Abstract
The unfolded protein response in the endoplasmic reticulum (UPRER) is involved in a number of metabolic diseases. Here, we characterize UPRER-induced metabolic changes in mouse livers in vivo through metabolic labeling and mass spectrometric analysis of lipid and proteome-wide fluxes. We induced UPRER by tunicamycin administration and measured synthesis rates of proteins, fatty acids and cholesterol, as well as RNA-seq. Contrary to reports in isolated cells, hepatic de novo lipogenesis and cholesterogenesis were markedly reduced, as were mRNA levels and synthesis rates of lipogenic proteins. H&E staining showed enrichment with lipid droplets while electron microscopy revealed ER morphological changes. Interestingly, the pre-labeling of adipose tissue prior to UPRER induction resulted in the redistribution of labeled fatty acids from adipose tissue to the liver, with replacement by unlabeled glycerol in the liver acylglycerides, indicating that the liver uptake was of free fatty acids, not whole glycerolipids. The redistribution of adipose fatty acids to the liver was not explicable by altered plasma insulin, increased fatty acid levels (lipolysis) or by reduced food intake. Synthesis of most liver proteins was suppressed under UPRER conditions, with the exception of BiP, other chaperones, protein disulfide isomerases, and proteins of ribosomal biogenesis. Protein synthesis rates generally, but not always, paralleled changes in mRNA. In summary, this combined approach, linking static changes with fluxes, revealed an integrated reduction of lipid and cholesterol synthesis pathways, from gene expression to translation and metabolic flux rates, under UPRER conditions. The reduced lipogenesis does not parallel human fatty liver disease. This approach provides powerful tools to characterize metabolic processes underlying hepatic UPRER in vivo.
Highlights
Proteostasis, or protein homeostasis, is important in maintaining a healthy cellular environment under stressful conditions [1]
Upregulated ontologies included genes involved in the response to ER stress, ER associated degradation (ERAD), and ribosome biogenesis
Upregulated ontologies included genes involved in the response to ER stress, ER associated degradation (ERAD), and ribosome biogenesis (Fig‐ u(rFeig1ubr–eg)1.bT–hge).exTphreesesixopnreosfsgioenneosfingveonlevsedinivnotlhveedUiPnRtEhR eshUifPteRdERovsehritfitmede opvoesrt EtiRm‐setrpeossst inEdRu-scttrieosns(iFnidguucrteio1nb–(Ff)i.gure 1b–f)
Summary
Proteostasis, or protein homeostasis, is important in maintaining a healthy cellular environment under stressful conditions [1]. In times of UPRER, a binding-immunoglobulin protein, or GRP-78, (BiP), a key chaperone, moves away from the ER membrane to combat the accumulation of misfolded proteins and activates these three arms [9]. A hallmark of the UPRER is suppressed global protein translation, with the exception of key ER stress responders, such as chaperones [10]. These responses have been characterized at the level of mRNA and protein concentrations, rates of protein synthesis (translation rates) in response to the induction of the UPRER in vivo have yet to be characterized. Dynamic proteomic techniques that we have developed [11], using heavy water labeling and tandem mass spectrometric kinetic analysis based on combinatorial analysis of deuterium incorporation patterns, can be applied to measure hepatic protein translation rates after tunicamycin-induced UPRER in living mice
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