Orange juice neutralizes the proinflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin increase and Toll-like receptor expression

Abstract

Background: The intake of glucose or a high-fat, high-carbohydrate (HFHC) meal, but not orange juice, induces an increase in inflammation and oxidative stress in circulating mononuclear cells (MNCs) of normal-weight subjects.

Objective: We investigated the effect of orange juice on HFHC meal-induced inflammation and oxidative stress and the expression of plasma endotoxin and Toll-like receptors (TLRs).

Design: Three groups (10 subjects in each group) of normal, healthy subjects were asked to drink water or 300 kcal glucose or orange juice in combination with a 900-kcal HFHC meal. Blood samples were obtained before and 1, 3, and 5 h after the drinks and meal combinations were consumed.

Results: Protein expression of the NADPH oxidase subunit p47(phox), phosphorylated and total p38 mitogen-activated protein kinase, and suppressor of cytokine signaling-3; TLR2 and TLR4 messenger RNA (mRNA) and protein expression; mRNA expression of matrix metalloproteinase (MMP)-9 in MNCs; and plasma concentrations of endotoxin and MMP-9 increased significantly after glucose or water were consumed with the meal but not when orange juice was consumed with the meal. The generation of reactive oxygen species by polymorphonuclear cells was significantly lower when orange juice was added to the meal than when water or glucose was added to the meal.

Conclusions: The combination of glucose or water and the HFHC meal induced oxidative and inflammatory stress and an increase in TLR expression and plasma endotoxin concentrations. In contrast, orange juice intake with the HFHC meal prevented meal-induced oxidative and inflammatory stress, including the increase in endotoxin and TLR expression. These observations may help explain the mechanisms underlying postprandial oxidative stress and inflammation, pathogenesis of insulin resistance, and atherosclerosis.

Figures

FIGURE 1

Mean (±SE) change in plasma glucose (A) and insulin (B) concentrations after intake of a 900-kcal high-fat, high-carbohydrate meal and a 300-kcal drink of orange juice (OJ+meal), glucose (Glu+meal), or water (W+meal) in normal subjects. *,**,#P < 0.05 [repeated-measures ANOVA (RMANOVA)] compared with baseline values after W+meal, Glu+meal, or OJ+meal, respectively; +,$P < 0.05 (2-factor RMANOVA) for comparison between W+meal or Glu+meal and OJ+meal treatments, respectively. P for interaction (treatment × time) = 0.011 for glucose with differences at 1 h between groups; P for interaction (treatment × time) = 0.027 for insulin with differences at 1 h between groups. n = 10 each.

FIGURE 2

Mean (±SE) change in reactive oxygen species (ROS) generation by mononuclear cells (MNC) (A) and polymorphonuclear cells (PMN) (B) after intake of a 900-kcal high-fat, high-carbohydrate meal and a 300-kcal drink of orange juice (OJ+meal), glucose (Glu+meal), or water (W+meal) in normal subjects. *,**,#P < 0.05 [repeated measures ANOVA (RMANOVA)] compared with baseline values after W+meal, Glu+meal, or OJ+meal, respectively; +,$P < 0.05 (2-factor RMANOVA) for comparison between W+meal or Glu+meal and OJ+meal treatments, respectively. P for interaction (treatment × time) = 0.151 for ROS generation by MNCs; P for interaction (treatment × time) = 0.039 for ROS generation by PMNs with differences between groups at 1 and 3 h. n = 10 each.

FIGURE 3

Western blotting representative gel and densitometry for the change in NADPH oxidase subunit (p47phox) (A and B), p38 mitogen-activated protein kinase (MAPK) (A and C), and phosphorylated p38 MAPK protein (A and D) after intake of a 900-kcal high-fat, high-carbohydrate meal and a 300-kcal drink of orange juice (OJ+meal), glucose (Glu+meal), or water (W+meal) in normal subjects. Values in B–D are means ± SEs. *,**,#P < 0.05 [repeated measures ANOVA (RMANOVA)] compared with baseline values after W+meal, Glu+meal, or OJ+meal, respectively; ^P < 0.05 (2-factor RMANOVA) for comparisons between W+meal and Glu+meal; +,$P < 0.05 (2-factor RMANOVA) for comparisons between W+meal or Glu+meal and OJ+meal treatments, respectively. P interaction (treatment × time) = 0.21 for p47phox; P for interaction (treatment × time) = 0.031 for p38 with differences between the groups at 3 and 5 h; P for interaction (treatment × time) = 0.28 for phosphorylated p38. n = 10 each. MNC, mononuclear cells.

