Palacios, H. | bioRxiv Science

bioRxiv2020

Metabolic flux signatures of the ER unfolded protein response in vivo reveal decreased hepatic de novo lipogenesis and mobilization of lipids from adipose tissue to liver

The unfolded protein response in the endoplasmic reticulum (UPRER) is involved in a number of metabolic diseases, including non-alcoholic fatty liver disease. Here, we characterize the UPRER induced metabolic changes in mouse liver through in vivo metabolic labeling and mass spectrometric analysis of proteome and lipid fluxes. We induced ER stress in vivo via tunicamycin treatment and measured rates of proteome-wide protein synthesis, de novo lipogenesis and cholesterol synthesis serially over a three-day period, thereby generating a metabolic "signature" of the UPRER over time. Synthesis of most proteins was suppressed under ER stress conditions, including proteins involved in lipogenesis, consistent with reduced de novo lipogenesis at 48 and 72 hours. Electron microscopy revealed striking morphological changes to ER and H&E staining showed lipid droplet enriched livers under ER stress. Pre-labeling of adipose tissue prior to ER stress induction revealed mobilization of lipids from adipose to the liver. Interestingly, the source of these lipids was uptake of free fatty acids, not whole triglycerides or phospholipids from lipoproteins, as demonstrated by replacement of the triglyceride-glycerol moiety in liver concurrently with increased incorporation of labeled palmitate from adipose. We also induced ER stress by a high-fat diet and observed similar metabolic flux signatures, suggesting that this mechanism may play a role in the progression of fatty liver disease. This flux-based approach provides a powerful tool to identify novel regulators of ER stress and potential targets for pharmacological intervention.

physiology↗

bioRxiv2020

Skeletal muscle DNMT3A plays a necessary role in endurance exercise by regulating oxidative capacity of red muscles

Exercise interventions alter the DNA methylation profile in skeletal muscle, yet little is known about the role of the DNA methylation machinery in exercise capacity. In this study, we found that in oxidative red muscle, DNMT3A expression increases greatly following a bout of endurance exercise. Mice lacking Dnmt3a in skeletal muscle fibers had reduced tolerance to endurance exercise, accompanied by reduced oxidative capacity and reduced mitochondrial counts. Moreover, during exercise, the knockout muscles overproduced reactive oxygen species (ROS), which are major contributors to muscle dysfunction. In mechanistic terms, we demonstrated that Aldh1l1 is a key target of repression by DNMT3A in red muscles. DNMT3A directly regulated the Aldh1l1 transcription by binding to the Aldh1l1 promoter region and altering DNA methylation and histone modification. Enforcing ALDH1L1 expression, leading to elevated NADPH, led to overproduction of ROS by the NADPH oxidase complex (NOX) in myotubes, ultimately resulting in mitochondrial defects. Moreover, both genetic inhibition of ALDH1L1 and pharmacological inhibition of NOX rescued oxidative stress and mitochondrial decline in Dnmt3a-deficient myotubes, confirming the essential role of ALDH1L1-dependent ROS generation as a downstream effector of DNMT3A loss of function. Together, our results reveal that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress.

cell biology↗