Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium

Abstract

This article is available online at http://www.jlr.orgMammalian cells manufacture fatty acids de novo using a distinct pathway to synthesize fatty acids from acetyl and malonyl esters of CoA catalyzed by the dimer of FASN (EC 2.3.1.85) ( 1 ). FASN is absolutely essential for production of de novo fatty acids in mammals ( 2 ). The biological requirement for FASN is highlighted by the fact that FASN is detected in most normal human tissues and that homozygous deletion of FASN in the mouse results in embryonic lethality ( 3, 4 ). The importance of the de novo pathway is underscored by the complex biological functions of its fatty acid products, including biosynthesis of phospholipids, energy storage via esterifi cation into triglycerides (TAGs), use as energy substrates for ␤ -oxidation, providing both endocrine and nuclear hormone signaling ligands, and for critical posttranslational modifi cations of proteins ( 5-12 ). Perhaps the most demanding setting for FASN catalysis is during lactation, when extremely high quantities of de novo fatty acids are transmitted to the offspring as substrate for the growth of the young ( 13-15 ).Regulation of the principle enzymes of de novo fatty acid synthesis, ATP citrate lyase (ACLY) (EC 2.3.3.8), acetyl-CoA carboxylase (ACC) (EC 6.4.1.2), and FASN, has been shown to occur at the level of enzyme gene expression ( 16 ), translation/degradation ( 17-19 ), phosphorylation ( 20-22 ), assembly into multimeric complexes ( 23-25 ), and allosteric activation/inhibition ( 18,23,26,27 ). Recently, evidence for the control of de novo fatty acid synthesis by small effector proteins has emerged. Thyroid hormone responsive protein "Spot 14" (THRSP) (Spot14, S14) and only family Abstract Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefi ned during lactation. Spot14-null mice produce milk defi cient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When

Constitutive expression of microRNA-150 in mammary epithelium suppresses secretory activation and impairs de novo lipogenesis

“…Interestingly, numerous other predicted targets in this same pathway were suppressed twofold or more by constitutive expression of miR-150 (Fig. 5) including Thrsp, required for MCFA synthesis in MECs (Rudolph et al, 2014), Acly, responsible for synthesis of the substrate acetyl-CoA used for de novo fatty acid synthesis (Linn and Srere, 1979), and Slc25a1, which shuttles substrate into the de novo fatty acid synthesis pathway (Kaplan et al, 1993). These targets, like FASN, might be suppressed even more strongly at the protein level.…”

“…Of the 22 listed lipid and cholesterol synthesis genes with a ≥twofold decrease, all but two are predicted to be direct targets of miR-150 (TargetScan and RNA22). Notably, these predicted targets include the primary enzymes of the de novo fatty acid synthesis pathway, including ATP citrate lyase (Acly) (Linn and Srere, 1979), acetyl-CoA carboxylase (Acaca, both isoforms) (Harada et al, 2007), Fasn (Smith et al, 2003), and thyroid hormone responsive protein spot 14 (Thrsp) (Rudolph et al, 2014). Further, mitochondrial citrate transporter (Slc25a1), required to shuttle substrate into the de novo fatty acid synthesis pathway (Kaplan et al, 1993) also decreased.…”

“…Samples were acidified with 0.525 ml of 1 M HCl, vortexed vigorously; fatty acids were extracted twice with 1.0 ml of isooctane, and taken to dryness under N 2 gas. Saponified fatty acids were derivatized at room temperature for 30 min by addition of 30 μl of 1% pentafluorobenzyl bromide in acetonitrile and 30 μl of 1% N,Ndiisopropylethylamine in acetonitrile according to Rudolph et al (2014), after which the samples were taken to dryness under N 2 gas. The resulting pentafluorobenzyl fatty acid esters were suspended in 200 μl isooctane, vortexed, and transferred into vials for gas chromatography mass spectrometry.…”

“…HPLC grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Total lipid extraction was performed as previously described (Rudolph et al, 2014) with modifications described in supplementary Materials and Methods.…”

FASNating Targets of Metformin in Breast Cancer Stem-Like Cells

“…Presumably, the amounts of de novo synthesized fatty acids correlate with the levels of FASN protein. In cancer cells, FASN expression is regulated by the HER2/Neu and PI3K signaling pathways, steroid hormones, and genomic amplification [20,34,37,42]; however, the spectrum of fatty acid products and the kinetics of the FASN reaction can be altered by effector proteins [21,32]. Interestingly, the effects of metformin on miR193b and FASN were only observed when cells were grown in media containing physiologic glucose levels (5 mM) compared to high glucose (17 mM).…”