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• W2331684995 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 undefined during lactation. Spot14-null mice produce milk deficient 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 [13C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed. 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 undefined during lactation. Spot14-null mice produce milk deficient 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 [13C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed. Mammalian 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) (1Smith S. The animal fatty acid synthase: one gene, one polypeptide, seven enzymes.FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (519) Google Scholar). FASN is absolutely essential for production of de novo fatty acids in mammals (2Smith S. Witkowski A. Joshi A.K. Structural and functional organization of the animal fatty acid synthase.Prog. Lipid Res. 2003; 42: 289-317Crossref PubMed Scopus (494) Google Scholar). 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 (3Chirala S.S. Chang H. Matzuk M. Abu-Elheiga L. Mao J. Mahon K. Finegold M. Wakil S.J. Fatty acid synthesis is essential in embryonic development: fatty acid synthase null mutants and most of the heterozygotes die in utero.Proc. Natl. Acad. Sci. USA. 2003; 100: 6358-6363Crossref PubMed Scopus (181) Google Scholar, 4Jayakumar A. Tai M.H. Huang W.Y. al-Feel W. Hsu M. Abu-Elheiga L. Chirala S.S. Wakil S.J. Human fatty acid synthase: properties and molecular cloning.Proc. Natl. Acad. Sci. USA. 1995; 92: 8695-8699Crossref PubMed Scopus (193) Google Scholar). 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 esterification into triglycerides (TAGs), use as energy substrates for β-oxidation, providing both endocrine and nuclear hormone signaling ligands, and for critical posttranslational modifications of proteins (5Rudolph M.C. Monks J. Burns V. Phistry M. Marians R. Foote M.R. Bauman D.E. Anderson S.M. Neville M.C. 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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 (13Rudolph M.C. Monks J. Burns V. Phistry M. Marians R. Foote M.R. Bauman D.E. Anderson S.M. Neville M.C. Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium.Am. J. Physiol. Endocrinol. Metab. 2010; 299: E918-E927Crossref PubMed Scopus (80) Google Scholar, 14Smith S. Watts R. Dils R. Quantitative gas-liquid chromatographic analysis of rodent milk triglycerides.J. Lipid Res. 1968; 9: 52-57Abstract Full Text PDF PubMed Google Scholar, 15Smith S. Mechanism of chain length determination in biosynthesis of milk fatty acids.J. Dairy Sci. 1980; 63: 337-352Abstract Full Text PDF PubMed Scopus (57) Google Scholar). 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 (16Horton J.D. Goldstein J.L. Brown M.S. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver.J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3776) Google Scholar), translation/degradation (17Gibson D.M. Lyons R.T. Scott D.F. Muto Y. Synthesis and degradation of the lipogenic enzymes of rat liver.Adv. Enzyme Regul. 1972; 10: 187-204Crossref PubMed Scopus (142) Google Scholar, 18Majerus P.W. Kilburn E. Acetyl coenzyme A carboxylase. The roles of synthesis and degradation in regulation of enzyme levels in rat liver.J. Biol. Chem. 1969; 244: 6254-6262Abstract Full Text PDF PubMed Google Scholar, 19Flick P.K. Chen J. Vagelos P.R. Effect of dietary linoleate on synthesis and degradation of fatty acid synthetase from rat liver.J. Biol. Chem. 1977; 252: 4242-4249Abstract Full Text PDF PubMed Google Scholar), phosphorylation (20Potapova I.A. El-Maghrabi M.R. Doronin S.V. Benjamin W.B. Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars.Biochemistry. 2000; 39: 1169-1179Crossref PubMed Scopus (125) Google Scholar, 21Ha J. Daniel S. Broyles S.S. Kim K.H. Critical phosphorylation sites for acetyl-CoA carboxylase activity.J. Biol. Chem. 1994; 269: 22162-22168Abstract Full Text PDF PubMed Google Scholar, 22Jin Q. Yuan L.X. Boulbes D. Baek J.M. Wang Y.N. Gomez-Cabello D. Hawke D.H. Yeung S.C. Lee M.H. Hortobagyi G.N. et al.Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells.Breast Cancer Res. 2010; 12: R96Crossref PubMed Scopus (87) Google Scholar), assembly into multimeric complexes (23Guy P.S. Cohen P. Hardie D.G. Purification and physicochemical properties of ATP citrate (pro-3S) lyase from lactating rat mammary gland and studies of its reversible phosphorylation.Eur. J. Biochem. 1981; 114: 399-405Crossref PubMed Scopus (52) Google Scholar, 24Landman A.D. Darkshinamurti K. Acetyl-Coenzyme A carboxylase. Role of the prosthetic group in enzyme polymerization.Biochem. J. 1975; 145: 545-548Crossref PubMed Scopus (10) Google Scholar, 25Joshi A.K. Rangan V.S. Smith S. Differential affinity labeling of the two subunits of the homodimeric animal fatty acid synthase allows isolation of heterodimers consisting of subunits that have been independently modified.J. Biol. Chem. 1998; 273: 4937-4943Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and allosteric activation/inhibition (18Majerus P.W. Kilburn E. Acetyl coenzyme A carboxylase. The roles of synthesis and degradation in regulation of enzyme levels in rat liver.J. Biol. Chem. 1969; 244: 6254-6262Abstract Full Text PDF PubMed Google Scholar, 23Guy P.S. Cohen P. Hardie D.G. Purification and physicochemical properties of ATP citrate (pro-3S) lyase from lactating rat mammary gland and studies of its reversible phosphorylation.Eur. J. Biochem. 1981; 114: 399-405Crossref PubMed Scopus (52) Google Scholar, 26Vagelos P.R. Alberts A.W. Martin D.B. Studies on the mechnism of activation of acetyl coenzyme A carboxylase by citrate.J. Biol. Chem. 1963; 238: 533-540Abstract Full Text PDF PubMed Google Scholar, 27Numa S. Ringelmann E. Lynen F. On inhibition of acetyl-CoA-carboxylase by fatty acid-coenzyme A compounds [article in German].Biochem. Z. 1965; 343: 243-257PubMed Google Scholar). 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 member Spot14-related protein (Spot14-R) (MIG12, Mid1IP1) are postulated to function as effector proteins for the de novo fatty acid synthesis pathway (28Colbert C.L. Kim C.W. Moon Y.A. Henry L. Palnitkar M. McKean W.B. Fitzgerald K. Deisenhofer J. Horton J.D. Kwon H.J. Crystal structure of Spot 14, a modulator of fatty acid synthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 18820-18825Crossref PubMed Scopus (50) Google Scholar, 29Knobloch M. Braun S.M. Zurkirchen L. von Schoultz C. Zamboni N. Arauzo-Bravo M.J. Kovacs W.J. Karalay O. Suter U. Machado R.A. et al.Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis.Nature. 2013; 493: 226-230Crossref PubMed Scopus (332) Google Scholar). Spot14 was discovered in 1981 and was established to be a small acidic protein of 17 kDa with an isoelectric point of 4.85 (30Seelig S. Liaw C. Towle H.C. Oppenheimer J.H. Thyroid hormone attenuates and augments hepatic gene expression at a pretranslational level.Proc. Natl. Acad. Sci. USA. 1981; 78: 4733-4737Crossref PubMed Scopus (138) Google Scholar). It has no known enzymatic function, the primary sequence predicts no established functional domains, and the crystal structure has identified three antiparallel α-helices in an asymmetrical dimer (28Colbert C.L. Kim C.W. Moon Y.A. Henry L. Palnitkar M. McKean W.B. Fitzgerald K. Deisenhofer J. Horton J.D. Kwon H.J. Crystal structure of Spot 14, a modulator of fatty acid synthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 18820-18825Crossref PubMed Scopus (50) Google Scholar, 31LaFave L.T. Augustin L.B. Mariash C.N. S14: insights from knockout mice.Endocrinology. 2006; 147: 4044-4047Crossref PubMed Scopus (58) Google Scholar). Spot14 mRNA is sharply induced in the liver of streptozotocin-induced diabetic rats by insulin treatment, as well as in livers of fasted rats refed carbohydrates and acutely treated with thyroid hormone (32Jump D.B. Bell A. Lepar G. Hu D. Insulin rapidly induces rat liver S14 gene transcription.Mol. Endocrinol. 