The assessment of mitochondrial function and metabolic activity in pancreatic progenitor derived hepatocytes

Abstract

Hepatocytes are the primary cell type of the liver, they play a key role in drug toxicity and therefore represent an ideal model for preclinical toxicity testing. However, current primary hepatocyte models resist in vitro proliferation and immortalised models are often not fully metabolically competent. One solution is the rodent B-13 cell line which forms hepatocyte-like B-13/H cells in response to glucocorticoid treatment. This thesis aimed to investigate the metabolic activity of B-13/H cells and assess the role of mitochondrial dysfunction in cytotoxicity. Cells were challenged with the anti-diabetic drug, troglitazone, which was withdrawn from the market following reports of liver injury, mitochondrial liabilities have since been associated with its toxicity. Extracellular flux analysis showed that basal levels of respiration were comparable between B-13 and B-13/H cells, however, reserve capacity was 5-fold greater in the B-13/H cells. In response to troglitazone, there was a concentration dependent decrease in oxygen consumption rate in B-13/H cells compared to a stimulation of respiration in B-13 cells and a concomitant increase in lactate levels and oxygen demand for ATP production. After 24 hours troglitazone treatment, there was a concentration dependent decrease in B-13/H viability. B-13 cell viability was unaffected. A larger baseline reserve capacity suggested a greater mitochondrial mass in B-13/H cells concomitant with a greater role in metabolism, similarly, B-13/H cells were more susceptible to troglitazone than B-13 cells. A drop in oxygen consumption rate suggested that there was mitochondrial dysfunction; this was supported by a drop in total ATP levels. In B-13 cells, troglitazone had a stimulatory effect on respiration and a concentration dependent increase in lactate suggested a switch from oxidative phosphorylation to glycolysis. The data presented indicate that B-13/H cells could potentially form the basis of a toxicity screening platform. This work could also underpin the development of a human equivalent model.