Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid

Abstract

We recently reported that levels of unsaturated lysophosphatidic acid (LPA) in the small intestine significantly correlated with the extent of aortic atherosclerosis in LDL receptor-null (LDLR⁻/⁻) mice fed a Western diet (WD). Here we demonstrate that WD increases unsaturated (but not saturated) LPA levels in the small intestine of LDLR⁻/⁻ mice and causes changes in small intestine gene expression. Confirmation of microarray analysis by quantitative RT-PCR showed that adding transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to WD prevented many WD-mediated small intestine changes in gene expression. If instead of feeding WD, unsaturated LPA was added to chow and fed to the mice: i) levels of LPA in the small intestine were similar to those induced by feeding WD; ii) gene expression changes in the small intestine mimicked WD-mediated changes; and iii) changes in plasma serum amyloid A, total cholesterol, triglycerides, HDL-cholesterol levels, and the fast-performance liquid chromatography lipoprotein profile mimicked WD-mediated changes. Adding Tg6F (but not control tomatoes) to LPA-supplemented chow prevented the LPA-induced changes. We conclude that: i) WD-mediated systemic inflammation and dyslipidemia may be in part due to WD-induced increases in small intestine LPA levels; and ii) Tg6F reduces WD-mediated systemic inflammation and dyslipidemia by preventing WD-induced increases in LPA levels in the small intestine.

Keywords: 6F peptide; apolipoprotein A-I mimetic peptides; atherosclerosis; genetically engineered tomato plants; lysophosphatidic acid.

Fig. 1.

Feeding WD increases the content of unsaturated (but not saturated) LPA in the small intestine. Female LDLR^−/− mice, age 8–10 months (n = 12 per group), were fed chow or WD for 2.5 weeks. The mice were fasted overnight, and after perfusion to remove all blood, the small intestine was harvested, the luminal contents were removed, and unsaturated (A–C) or saturated (D) LPA levels in the duodenum were determined by LC-ESI-MS/MS as described in Materials and Methods. The data shown are mean ± SD.

Fig. 2.

Feeding Tg6F reduces levels of unsaturated PA in the tissue of the small intestine. Female LDLR^−/− mice, age 6–8 months (n = 8 per group), were fed WD or WD plus 2.2% ground freeze-dried Tg6F or EV tomatoes for 3 weeks. The mice were fasted overnight, and after perfusion to remove all blood, the small intestine was harvested, the luminal contents were removed, and the levels in the tissue of the duodenum of saturated (A) or unsaturated (B, C) PA were determined as described in Materials and Methods. The data shown are mean ± SD.

Fig. 3.

Microarray analysis identifies WD-induced genes whose expression was prevented by addition of Tg6F. Female LDLR^−/− mice, 7–8 months of age (n = 4–6 per group), were fed chow, WD, WD plus 2.2% by weight ground freeze-dried EV tomatoes, or WD plus 2.2% by weight ground freeze-dried Tg6F. After 3 weeks the small intestine was harvested from each mouse and RNA was isolated from the jejunum and analyzed by microarray analysis as described in Materials and Methods. The data are a TukeyHSD plot for the genes Acadl, Acot1, and Angptl4. A, WD versus Chow; B, WD + EV versus Chow; C, WD + Tg6F versus Chow; D, WD + Tg6F versus WD + EV.

Fig. 4.

RT-qPCR confirms 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 i) significantly changed by WD compared with chow, and ii) 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.

Fig. 5.

Addition of unsaturated PA or unsaturated LPA to mouse chow produces changes similar to feeding WD in LDLR^−/− mice. Female LDLR^−/− mice, age 8–10 months (n = 8 per group), were fed mouse chow (Chow), WD, or chow supplemented with 1 μg of PA or LPA (18:2 or 20:4) per gram chow. Each night the mice were given 20 g of chow for each cage of four mice. The mice ate all of the diet each night. After 18 days, plasma levels of SAA (A), plasma total cholesterol (B), plasma triglycerides (C), plasma HDL-cholesterol (D), and LPL activity (E) were determined as described in Materials and Methods. *P < 0.03; **P < 0.01; ***P < 0.001.

Fig. 6.

Addition of unsaturated (but not saturated) PA or unsaturated LPA to mouse chow produces changes similar to feeding WD in LDLR^−/− mice. Female LDLR^−/− mice, age 7–8 months (n = 8 per group), were fed mouse chow (Chow), WD, or chow supplemented with 1.25 μg of PA or LPA (18:0, 18:2, or 20:4) per gram chow. Each night the mice were given 16 g of chow for each cage of four mice. The mice ate all of the diet each night. After 3 weeks, plasma levels of SAA (A), plasma total cholesterol (B), plasma triglycerides (C), and plasma HDL-cholesterol (D) were determined as described in Materials and Methods. Data are mean ± SEM. *P < 0.03; **P < 0.01; ***P < 0.001.

