Thyroid hormone responsive protein Spot14 modulates fatty acid synthase kinetics and fatty acid products

Abstract

The mammary gland synthesizes and secretes a complex fluid, milk, that is balanced in nutrient content and uniquely tailored to the growth and development of the neonate. Milk is comprised of three primary nutrients including carbohydrates, proteins, and fats. The fat component, triglycerides, is an amalgam of fatty acids either taken up by the mammary epithelial cell (MECs) from dietary and adipose sources, or synthesized by the MECs via the de novo fatty acid synthesis pathway. Fatty acids are synthesized in the cytosol using three enzymes, ATP citrate lyase (ACLY), Acetyl-CoA carboxylase (ACC), and Fatty acid synthase (FASN). Thyroid hormone responsive protein Spot 14 (Spot14) is required to achieve the necessary de novo fatty acids that sustain proper growth of the neonates; however, the molecular function of Spot14 remains undefined. Analysis of Spot14 null milk, mammary glands, and MECs exposed significant reductions in the medium chain fatty acids (MCFAs). Spot14 loss did not result in loss of metabolic gene expression, enzyme protein levels, or in the intermediate metabolites that support de novo MCFA synthesis, suggesting that FASN activity in the Spot14 null MECs is deficient. Evidence is presented indicating that Spot14 co-migrated with enzyme complexes of FASN, implying that Spot14 might influence the enzyme's activity in the production of MCFAs. To test this possibility, a novel FASN activity assay was designed to sensitively quantify enzyme products and to discriminate those products by acyl chain length. The new in vitro FASN assay revealed that Spot14 could modulate FASN kinetics to increase the amount of MCFA products synthesized in the reaction. Collectively, these data suggest Spot14 may function to influence the activity of FASN, and is suggestive of a molecular role in vivo for Spot14 function in the lactating mammary gland. The de novo fatty acid synthesis pathway plays a pivotal role in obesity and multiple forms of cancer, and both Spot14 and FASN have been linked to a poor outcome. Given that inhibitors FASN often result in whole body complications, we posit that understanding the role of proteins that can modulate FASN might expand the repertoire of drug-based therapies.