1990; 4: 1655-1660Crossref PubMed Scopus (39) Google Scholar, 33Mariash C.N. Seelig S. Schwartz H.L. Oppenheimer J.H. Rapid synergistic interaction between thyroid hormone and carbohydrate on mRNAS14 induction.J. Biol. Chem. 1986; 261: 9583-9586Abstract Full Text PDF PubMed Google Scholar). Its gene expression in the liver was decreased in rats fed a diet containing long chain PUFAs, and in the lactating mammary gland following administration of trans-10 cis-12 conjugated linoleic acid (34Foretz M. Foufelle F. Ferre P. Polyunsaturated fatty acids inhibit fatty acid synthase and spot-14-protein gene expression in cultured rat hepatocytes by a peroxidative mechanism.Biochem. J. 1999; 341: 371-376Crossref PubMed Scopus (30) Google Scholar, 35Kadegowda A.K. Connor E.E. Teter B.B. Sampugna J. Delmonte P. Piperova L.S. Erdman R.A. Dietary trans fatty acid isomers differ in their effects on mammary lipid metabolism as well as lipogenic gene expression in lactating mice.J. Nutr. 2010; 140: 919-924Crossref PubMed Scopus (32) Google Scholar, 36Harvatine K.J. Bauman D.E. SREBP1 and thyroid hormone responsive spot 14 (S14) are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with CLA.J. Nutr. 2006; 136: 2468-2474Crossref PubMed Scopus (217) Google Scholar). In 2003, we observed that Spot14 was induced in unison with 75 metabolic genes in the mouse mammary gland at secretory activation (37Rudolph M.C. McManaman J.L. Hunter L. Phang T. Neville M.C. Functional development of the mammary gland: use of expression profiling and trajectory clustering to reveal changes in gene expression during pregnancy, lactation, and involution.J. Mammary Gland Biol. Neoplasia. 2003; 8: 287-307Crossref PubMed Scopus (180) Google Scholar). More recently, we showed regulation of Spot14 mRNA by sterol response element binding protein 1 in mammary epithelial cells (MECs) depleted of the adipose compartment, and that Spot14 mRNA in MECs was not suppressed by provision of a high fat diet (HFD) to the dam (13Rudolph M.C. Monks J. Burns V. Phistry M. Marians R. Foote M.R. Bauman D.E. Anderson S.M. Neville M.C. Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium.Am. J. Physiol. Endocrinol. Metab. 2010; 299: E918-E927Crossref PubMed Scopus (80) Google Scholar). Others have shown evidence, independent of rodent models, for Spot14 involvement during milk fat production in dairy cows (36Harvatine K.J. Bauman D.E. SREBP1 and thyroid hormone responsive spot 14 (S14) are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with CLA.J. Nutr. 2006; 136: 2468-2474Crossref PubMed Scopus (217) Google Scholar, 38Bauman D.E. Harvatine K.J. Lock A.L. Nutrigenomics, rumen-derived bioactive fatty acids, and the regulation of milk fat synthesis.Annu. Rev. Nutr. 2011; 31: 299-319Crossref PubMed Scopus (217) Google Scholar). Subsequent analysis of the Spot14-null mouse firmly established Spot14 as necessary for the de novo synthesis of medium chain fatty acids (MCFAs) characteristic of milk TAG (39Zhu Q. Anderson G.W. Mucha G.T. Parks E.J. Metkowski J.K. Mariash C.N. The Spot 14 protein is required for de novo lipid synthesis in the lactating mammary gland.Endocrinology. 2005; 146: 3343-3350Crossref PubMed Scopus (74) Google Scholar). Hence, in lactation, Spot14 appears to enhance de novo synthesis of the MCFAs that contribute to the total milk energy content needed for neonatal growth. Both activation and inhibition of de novo fatty acid synthesis by Spot14 have been reported, depending upon the lipogenic tissue type and the cellular context (28Colbert C.L. Kim C.W. Moon Y.A. Henry L. Palnitkar M. McKean W.B. Fitzgerald K. Deisenhofer J. Horton J.D. Kwon H.J. Crystal structure of Spot 14, a modulator of fatty acid synthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 18820-18825Crossref PubMed Scopus (50) Google Scholar, 29Knobloch M. Braun S.M. Zurkirchen L. von Schoultz C. Zamboni N. Arauzo-Bravo M.J. Kovacs W.J. Karalay O. Suter U. Machado R.A. et al.Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis.Nature. 2013; 493: 226-230Crossref PubMed Scopus (332) Google Scholar, 31LaFave L.T. Augustin L.B. Mariash C.N. S14: insights from knockout mice.Endocrinology. 