Mitochondrial DNA Unwinding Enzyme Required for Liver Regeneration

A model of liver regeneration following injection of wild type and TOP1mt knockout mice with the liver toxin carbon tetrachloride (CCl4). Damage from CCl4 triggers the growth of liver cells. Lack of Top1mt limits mitochondrial replication, reducing the number of mitochondria in daughter cells, which, in turn, reduces adenosine triphosphate (ATP) production and delays liver regeneration.

A model of liver regeneration following injection of wild type and TOP1mt knockout mice with the liver toxin carbon tetrachloride (CCl4). Damage from CCl4 triggers the growth of liver cells. Lack of Top1mt limits mitochondrial replication, reducing the number of mitochondria in daughter cells, which, in turn, reduces adenosine triphosphate (ATP) production and delays liver regeneration.

The liver has an exceptional capacity to proliferate. This ability allows the liver to regenerate its mass after partial surgical removal or injury and is the key to successful partial liver transplants. Liver cells, called hepatocytes, are packed with mitochondria, and regulating mitochondrial DNA (mtDNA) copy number is crucial to mitochondrial function, including energy production, during proliferation. Yves Pommier, M.D., Ph.D., of CCR’s Developmental Therapeutics Branch, and his colleagues recently showed that the vertebrate mitochondrial topoisomerase, Top1mt, was critical in maintaining mitochondrial function in the heart after doxorubicin-induced damage. The group wondered whether Top1mt might play a similar role in liver regeneration.

The researchers treated seven-week-old wild-type (WT) and Top1mt knockout (KO) mice with a single injection of carbon tetrachloride (CCl4), a potent liver toxin, or a placebo and examined the livers from treated mice two or four days later. Normally, following damage, liver weight increases rapidly due to regeneration. While mice given the placebo showed no differences in liver size, KO mice treated with CCl4 showed a smaller increase in liver size compared to WT mice two days after treatment. The investigators examined sections of liver tissue and observed persistent dead and inflamed areas in livers from CCl4-treated KO mice, which had already been cleared in livers from WT mice. There were also fewer proliferating cells in livers from mice lacking Top1mt at day two, but cell proliferation was increased at day four, suggesting a delay in tissue regeneration.

The scientists next evaluated mtDNA levels and mitochondrial activity in the livers of treated mice. Tissue from KO mice treated with CCl4 showed reduced mtDNA copy number whereas WT tissue showed a slight increase, suggesting that Top1mt is required for expansion of mtDNA after liver damage. To study the levels of reactive oxygen species (ROS) in the liver, the research team measured reduced glutathione (GSH), a known quencher of ROS. CCl4 treatment reduced GSH levels in WT and KO mouse livers at day two. By day four, however, GSH levels completely recovered in WT livers but remained low in those from KO mice, indicating a persistent elevation of ROS. The investigators then isolated mitochondria from liver tissue and assessed the enzyme activities of complexes I and IV, which participate in mitochondrial energy production. Tissue from KO mice revealed significantly reduced activities for both enzymes compared to tissue from WT mice at day four after CCl4 administration. There was also a slight increase in caspase-3 activity, a marker of apoptosis, and Oil Red O staining, which indicates tissue damage, in livers from the KO mice.

The scientists isolated hepatocytes from WT and KO placebo or CCl4-treated mice. Interestingly, fewer cells were able to grow from the toxin-treated KO livers, and these cells showed reduced mtDNA and energy levels compared to WT cells. CCl4-treated KO hepatocytes consumed less oxygen, a measure of oxidative phosphorylation-mediated energy production, and elevated glycolysis, a less efficient means of producing energy, demonstrating the cells’ reliance on the latter. In contrast, cells from WT mice showed elevated oxygen consumption and glycolysis, likely representing the high metabolic activity required for liver regeneration. The researchers then used electron microscopy to evaluate the structure and density of mitochondria in the isolated hepatocytes. They saw no obvious differences in cells from placebo-treated WT and KO mice. However, after CCl4 treatment, the investigators observed reduced mitochondrial density and more extensive structural damage in the KO hepatocytes.

Lastly, the scientists examined the role of Top1mt in mouse embryonic fibroblasts (MEFs) and human cancer cells. They treated WT and KO MEFs with ethidium bromide to deplete mtDNA and then allowed the cells to recover. Initially, there was a similar reduction in mtDNA in both cell types. mtDNA from WT MEFs, however, recovered much more rapidly than mtDNA in KO MEFs. Similarly, human colon cancer cells lacking TOP1mt had delayed recovery of mtDNA and defective mitochondrial energy production following ethidium bromide treatment.

Together these results show the importance of Top1mt for mtDNA synthesis and mitochondrial function following cellular damage. The fact that KO mice do not show significant mtDNA copy number depletion in the liver under normal conditions suggests that other topoisomerase enzymes may compensate for Top1mt loss under baseline conditions. Under acute stress, though, Top1mt becomes essential. Further studies are needed to learn whether genetic variants of Top1mt in humans may be responsible for the success of liver transplants or the extent of drug-induced liver injuries, which is consistent with the critical importance of Top1mt for cardiomyocyte response to doxorubicin.

Summary Posted: 09/2015

Reference

Khiati S, Baechler SA, Factor VM, Zhang H, Huang SN, Rosa ID, Sourbier C, Neckers L, Thorgeirsson SS, and Pommier Y. Lack of mitochondrial topoisomerase I (TOP1mt) impairs liver regeneration. PNAS. August 24, 2015 PubMed Link