Tg6F ameliorates the increase in oxidized phospholipids in the jejunum of mice fed unsaturated LysoPC or WD

Abstract

Mouse chow supplemented with lysophosphatidylcholine with oleic acid at sn-1 and a hydroxyl group at sn-2 (LysoPC 18:1) increased LysoPC 18:1 in tissue of the jejunum of LDL receptor (LDLR)-null mice by 8.9 ± 1.7-fold compared with chow alone. Western diet (WD) contained dramatically less phosphatidylcholine 18:1 or LysoPC 18:1 compared with chow, but feeding WD increased LysoPC 18:1 in the jejunum by 7.5 ± 1.4-fold compared with chow. Feeding LysoPC 18:1 or feeding WD increased oxidized phospholipids in the jejunum by 5.2 ± 3.0-fold or 8.6 ± 2.2-fold, respectively, in LDLR-null mice (P < 0.0004), and 2.6 ± 1.5-fold or 2.4 ± 0.92-fold, respectively, in WT C57BL/6J mice (P < 0.0001). Adding 0.06% by weight of a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) decreased LysoPC 18:1 in the jejunum of LDLR-null mice on both diets (P < 0.0001), and prevented the increase in oxidized phospholipids in the jejunum in LDLR-null and WT mice on both diets (P < 0.008). Tg6F decreased inflammatory cells in the villi of the jejunum, decreased dyslipidemia, and decreased systemic inflammation in LDLR-null and WT mice on both diets. We conclude that Tg6F reduces diet-induced inflammation by reducing the content of unsaturated LysoPC and oxidized phospholipids in the jejunum of mice.

Keywords: E06 antibody; Western diet; apolipoprotein A-I mimetic peptides; atherosclerosis; lysophosphatidic acid; lysophosphatidylcholine; transgenic tomatoes expressing the 6F peptide.

Fig. 1.

Feeding LDLR-null mice standard mouse chow supplemented with LysoPC 18:1 or feeding the mice a WD increased levels of oxidized phospholipids in the villi of the jejunum. Female LDLR-null mice 5–7 months of age (n = 20 per group) were fed standard mouse chow (Chow), standard mouse chow supplemented with 1 mg LysoPC 18:0 per gram chow, standard mouse chow with the same dose of LysoPC 18:0 plus 0.06% by weight of tomato concentrate from Tg6F, standard mouse chow with 1 mg LysoPC 18:1 per gram chow, standard mouse chow with the same dose of LysoPC 18:1 per gram chow plus 0.06% by weight of tomato concentrate from transgenic control tomatoes (EV) or Tg6F, WD, WD + 0.06% by weight EV (WD + EV), or Tg6F (WD + Tg6F). After 2 weeks the mice were fasted overnight and blood was collected, the mice were extensively perfused with cold saline to remove blood, the jejunum was harvested, and luminal contents were removed by washing, as described in the Materials and Methods. A: Representative photomicrographs for E06. B: Quantification of the area staining positively for E06 in the villi of the jejunum. The data shown are the percent of villous area stained for E06 (mean ± SEM) and are representative of two of two separate experiments. NS, not significant.

Fig. 2.

Determination of E06 by ELISA confirmed immunohistochemistry. Female LDLR-null mice 3–4 months of age (n = 12 per group) were fed standard mouse chow (Chow), standard mouse chow supplemented with 1 mg LysoPC 18:0 per gram chow, standard mouse chow with the same dose of LysoPC 18:0 plus 0.06% by weight of tomato concentrate from Tg6F, standard mouse chow with 1 mg LysoPC 18:1 per gram chow, standard mouse chow with the same dose of LysoPC 18:1 per gram chow plus 0.06% by weight of tomato concentrate from transgenic control tomatoes (EV) or Tg6F, WD, WD + 0.06% by weight EV (WD + EV), or Tg6F (WD + Tg6F). After 2 weeks the mice were fasted overnight and blood was collected, the mice were extensively perfused with cold saline to remove blood, the jejunum was harvested, luminal contents were removed by washing with cold saline, and the jejunum was everted and processed to determine E06 reactive material as described in the Materials and Methods. The data shown are mean ± SEM.

Fig. 3.

