Strategies for measuring induction of fatty acid oxidation in intestinal stem and progenitor cells

Abstract

© Springer Science+Business Media, LLC, part of Springer Nature 2020. This protocol describes a multipronged approach that we have created to determine the transcriptional induction of fatty acid oxidation (FAO) genes in Lgr5high intestinal stem cells and a subsequent metabolomics-based approach for assessing fatty acid utilization in the mammalian intestinal crypt. More specifically, we describe methods for crypt isolation followed by a FACS-based purification of stem and progenitor populations and RNA-sequencing analysis. Using this workflow, we can determine both basal gene expression profiles of key metabolic genes as well as corresponding changes in response to altered metabolic states, such as fasting. Subsequently, we describe a complementary metabolomics-based approach that we have developed to assess fatty acid uptake and utilization in the crypt using 13C stable isotope tracing. Combining these approaches, one can gain a better understanding of substrate utilization and the preceding transcriptional changes that accommodate these reactions in physiologic states of low carbohydrate utilization or during overabundance of dietary lipids.