2006; 147: 4044-4047Crossref PubMed Scopus (58) Google Scholar, 39Zhu Q. Anderson G.W. Mucha G.T. Parks E.J. Metkowski J.K. Mariash C.N. The Spot 14 protein is required for de novo lipid synthesis in the lactating mammary gland.Endocrinology. 2005; 146: 3343-3350Crossref PubMed Scopus (74) Google Scholar, 40Zhu Q. Mariash A. Margosian M.R. Gopinath S. Fareed M.T. Anderson G.W. Mariash C.N. Spot 14 gene deletion increases hepatic de novo lipogenesis.Endocrinology. 2001; 142: 4363-4370Crossref PubMed Scopus (44) Google Scholar, 41Aipoalani D.L. O'Callaghan B.L. Mashek D.G. Mariash C.N. Towle H.C. Overlapping roles of the glucose-responsive genes, S14 and S14R, in hepatic lipogenesis.Endocrinology. 2010; 151: 2071-2077Crossref PubMed Scopus (26) Google Scholar, 42Donnelly C. Olsen A.M. Lewis L.D. Eisenberg B.L. Eastman A. Kinlaw W.B. Conjugated linoleic acid (CLA) inhibits expression of the Spot 14 (THRSP) and fatty acid synthase genes and impairs the growth of human breast cancer and liposarcoma cells.Nutr. Cancer. 2009; 61: 114-122Crossref PubMed Scopus (36) Google Scholar). For example, the rate of de novo fatty acid synthesis in the Spot14-null mammary gland was decreased over 2-fold, implicating Spot14 as an activator of de novo fatty acid synthesis in this tissue (39Zhu Q. Anderson G.W. Mucha G.T. Parks E.J. Metkowski J.K. Mariash C.N. The Spot 14 protein is required for de novo lipid synthesis in the lactating mammary gland.Endocrinology. 2005; 146: 3343-3350Crossref PubMed Scopus (74) Google Scholar). Conversely, Spot14-null hepatic de novo fatty acid synthesis was increased 2-fold under lipogenic stimuli, suggesting Spot14 is an inhibitor of this process in the liver (40Zhu Q. Mariash A. Margosian M.R. Gopinath S. Fareed M.T. Anderson G.W. Mariash C.N. Spot 14 gene deletion increases hepatic de novo lipogenesis.Endocrinology. 2001; 142: 4363-4370Crossref PubMed Scopus (44) Google Scholar). Coexpression of Spot14 with Spot14-R was determined to be inhibitory, as Spot14 heterodimerizes with Spot14-R to prevent Spot14-R association with ACC and decrease activation of the hepatic de novo fatty acid synthesis pathway (43Kim C.W. Moon Y.A. Park S.W. Cheng D. Kwon H.J. Horton J.D. Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 9626-9631Crossref PubMed Scopus (92) Google Scholar). In adult neural stem cells, Knobloch et al. (29Knobloch M. Braun S.M. Zurkirchen L. von Schoultz C. Zamboni N. Arauzo-Bravo M.J. Kovacs W.J. Karalay O. Suter U. Machado R.A. et al.Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis.Nature. 2013; 493: 226-230Crossref PubMed Scopus (332) Google Scholar) demonstrated cytosolic localization and coimmunoprecipitation of Spot14 and Spot14-R, and that Spot14 overexpression decreased de novo synthesis of fatty acids in low proliferating cells. Colbert et al. (28Colbert C.L. Kim C.W. Moon Y.A. Henry L. Palnitkar M. McKean W.B. Fitzgerald K. Deisenhofer J. Horton J.D. Kwon H.J. Crystal structure of Spot 14, a modulator of fatty acid synthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 18820-18825Crossref PubMed Scopus (50) Google Scholar) used liver-specific antisense Spot14 knock down in chow-fed mice resulting in approximately 30% decreased 3H2O incorporation into de novo lipid. This work was supported by efforts from Aipoalani et al. (41Aipoalani D.L. O'Callaghan B.L. Mashek D.G. Mariash C.N. Towle H.C. Overlapping roles of the glucose-responsive genes, S14 and S14R, in hepatic lipogenesis.Endocrinology. 2010; 151: 2071-2077Crossref PubMed Scopus (26) Google Scholar), who knocked down both Spot14 and Spot14-R in primary hepatocyte culture and observed a 65% reduction of [14C]-acetate incorporation into de novo fatty acids. Thus, overlapping and conditional roles for Spot14 regulation of fatty acid synthesis have been demonstrated (29Knobloch M. Braun S.M. Zurkirchen L. von Schoultz C. Zamboni N. Arauzo-Bravo M.J. Kovacs W.J. Karalay O. Suter U. Machado R.A. et al.Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis.Nature. 