Transgenic 6F tomatoes act on the small intesti..


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3418Journal of Lipid Research Volume 24 , J. R. , A. M. Fogelman , M. E. , and J. A. . Diesel exhaust ammatory high-density lipoprotein. 1153 – 1161 . 25 , D. L. , D. M. , D. D. , and G. J. Tigyi . 2001 . Direct quantitative analysis of lysophosphatidic acid molecu- www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: Transgenic 6F tomatoes and intestinal lysophosphatidic acid3417 and 4F peptide reduce systemic in ammation by modulating in- www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: 3416Journal of Lipid Research Volume Addition of Tg6F to chow supplemented with LPA pre- vents the increase in unsaturated LPA levels in the small intestine and prevents systemic in ammation and dyslipidemia. Female / g of LPA (18:0 or www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: Transgenic 6F tomatoes and intestinal lysophosphatidic acid3415 ammation and dyslipidemia. The 64 genes shown in Tables 2 and 4 are involved in several pathways including PPAR signaling, lipid and www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: 3414Journal of Lipid Research Volume The precise mechanism(s) by which intestinally derived LPA modulates these remarkable changes in plasma lipids is www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: Transgenic 6F tomatoes and intestinal lysophosphatidic acid3413 LPA per gram chow produced levels of LPA in the plasma cantly greater than chow or chow supplemented saturated LPA plasma levels that were less than those achieved on feeding WD ( Fig. 9 ); the levels fell within the Addition of unsaturated PA or unsaturated LPA to mouse / / g of PA or LPA (18:0, 18:2, or 20:4) per gram chow. After 3 weeks the small intestine was harvested from each mouse, RNA was isolated from the jejunum, and RT-qPCR for the genes shown in performed. The data shown are mean ± SD. * 0.001. www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: 3412Journal of Lipid Research Volume intestine ( Figs. 1, 12B ), one might think that the doses of PA and LPA added to chow or administered SQ in the experiments reported here might overwhelm all of the g of unsaturated explained by the PA and LPA content of WD. Thus, it seems likely that the increased unsaturated LPA content of the small intestine on WD is largely derived from local formation, which is stimulated by feeding WD through www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: Transgenic 6F tomatoes and intestinal lysophosphatidic acid3411 RT-qPCR con rms microarray analysis. The RNA isolated from the mice described in Fig. 3 was analyzed by RT-qPCR for some of the genes in Tables 2 and 4 whose expression was cantly changed by WD compared with chow, and ) changed by Tg6F in a direction that was opposite to the WD-induced change. A–C: Genes whose expression was increased by WD and prevented by adding Tg6F to WD. D: A gene whose expression was decreased by WD and prevented by adding Tg6F to WD. Data shown are mean ± SD. www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: 3410Journal of Lipid Research Volume that adding LPA 18:2 to chow signi cantly increased the levels of LPA 18:2 in the small intestine compared with adding LPA 18:0, and adding Tg6F (but not EV) to LPA cantly reduced LPA 18:2 levels in the tissue of the small intes- tine (duodenum). The data in Fig. 12D–G show that add- ing Tg6F (but not EV) to LPA 18:2-supplemented mouse chow or WD signi cantly prevented the resulting sys- temic in ammation and dyslipidemia. The data in sup- plementary Fig. VIIA–C demonstrate a highly signi cant Srebf1 www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: Transgenic 6F tomatoes and intestinal lysophosphatidic acid3409 cantly different ( Fig. 9 ), consistent with known pro- acid species ( 9, 10 ). Interestingly, feeding the same quan- ( Fig. 9 ). cantly ammation, SAA ( Plasma levels of LPA 20:4 also positively correlated with plasma total cholesterol Fig. 10B ) and plasma triglyceride ( Fig. 10C ) levels, and inversely correlated with plasma HDL-cholesterol levels ( Fig. 10D ). Oral administration of LPA is modestly but signiÞ more potent than administering the same dose by cantly, caused greater systemic in ammation and a ). decreased instead of increasing ( Fig. 8A–C , supplemen- onstrated decreased expression on feeding WD ( Fig. 4D or LPA to chow ( Fig. 8D , supplementary Fig. VE). These compared with feeding the unsaturated species ( Fig. 8D supplementary Fig. VE). The data in Fig. 8 and supple- Adding 1 g per gram of unsaturated (but not saturated) LPA to mouse chow increases plasma LPA levels to values 6.31Stearoyl-CoA desaturase 1 5.89Solute carrier family 6 (neurotransmiter transporter, 4.95Acyl-CoA thioesterase 1 Srebf1 3.52Sterol regulatory element binding transcription factor 1 3.47Cyp4a10, Cyp4a31, Cyp4a32, Gm10774, Gm13015, www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: 3408Journal of Lipid Research Volume