FIGURE 4

Mean (±SE) change in matrix metalloproteinase (MMP)-9 messenger RNA (mRNA) expression in mononuclear cells (MNC) (A) and plasma concentration (B) after intake of a 900-kcal high-fat, high-carbohydrate meal and a 300-kcal drink of orange juice (OJ+meal), glucose (Glu+meal), or water (W+meal) in normal subjects. *,**P < 0.05 [repeated measures ANOVA (RMANOVA)] compared with baseline values after W+meal, Glu+meal, or OJ+meal, respectively; ^P < 0.05 (2-factor RMANOVA) for comparisons between W+meal and Glu+meal; +,$P < 0.05 (2-factor RMANOVA) for comparison between W+meal or Glu+meal and OJ+meal treatments, respectively. P for interaction (treatment × time) = 0.032 for MMP-9 mRNA with differences between the groups at 1 and 3 h; P for interaction (treatment × time) = 0.028 for plasma MMP-9 with differences between the groups at 3 h. n = 10 each.

FIGURE 5

Mean (±SE) change in Toll-like receptor (TLR) 2 expression (A) and TLR4 messenger RNA (mRNA) expression (B) in mononuclear cells (MNC) after intake of a 900-kcal high-fat, high-carbohydrate meal and a 300-kcal drink of orange juice (OJ+meal), glucose (Glu+meal), or water (W+meal) in normal subjects. *,**P < 0.05 [repeated measures ANOVA (RMANOVA)] compared with baseline values after W+meal, Glu+meal, or OJ+meal, respectively; ^P < 0.05 (2-factor RMANOVA) for comparisons between W+meal and Glu+meal; +,$P < 0.05 (2-factor RMANOVA) for comparison between W+meal or Glu+meal and OJ+meal treatments, respectively. P for interaction (treatment × time) = 0.034 for TLR2 mRNA with differences between the groups at 1 and 3 h; P for interaction (treatment × time) = 0.027 for TLR4 mRNA with differences between the groups at 1, 3, and 5 h. n = 10 each.

FIGURE 6

Western blotting representative gel and densitometry for the change in Toll-like receptor (TLR) 2 protein (A and B) and TLR4 protein (A and C) in mononuclear cells (MNC) after intake of a 900-kcal high-fat, high-carbohydrate meal and a 300-kcal drink of orange juice (OJ+meal), glucose (Glu+meal), or water (W+meal) in normal subjects. Values in B and C are means ± SEs. *,**P < 0.05 [repeated measures ANOVA (RMANOVA)] compared with baseline values after W+meal, Glu+meal, or OJ+meal, respectively; +,$P < 0.05 (2-factor RMANOVA) for comparison between W+meal or Glu+meal and OJ+meal treatments, respectively. P for interaction (treatment × time) = 0.041 for TLR2 protein with differences between the groups at 1 h; P for interaction (treatment × time) = 0.044 for TLR4 protein with differences between the groups at 3 and 5 h. n = 10 each.

FIGURE 7

Change in suppressor of cytokine signaling (SOCS)-3 (A and B) protein in mononuclear cells (MNC) after intake of a 900-kcal high-fat, high-carbohydrate meal and a 300-kcal drink of orange juice (OJ+meal), glucose (Glu+meal), or water (W+meal) in normal subjects. Values in B are means ± SEs. *,**P < 0.05 [repeated measures ANOVA (RMANOVA)] compared with baseline values after W+meal, Glu+meal, or OJ+meal, respectively. P for interaction (treatment × time) = 0.364. n = 10 each.

FIGURE 8

Mean (±SE) change in plasma endotoxin concentrations after intake of a 900-kcal high-fat, high-carbohydrate meal and a 300-kcal drink of orange juice (OJ+meal), glucose (Glu+meal), or water (W+meal) in normal subjects. *,**P < 0.05 [repeated measures ANOVA (RMANOVA)] compared with baseline values after W+meal, Glu+meal, or OJ+meal, respectively; +,$P < 0.05 (2-factor RMANOVA) for comparisons between W+meal or Glu+meal and OJ+meal treatments, respectively. P for interaction (treatment × time) = 0.134. n = 10 each.