UC Berkeley

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 first 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. The reduction in de novo lipogenesis was explicable by reduced food intake, shown in pair-feeding studies. Despite the lower de novo lipogenesis rates, 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 in liver 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 tissue. 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. Using these same stable isotope labeling approaches, several changes in protein synthesis across ontologies were noted with age, including a more dramatic suppression of translation under ER stress in aged mice as compared to young mice. De novo lipogenesis rates decreased under ER stress conditions in aged mice, including both triglyceride and phospholipid fractions. In young mice, a significant reduction was only seen in the triglyceride fraction. These data indicate that aged mice have an exaggerated response to ER stress, which may indicate less effective mechanisms of protein clearance after inducing the UPRER. These flux-based approaches provide a powerful tool to identify novel regulators of ER stress and potential targets for pharmacological intervention. We applied the same stable isotope labeling techniques to generate region-specific metabolic flux signatures of mouse brains. Assessing how metabolic flux signatures of the brain are perturbed through different interventions, including anti-aging therapeutics and preventative lifestyle interventions, could provide a powerful tool to assess cell-specific and region-specific changes with age, and provide a kinetic metric to assess intervention effectiveness.