Deficiency of PDK4 Sensitizes Mouse Liver to Diethylnitrosamine and Arsenic Toxicity Through Inducing Apoptosis
Authors: Choiniere Jonathan , Wu Jianguo , Lin Matthew , Wang Li 
Published: June 2021

Background

Pyruvate dehydrogenase kinase 4 (PDK4) is a metabolic switch that regulates glucose oxidation and the TCA cycle in the mitochondria. The liver detoxifies xenobiotics and is constantly challenged by various injuries. How the loss of this metabolic regulator, PDK4, contributes to liver injuries remains elusive. Previous studies from our lab have revealed that knockout of Pdk4 in mice resulted in increased cellular proliferation via E2F1 transcription factor‐mediated increase in cyclins.

Objective

The objective of this study was to elucidate a novel function of PDK4 in toxin‐induced liver injury.

Methods

Two week‐old wild‐type (WT) (C57BL/6J, male) and Pdk4−/− mice were i.p. injected with a single dose of diethylnitrosamine (DEN) (25 mg/kg body weight) or two month‐old mice were fed with arsenic at 50 ppm (one month) via distilled drinking water. DEN treated mouse samples were collected at day 6 after DEN injection due to unexpected death of Pdk4−/− mice. Liver tissues were collected after both treatments for western blot (WB), IHC, and qPCR. Pdk4 shRNA was used to generate Pdk4 knockdown cells for mitochondrial respiration analysis to measure cellular oxygen consumption rate and extracellular acidification rate (Seahorse Bioscience). Electron microscopy was applied in order to visualize mitochondria.

Results

TUNEL analysis revealed a significant level of spontaneous apoptotic cells in Pdk4−/− mouse livers, which increased with age in the absence of treatment indicating an age‐dependent progression. Microscopy and staining revealed extensive hepatic damage in DEN treated Pdk4−/− mouse livers while WT mouse livers showed no sign of damage which suggests loss of Pdk4 increases hepatic sensitivity to chemical toxicant DEN administration. TUNEL analysis of Arsenic treated mouse liver sections revealed increased apoptosis in Pdk4−/− mice compared to WT mice. Caspase‐3 activity was analyzed via western blot and activity assays revealing a caspase‐3 dependent induction of apoptosis in Pdk4−/− mouse livers. Mitochondrial respiration analysis revealed shPDK4 knockdown cells had significantly higher oxygen consumption rate and a higher respiratory capacity than control cells. Mitochondrial respiration analysis also revealed that shPDK4 knockdown cells had higher extracellular acidification rates suggesting increased glycolytic capacity.

Conclusions

In the present study, we showed that age‐dependent spontaneous hepatic apoptosis happened in systematic Pdk4 knockout (Pdk4−/−) mice and that PDK4‐deficiency increased the sensitivity of adult mice to arsenic insult, as well as juvenile mice to toxicity of DEN, which correlated with a lethal consequence and massive hepatic apoptosis and abnormal mitochondrial respiration.

Support or Funding Information

NIH R01ES025909, R01DK104656, R01DK080440, R21AA022482, R21AA024935, VA Merit Award 1I01BX002634, P30 DK34989 (Yale Liver Center).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.