ble to WD ( Fig. 7B ). There was a signi cant dose response resulting in lowered plasma HDL-cholesterol levels on g LPA 18:2 per gram chow ( Fig. 7C ). The data in Figs. 5–7 and supplementary Fig. III were / mentary Fig. IV show that similar results were obtained in Adding unsaturated (but not saturated) PA or LPA to mouse chow produces changes in gene expression in the / mice / in Fig. 4 and supplementary Fig. I that ) was prevented when WD was supplemented with Tg6F, also showed increased expression when fed chow supple mented Fig. VA–D). When saturated PA or LPA was added to chow, either the changes were not signi cant, or the increased expression was signi cantly less than when the unsatu- rated species were added, or gene expression actually rated PA or saturated LPA to mouse chow produced either no signi cant change (total plasma cholesterol, plasma trig- , respectively) or a change that was quantitatively much less compared with adding the unsaturated species (SAA, Fig. 6A ). In the experiments described in Figs. 5 and 6 , the PA and LPA were added to the surface of frozen chow just before the mice were allowed to eat. Because unsaturated PA and LPA were so much more effective than saturated PA or LPA, we asked if air oxidation of the unsaturated compounds may have in uenced the results. To answer this question, a third experiment was conducted as shown in supplementary Fig. III. In this experiment LPA 18:0, LPA 18:2, or LPA 20:4 were mixed into the chow (or were added to the surface as was the case in Figs. 5 and 6 ) or LPA 18:2 or LPA 20:4 were deliber- ately air oxidized prior to mixing into the chow . In the case of the air oxidized LPA, essentially all of the LPA was altered by . ) or Sprr2a Catalyzes the  rst oxygenation step in sterol biosynthesis Small c glycosylphosphatidylinositol-anchored protein that Belongs to the FAM177 family Receptor on NK cells for class I MHC May be involved in ovary development May decrease apoptosis www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: Transgenic 6F tomatoes and intestinal lysophosphatidic acid3407 decreased expression on WD, which was prevented with addition of Tg6F to WD, but not EV ( Fig. 4D , supple- , Fig. 4D was previously shown to have decreased expression on a These data indicate that Tg6F acts in the small intestine mediated changes in gene expression. Adding unsaturated PA or LPA to mouse chow produces / mice WD sue of the small intestine ( Fig. 1 ), and unsaturated LPA g / mice for 18 days. Adding this amount of unsaturated PA or LPA to mouse chow produced signi cant changes in plasma levels of plasma total cholesterol ( Fig. 5B ), plasma triglycerides ( Fig. 5C ), plasma HDL-cholesterol levels Fig. 5D ), and LPL activity ( Fig. 5E ) that were similar in direction to those observed after feeding WD. The changes in plasma cholesterol ( Fig. 5B ) in this experiment were highly signi cant, but were not quantitatively as similar to Fold Change Gene Name Reg3g Tia1 nger protein 692 ; prothymosin www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: 3406Journal of Lipid Research Volume content of unsaturated PA by about 3- to 5-fold ( Figs. 2B, C ). Comparing Figs. 1 and 2 shows that the content of PA (even saturated PA) in the small intestine was much less than that of LPA. Both saturated and unsaturated PA www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: Transgenic 6F tomatoes and intestinal lysophosphatidic acid3405 switched to WD (Teklad, Harlan, catalog #TD88137). Tg6F or www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: 3404Journal of Lipid Research Volume cacy ( 5, 6 ). The high dose requirement provides a bar- matoes ( 7 ). Feeding LDL receptor-null (LDLR ) mice a Western www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at: This article is available online at http://www.jlr.org Journal of Lipid Research Volume 54, 2013 We conclude ) WD-mediated systemic inß ammation and dyslipi- demia may be in part due to WD-induced increases in small intestine LPA levels; and ) Tg6F reduces WD-mediated systemic inß ammation and dyslipidemia by preventing WD- induced increases in LPA levels in the small intestine. — Navab, M., G. Hough, G. M. Buga, F. Su, A. C. Wagner, This work was supported in part by US Public Health Service Research Grants HL-30568 and HL-34343; the Laubisch, Castera, and M. K. Grey Funds at Mohamad Navab , * Greg Hough , * Abbreviations: CXCL1, chemokine (CXC motif) ligand 1; EV, gene; 6F, the peptide D-W-L-K-A-F-Y-D-K- -amino acids; FPLC, fast-performance activated receptor; RT-qPCR, quantitative RT-PCR ; SAA, serum amyloid A; SQ, subcutaneous; Tg6F, transgenic tomatoes constructed with the The online version of this article (available at http://www.jlr. org) contains supplementary data in the form of seven  gures and two tables. www.jlr.org Downloaded from http://www.jlr.org/content/suppl/2013/10/01/jlr.M042051.DC1 Supplemental Material can be found at:

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