Mitochondria play pivotal roles in cellular energy metabolism. Most of the intracellular adenosine triphosphate (ATP) is generated by mitochondrial respiration. The Cell Mito Stress Test is a common method to measure the key parameters of mitochondrial respiration. Here, we use the human cell line HK-2 as an example to present the procedures to quantify the oxygen consumption rate using a Seahorse XFe96 extracellular flux analyzer.

Under normal conditions, insulin promotes hepatic de novo lipogenesis (DNL). However, during insulin resistance (IR), when insulin signalling is blunted and accompanied by hyperinsulinaemia, the promotion of hepatic DNL continues unabated and hepatic steatosis increases. Here, we show that WD40 repeat-containing protein 6 (WDR6) promotes hepatic DNL during IR. Mechanistically, WDR6 interacts with the beta-type catalytic subunit of serine/threonine-protein phosphatase 1 (PPP1CB) to facilitate PPP1CB dephosphorylation at Thr316, which subsequently enhances fatty acid synthases transcription through DNA-dependent protein kinase and upstream stimulatory factor 1. Using molecular dynamics simulation analysis, we find a small natural compound, XLIX, that inhibits the interaction of WDR6 with PPP1CB, thus reducing DNL in IR states. Together, these results reveal WDR6 as a promising target for the treatment of hepatic steatosis.