2013; 493: 226-230Crossref PubMed Scopus (332) Google Scholar, 41Aipoalani D.L. O'Callaghan B.L. Mashek D.G. Mariash C.N. Towle H.C. Overlapping roles of the glucose-responsive genes, S14 and S14R, in hepatic lipogenesis.Endocrinology. 2010; 151: 2071-2077Crossref PubMed Scopus (26) Google Scholar). In mammary cancer cells, antisense Spot14 knock down decreased 14C-acetate incorporation into lipid nearly 60%, supporting a role for Spot14 in de novo fatty acid synthesis activation (44Martel P.M. Bingham C.M. McGraw C.J. Baker C.L. Morganelli P.M. Meng M.L. Armstrong J.M. Moncur J.T. Kinlaw W.B. S14 protein in breast cancer cells: direct evidence of regulation by SREBP-1c, superinduction with progestin, and effects on cell growth.Exp. Cell Res. 2006; 312: 278-288PubMed Google Scholar). Cumulatively, these published findings indicate that Spot14 may both positively or negatively regulate the de novo fatty acid synthesis pathway depending on the tissue-specific or cellular context, as well as the presence of Spot14-R. The lactating mammary gland lacks expression of Spot14-R, meaning that the MEC-specific function of Spot14 is independent from Spot14-R. In the lactating mammary gland, Spot14 is considered an activator of de novo fatty acid synthesis because the milk produced by Spot14-null dams contains 60% less MCFAs (39Zhu Q. Anderson G.W. Mucha G.T. Parks E.J. Metkowski J.K. Mariash C.N. The Spot 14 protein is required for de novo lipid synthesis in the lactating mammary gland.Endocrinology. 2005; 146: 3343-3350Crossref PubMed Scopus (74) Google Scholar), which are unique products of mammary alveolar de novo fatty acid synthesis during lactation (15Smith S. Mechanism of chain length determination in biosynthesis of milk fatty acids.J. Dairy Sci. 1980; 63: 337-352Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Zhu et al. (39Zhu Q. Anderson G.W. Mucha G.T. Parks E.J. Metkowski J.K. Mariash C.N. The Spot 14 protein is required for de novo lipid synthesis in the lactating mammary gland.Endocrinology. 2005; 146: 3343-3350Crossref PubMed Scopus (74) Google Scholar) determined that the activities of enzymes upstream of FASN in the biosynthetic pathway were present and functioning in the Spot14-null mammary gland during lactation, including malic enzyme, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, ACC, and FASN. Importantly, in vivo tracer studies using 3H2O incorporation into milk MCFA revealed that the rate of fatty acid synthesis in Spot14-null mice was decreased approximately 2.5-fold (39Zhu Q. Anderson G.W. Mucha G.T. Parks E.J. Metkowski J.K. Mariash C.N. The Spot 14 protein is required for de novo lipid synthesis in the lactating mammary gland.Endocrinology. 2005; 146: 3343-3350Crossref PubMed Scopus (74) Google Scholar). Taken together, these observations indicate that the catalytic rate of FASN in the lactating mammary gland is diminished in the Spot14-null mouse. Here we report that tissue-specific overexpression of Spot14 in MECs increased the amount of MCFA known to originate from de novo synthesis, without altering the percent milk fat by volume or influencing offspring growth. Spot14-null milk production was not diminished, but milk from Spot14-null dams had significantly reduced fat percentage leading to restricted pup growth. We found that this loss in total milk fat and subsequent inability to support litters was rescued by provision of a diet high in fat to the Spot14-null dams. Further, we tested the hypothesis that Spot14 increases the in vitro catalytic rate of both recombinant FASN and of native FASN in MEC cytosolic lysates using a recently developed method that specifically quantifies synthesis of [13C] fatty acid products (45Rudolph M.C. Karl Maluf N. Wellberg E.A. Johnson C.A. Murphy R.C. Anderson S.M. Mammalian fatty acid synthase activity from crude tissue lysates tracing (13)C-labeled substrates using gas chromatography-mass spectrometry.Anal. Biochem. 