Thyroid Hormone Responsive Protein (Spot14) Increases Fatty Acid Synthase Activity in vitro

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 723-745-Lipids: Fatty Liver Disease & Lipodystrophies
Basic/Clinical
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-738
Michael C. Rudolph*1, Elizabeth Ann Wellberg1, Andrew S Lewis1, Chris A Johnson2, Robert C Murphy2 and Steven Mathias Anderson1
1University of Colorado Anschutz Medical Campus, Aurora, CO, 2University of Colorado Anschutz Medical Campus
The de novo fatty acid synthesis pathway plays a pivotal role in obesity and multiple forms of cancer, and both THRSP (Spot14) and fatty acid synthase (FASN) have been linked to a poor outcome for patients with breast cancer.  The de novo fatty acid synthesis pathway also contributes prominently to the triglyceride fat (TAG) component of milk during lactation via synthesis of medium chain fatty acids (MCFAs). Effector proteins are present in mammary epithelial cells (MECs) that modify the de novo pathway output such that the distribution of FASN products is converted from primarily 16 carbon fatty acids to MCFAs that contain 8-14 acyl carbons.  The Spot14 genomic knockout mice exhibit a lactation defect, and analysis of Spot14 null milk, whole mammary glands, and adipose depleted MECs revealed significant decreases specifically in the de novo synthesized MCFAs.  Spot14 loss did not result in a decrease in the expression of metabolic genes, enzyme protein levels, or in a variety of intermediate metabolites that support de novo MCFA synthesis.  Further, mRNA and protein levels for the MEC specific enzyme required for termination of MFCAs, thioesterase 2, was present at equivalent levels in Spot14 null and control mammary epithelial cells.  Together, these results suggested that the activity of FASN is deficient in MECs from Spot14 knockout mice during lactation.  Spot14 protein co-migrated with native enzyme complexes of FASN in non-denaturing gels, implying that Spot14 might associate with FASN and thereby alter enzyme activity for the synthesis of MCFAs. To test this possibility, a novel FASN activity assay was designed to sensitively quantify FASN 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.  Recombinant FASN activity was increased 1.6-fold in the presence of S14 relative to FASN only controls.  Here we present in vitro data that Spot14 directly increases the rate and yield of fatty acids synthesized by altering FASN enzyme kinetics.  Given that inhibitors FASN often result in whole body complications, we posit that understanding the role of proteins that can modulate FASN activity might expand the repertoire of drug-based therapies.

(1) Rudolph, M. C., Karl Maluf, N., Wellberg, E. A., Johnson, C. A., Murphy, R. C. & Anderson, S. M. (2012). Mammalian fatty acid synthase activity from crude tissue lysates tracing (13)C-labeled substrates using gas chromatography-mass spectrometry. Anal Biochem 428, 158-66.

Nothing to Disclose: MCR, EAW, ASL, CAJ, RCM, SMA

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: MCR = BC810596 DoD BCRP Fellowship; EAW = BC098051 DoD BCRP Fellowship; RCM = 5UL1RR025780 CCTSI; SMA = PO1-HD38129 NIH