Ex vivo incubation of jejunum with LysoPC 18:1 resulted in a dramatically greater time-dependent increase in E06 staining of the villi compared with incubating jejunum with LysoPC 18:0. Female LDLR-null mice 6–9 months of age (n = 5 per group) were maintained on standard mouse chow; the jejunums were removed, everted, and incubated ex vivo with LysoPC 18:1 or LysoPC 18:0 (50 μg/ml), and the segments were analyzed by quantitative immunohistochemistry as described in the Materials and Methods. The data shown are the percent of villous area stained for E06 (mean ± SEM) and are representative of two of two separate experiments.

Fig. 4.

Feeding LDLR-null mice standard mouse chow supplemented with LysoPC 18:1 or feeding them WD significantly increased the content of inflammatory cells in the villi of the jejunum. The jejuna from the mice described in Fig. 1 were processed as described in the Materials and Methods. A: F4/80 staining. B: CD68 staining. C: Ly6G staining. D: CD8 staining. E: CD103 staining. Quantification of the area staining positively for each marker was performed as described in the Materials and Methods. The data shown are the percent of villous area stained for each marker (mean ± SEM) and are representative of two of two separate experiments.

Fig. 5.

Flow cytometry of macrophages isolated from the lamina propria of the jejunum confirmed findings with immunohistochemistry. Female LDLR-null mice 4 months of age (n = 16 per group) were fed standard mouse chow (Chow), standard mouse chow supplemented with 1 mg LysoPC 18:1 per gram chow, standard mouse with the same dose of LysoPC 18:1 plus 0.06% by weight of tomato concentrate from Tg6F, WD, or WD + 0.06% by weight Tg6F (WD + Tg6F). After 2 weeks, the mice were fasted overnight and blood was collected, the mice were extensively perfused with cold saline to remove blood, the jejunum was harvested, and luminal contents were removed by washing, as described in the Materials and Methods. The lamina propria was isolated and the cells subjected to flow cytometry to determine the percent of live cells that were positive for the F4/80 antigen, as described in the Materials and Methods. The data shown are mean ± SEM.

Fig. 6.

Feeding LDLR-null mice standard mouse chow supplemented with LysoPC 18:1 or feeding the mice WD caused dyslipidemia and systemic inflammation. Plasma lipids, PON activity, and SAA and IL-6 levels in the mice described in Fig. 1 were determined as described in the Materials and Methods. A: Plasma total cholesterol levels. B: Plasma triglyceride levels. C: Plasma HDL-cholesterol levels. D: Plasma PON activity. E: Plasma SAA levels. F: Plasma IL-6 levels. The data shown are mean ± SEM and are representative of two of two separate experiments.

Fig. 7.

Standard mouse chow contained dramatically higher levels of oleic acid (18:1)-containing PC and higher levels of LysoPC 18:1 compared with WD. Lipids from 200 mg of standard mouse chow or WD (three samples from each diet) were extracted and subjected to LC-MS/MS as described in the Materials and Methods to determine levels of 18:1-containing PC (A) or LysoPC 18:1 (B). The data shown are mean ± SEM.

Fig. 8.

Despite low levels of 18:1-containing PC and low levels of LysoPC 18:1, feeding WD to LDLR-null mice increased the content of LysoPC 18:1 in the tissue of the jejunum, similar to mouse chow supplemented with 1 mg LysoPC 18:1 per gram chow. Adding Tg6F to both diets decreased the levels of LysoPC 18:1 in the jejunum. Female LDLR-null mice 5–8 months of age (n = 10 per group) were fed standard mouse chow (Chow), standard mouse chow supplemented with 1 mg LysoPC 18:0 per gram chow, standard mouse chow with the same dose of LysoPC 18:0 plus 0.06% by weight of tomato concentrate from Tg6F, standard mouse chow with 1 mg LysoPC 18:1 per gram chow, standard mouse chow with the same dose of LysoPC 18:1 per gram chow plus 0.06% by weight of tomato concentrate from transgenic control tomatoes (EV) or Tg6F, WD, WD + 0.06% by weight EV (WD + EV), or Tg6F (WD + Tg6F). After 2 weeks the mice were fasted overnight and extensively perfused with cold saline to remove blood. The jejunum was harvested. The luminal contents were washed out and the content of LysoPC 18:1 and LysoPC 18:0 in the tissues was determined by LC-MS/MS as described in the Materials and Methods. A: Data for LysoPC 18:1 in the tissue of the jejunum. B: Data for LysoPC 18:0 in the tissue of the jejunum. The data shown are mean ± SEM and are representative of two of two separate experiments.

Fig. 9.