2012; 428: 158-166Crossref PubMed Scopus (17) Google Scholar). Together, these results suggest the molecular function of Spot14 in MECs is to enhance FASN catalysis during lactation when de novo fatty acid synthesis is needed to achieve optimal MCFA content in the milk. Mice were maintained in the Center for Comparative Medicine. The Institutional Animal Care and Use Committee of the University of Colorado Denver approved all animal procedures. C57Bl/6 and FVB control mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Spot14-null mice maintained on the C57B6/J background were a kind gift of Dr. Cary Mariash, and details regarding generation of these mice are described elsewhere (40Zhu Q. Mariash A. Margosian M.R. Gopinath S. Fareed M.T. Anderson G.W. Mariash C.N. Spot 14 gene deletion increases hepatic de novo lipogenesis.Endocrinology. 2001; 142: 4363-4370Crossref PubMed Scopus (44) Google Scholar). The Transgenic and Knockout Core Facility at the University of Colorado Denver generated the transgenic mice overexpressing Spot14 driven by the mouse mammary tumor virus long terminal repeat (MMTV-Spot14). The pCMV-SPORT6 vector containing mouse Spot14 was purchased from Open Biosystems (Thermo Fisher Scientific, Waltham, MA), and PCR was used to add restriction enzyme sites and the C-terminal hemaglutinin A tag (forward, 5′-CAGT-GGA-TCC-GCC-ACC-ATG-CAA-GTG-CTA-ACG-AAA-CGC-TAT-CC; reverse, 5′-CAG-TGC-GAA-TTC-TCA-AGC-GTA-GTC-TGG-GAC-GTC-GTA-TGG-GTA-CAG-GAC-CTG-CCC-TGT-CAT-TTC-C). The PCR product was ligated into pcDNA3.1 with BamHI and EcoRI, excised with HindIII and EcoRI, and ligated into MMTV-SV40-Bssk, a gift from Dr. William Muller (McGill University). MMTV-Spot14 mice were genotyped using tail DNA with the following primers: forward, 5′-CAG-CGA-GGC-TGA-GAA-CGA-C-3′ reverse, 5′-TAG-TCT-GGG-ACG-TCG-TAT-GGG-TA-3′ to generate a product of 200 base pairs. Mice were maintained on a standard day/night cycle. FVB, C57BL/6, Spot14-null, and MMTV-Spot14 females were mated with corresponding males of the same genetic background. Pregnancy day 1 was designated as the day vaginal plug was observed, and lactation day 1 was the day that pups were born. Litters were standardized to eight pups for all studies. For pup growth studies, litters from both backgrounds were cross-fostered to avoid genetic background differences. Litters were weighed daily between 9:00 AM and noon. FVB, C57B6 control, and Spot14-null mice were maintained on standard laboratory chow (5020, Lab Diets) that contained 9% (21.5% kcal) as fat. Specialized diets were purchased from Research Diets and contained 3.9% kcal fat, 12% kcal fat, and 46% kcal fat (Research Diets, Inc., New Brunswick, NJ), and dams were provided specialized diets 1 day prior to parturition and subsequently fed ad libitum. Milk was collected at lactation days 4 and 10 using vacuum suction, and milk fat analysis was conducted as previously described (46Schwertfeger K.L. McManaman J.L. Palmer C.A. Neville M.C. Anderson S.M. Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation.J. Lipid Res. 2003; 44: 1100-1112Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Weigh-suckle-weigh experiments were conducted according to Dempsey et al. (47Dempsey C. McCormick N.H. Croxford T.P. Seo Y.A. Grider A. Kelleher S.L. Marginal maternal zinc deficiency in lactating mice reduces secretory capacity and alters milk composition.J. Nutr. 2012; 142: 655-660Crossref PubMed Scopus (42) Google Scholar). Milk protein concentrations were determined using the Pierce 660 protein assay (Thermo Fisher Scientific). Milk lactose concentrations were quantified using the lactose assay kit according to the manufacturer's instructions (Abcam, ab83384). HPLC grade reagents were purchased from Sigma-Aldrich, St. Louis, MO. Neutral lipid extraction was adapted from Hutchins et al. (48Hutchins P.M. Barkley R.M. Murphy R.C. Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ioniz" @default.
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• W2331684995 date "2014-06-01" @default.
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• W2331684995 title "Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium" @default.
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