Fig. 7.

Plasma levels of total cholesterol, triglycerides, and HDL-cholesterol after addition of increasing doses of LPA 18:2 to chow. Female LDLR^−/− mice, age 6–8 months (n = 12 per group), were fed chow, chow with LPA 18:2 mixed in (at a dose of 1, 2, or 4 μg per gram chow), or WD. After 2 weeks the mice were bled and plasma levels of total cholesterol, triglycerides, and HDL-cholesterol were determined as described in Materials and Methods.

Fig. 8.

Addition of unsaturated PA or unsaturated LPA to mouse chow produces changes in gene expression in the small intestine similar to those seen after feeding WD in LDLR^−/− mice. Female LDLR^−/− mice, age 7–8 months (n = 4–6 per group), were fed mouse chow (Chow), WD, or chow supplemented with 1 μg 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 Fig. 4 was performed. The data shown are mean ± SD. *P < 0.05; **P < 0.01; ***P ≤ 0.001.

Fig. 9.

Addition of 1 μg/g unsaturated LPA to mouse chow produces plasma levels of LPA 20:4 in LDLR^−/− mice that are between the levels seen in mice fed chow and mice fed WD. Female LDLR^−/− mice, age 7–8 months (n = 8 per group), were fed mouse chow (Chow), WD, or chow supplemented with 1 μg of LPA (18:0, 18:2, or 20:4) per gram chow for 3 weeks. After fasting overnight, plasma was obtained from the mice and levels of LPA 20:4 were determined by LC-ESI-MS/MS as described in Materials and Methods. The data shown are mean ± SD. *P < 0.02; **P < 0.001; ***P < 0.0001.

Fig. 10.

Plasma levels of LPA 20:4 correlate with plasma SAA levels and with plasma lipid levels. SAA levels (A), total cholesterol (B), triglycerides (C), and HDL-cholesterol levels (D) were determined in the plasma from the mice described in Fig. 9 and linear regression analysis was performed as described in Materials and Methods.

Fig. 11.

LPA administered orally to LDLR^−/− mice is modestly but significantly more potent than the same dose administered by SQ injection. Female LDLR^−/− mice, age 3 months (n = 8 per group), were fed mouse chow (Chow), WD, or chow supplemented with 4 μg of LPA (18:0, 18:2, or 20:4) per gram chow. Each night the mice were given 16 g of chow for each cage of four mice. The mice ate all of the diet each night. Other mice received mouse chow without LPA and received daily SQ injections on the back of 16 μg of LPA (18:0, 18:2, or 20:4) in saline, or they received saline alone. After 2 weeks, plasma levels of SAA (A), total cholesterol (B), plasma triglycerides (C), and plasma HDL-cholesterol (D) were determined as described in Materials and Methods. Data are mean ± SD.

Fig. 12.

Addition of Tg6F to chow supplemented with LPA prevents the increase in unsaturated LPA levels in the small intestine and prevents systemic inflammation and dyslipidemia. Female LDLR^−/− mice, age 3–4 months (n = 10–20 per group), were fed chow only (A, B), chow supplemented with 1 μg of LPA (18:0 or 18:2) per gram chow that was mixed into normal mouse chow as described in Materials and Methods (A–G), chow supplemented with 1 μg of LPA (18:0 or 18:2) per gram chow plus 2.2% by weight freeze-dried tomato powder that was also mixed into the chow using either EV tomatoes or Tg6F tomatoes (A–G), or WD or WD plus 2.2% by weight Tg6F (A–G). After 2 weeks the mice were bled and perfused to remove all blood, the small intestine was harvested, and luminal contents were removed. LPA 18:0 levels in the tissue of the duodenum (A), LPA 18:1 levels in the tissue of the duodenum (B), and LPA 18:2 levels in the tissue of the duodenum (C) were determined as described in Materials and Methods. Plasma SAA (D), plasma total cholesterol (E), plasma triglycerides (F), and HDL-cholesterol (G) were determined as described in Materials and Methods for all groups except the chow only group.

Fig. 13.

FPLC cholesterol profiles of plasma taken from the mice described in Fig. 12. Plasma was collected from five randomly selected mice from each of the groups described in Fig. 12. An equal volume of plasma (200 μl) from each mouse was pooled in each group and FPLC cholesterol profiles were determined as described in Materials and Methods.