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Systems proteomics of the intermittent fasting response highlights the importance of hnf4a

Mark Larance

Charles Perkins Centre and School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales, Australia

Every-other-day-fasting (EODF) is an intermittent fasting regime that improves insulin sensitivity and lifespan in model animals without weight loss. However, the mechanisms mediating these beneficial phenotypes remain to be uncovered. Here, we have employed proteome analysis of mouse liver, a key fasting-responsive organ, to identify protein abundance changes after EODF compared to ad libitum fed animals. From >6,000 proteins quantified, more than 250 proteins were significantly altered by EODF. Among the most up-regulated proteins after EODF was acyl-CoA thioesterase 2 (ACOT2), which can accelerate liver fatty-acid oxidation that benefits whole-body metabolism. Surprisingly, alpha1-antitrypsin (SERPINA1) was the most down-regulated protein (>16-fold) after the EODF intervention. SERPINA1 function has previously been linked with lipoprotein particle metabolism in mice and humans. Given that SERPINA1 is among the 10 most abundant proteins in blood plasma and only synthesized in the liver, we performed single-shot plasma proteome analysis to quantify the top 200 most abundant proteins. This showed SERPINA1 was down-regulated ~3-fold in plasma of EODF animals, in addition to other significant protein changes such as increased abundance of apolipoprotein A-IV (APOA4), which was also increased in plasma of humans undergoing EODF and likely provides beneficial lower plasma triglycerides. To identify how the decrease in liver SERPINA1 protein abundance was mediated, we measured its mRNA abundance and chromatin interactions. We observed a significant decrease in liver SERPINA1 mRNA after EODF and using CHIP-qPCR, and reduced association between HNF4A and the promoter of either SERPINA1, or an unrelated canonical HNF4A-target gene (ABCC6). These data suggest that HNF4A is globally inhibited by EODF, however the HNF4A liver protein abundance was not changed by EODF. Therefore, we hypothesise that HNF4A is EODF-regulated by either post-translational modification and/or changes in protein-protein interactions. Immunoprecipitations of HNF4A from the liver tissue are on-going to characterise any significant differences.