Restoration of oxidative stress by HNF4α deletion in VWA8-null hepatocytes.

VWA8 is a mitochondrial protein expressed at high levels in mitochondria-enriched organs such as the liver, kidney, and heart. Deletion of VWA8 (VWA8-KO) in AML12 mouse hepatocytes leads to oxidative stress characterized by elevated production of reactive oxygen species (ROS) and, subsequently, higher oxidative capacity. Concurrently, there is a substantial rise in the expression of the transcription factor HNF4α in VWA8-KO hepatocytes. Thus, exploring the potential significance of HNF4α in the observed effects warrants investigation. In this study, we deleted the HNF4α gene using CRISPR/Cas9 in VWA8-KO AML12 mouse hepatocytes. Measurements of ROS production rates, MitoStress Assays in permeabilized cells, and MitoStress Assays in intact cells were performed. Our results show that Deleting HNF4α in VWA8-KO AML12 mouse hepatocytes (V&H-KO) rescued the increased rate of ROS production observed in VWA8-KO cells, returning ROS to levels comparable to wild-type cells, thereby reversing the oxidative stress associated with VWA8-KO. Furthermore, V&H-KO recovered the elevated respiration rates observed in VWA8-KO intact cells and permeabilized cells when fueled by palmitate, lowering them to wild-type levels. However, the increased respiration rate in VWA8-KO intact cells when using glucose as the fuel source remained unaltered by V&H-KO. This suggests that HNF4α plays an important role but is not the sole factor contributing to the phenotypic changes resulting from VWA8-KO. We conclude that deleting HNF4α in VWA8-KO AML12 mouse hepatocytes partially rescues the higher oxidative capacity and rescues the oxidative stress in VWA8-KO cells. Mechanisms involved in changes in oxidative stress remain to be determined.