In LDLR-null mice, LysoPC levels in plasma directionally changed the same as in the tissue of the jejunum. Female LDLR-null mice 3–4 months of age (n = 10 per group) were fed standard mouse chow (Chow), standard mouse chow supplemented with 1 mg LysoPC 18:0 per gram chow, standard mouse chow with the same dose of LysoPC 18:0 plus 0.06% by weight of tomato concentrate from Tg6F, standard mouse chow with 1 mg LysoPC 18:1 per gram chow, standard mouse chow with the same dose of LysoPC 18:1 per gram chow plus 0.06% by weight of tomato concentrate from transgenic control tomatoes (EV) or Tg6F, WD, WD + 0.06% by weight EV (WD + EV), or Tg6F (WD + Tg6F). After 2 weeks, the mice were fasted overnight and bled, and the content of LysoPC 18:1 and LysoPC 18:0 in plasma was determined by LC-MS/MS as described in the Materials and Methods. A: The data for LysoPC 18:1 in plasma. B: The data for LysoPC 18:0 in plasma. The data shown are mean ± SEM and are representative of two of two separate experiments.

Fig. 10.

FPLC lipoprotein profiles and LysoPC 18:1 content in LDLR-null mice. Female LDLR-null mice 3–5 months of age (n = 12 per group) were fed standard mouse chow (Chow), standard mouse chow supplemented with 1 mg LysoPC 18:0 per gram chow, standard mouse chow with the same dose of LysoPC 18:0 plus 0.06% by weight of tomato concentrate from Tg6F, standard mouse chow with 1 mg LysoPC 18:1 per gram chow, standard mouse chow with the same dose of LysoPC 18:1 per gram chow plus 0.06% by weight of tomato concentrate from transgenic control tomatoes (EV) or Tg6F, WD, WD + 0.06% by weight EV (WD + EV), or Tg6F (WD + Tg6F). After 2 weeks, the mice were fasted overnight, bled, and plasma was separated by FPLC and cholesterol and LysoPC levels were determined in the fractions as described in the Materials and Methods. A: The FPLC cholesterol profiles. B–D: The data for the LDL fractions, the HDL fractions, and the postHDL fractions (P-HDL) for each dietary condition. B: The content of LysoPC 18:1 in the FPLC fractions from mice receiving chow or chow supplemented with 1 mg LysoPC 18:0 per gram chow or the mice were fed chow with the same dose of LysoPC 18:0 plus 0.06% by weight of tomato concentrate from Tg6F. C: The content of LysoPC 18:1 in mice fed chow supplemented with 1 mg LysoPC 18:1 per gram chow or the same dose of LysoPC 18:1 plus 0.06% by weight of tomato concentrate from transgenic control tomatoes (EV) or Tg6F. D: The content of LysoPC 18:1 in the mice fed WD, WD + 0.06% by weight EV (WD + EV), or Tg6F (WD + Tg6F). B–D: The data are mean ± SEM and are representative of two of two experiments.

Fig. 11.

While the magnitude of the observed changes were less, the directionality of the changes for all parameters measured were the same in WT C57BL/6J mice fed chow supplemented with LysoPC 18:1 or WD, as was the case in LDLR-null mice. Female WT C57BL/6J mice 3 months of age (n = 20 per group) were fed standard mouse chow (Chow), standard mouse chow supplemented with 1 mg LysoPC 18:0 per gram chow, standard mouse chow with the same dose of LysoPC 18:0 plus 0.06% by weight of tomato concentrate from Tg6F, standard mouse chow with 1 mg LysoPC 18:1 per gram chow, standard mouse chow with the same dose of LysoPC 18:1 per gram chow plus 0.06% by weight of tomato concentrate from transgenic control tomatoes (EV) or Tg6F, WD, WD + 0.06% by weight EV (WD + EV), or Tg6F (WD + Tg6F). After 4 weeks, the mice were fasted overnight, bled, and processed as described in Figs. 1, 4, 6. Analyses were performed as described in the Materials and Methods. A: Quantification of E06 reactivity in the jejunum as determined by immunohistochemistry. B: Quantification for macrophage content in the jejunum as determined by immunohistochemistry for F4/80. C: Plasma total cholesterol. D: Plasma triglyceride levels. E: Plasma HDL-cholesterol levels. F: PON activity. G: Plasma SAA levels. The data are mean ± SEM.

Fig. 12.

Schematic representation of a hypothesis for the proposed location and sequence of events leading to the development of dyslipidemia and systemic inflammation in mice fed a WD. The details of this hypothesis are given in the text.