by a dramatic formation of MM, suggesting this as a mechanism to avoid death. We also show for the first time the involvement of MSP in this pathway, raising the potentiality for the use of these proteins as therapeutic targets and biomarkers in ALD.
Altamirano, et al.Gastroenterology, 2014;146(5):1231–1239
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