In Western diets, vegetable, canola and palm oils are common components of the diet and to a lesser extent, long chain n-3 PUFA (DHA and EPA) oils from algal or marine sources [26, 27]. In recent years, the development of obesity, inflammation, atherosclerosis and other metabolic diseases has been linked to low grade endotoxemia associated with high dietary fat and energy intake [3, 14, 28–30]. However, these studies and others have raised questions on whether this diet induced endotoxemia reflects changes in energy and fat content of the diet, intestinal permeability or diet induced changes in gut microbiota. In the current study, we used ex vivo and in vivo methods to examine intestinal permeability to endotoxin as it relates to dietary oil composition. All pigs were clinically healthy and raised on typical commercial swine corn-soybean diets. We observed no differences in intestinal integrity due to our ex vivo treatments. Even though some cell culture experiments have been shown to indicate bile acids affecting the intestinal permeability, we didn’t observe any negative impacts from bile acids on intestinal integrity and permeability. Further, Lang et al. reported that bile alone may not be sufficient to induce barrier function in the Ex-vivo experiments . Importantly, we only examined the acute actions of a meal or oil bolus treatment and did not conduct a prolonged feeding trial in an attempt to change the pig microbiota populations or the fatty acid profiles of tissues due to diet.
We hypothesized that dietary intake of oils rich in DHA and EPA would attenuate intestinal endotoxin transport and postprandial circulating endotoxin. We found that dietary cod liver and fish oils attenuated serum endotoxin concentrations compared to the coconut oil and the endotoxin levels in these pigs were similar to the control group (Figure 1). To the best of our knowledge, there are no other studies that have shown this effect of DHA and EPA on endotoxin transport and blood endotoxemia.
Interestingly, only one paper has examined the effects of dietary oil composition on endotoxin uptake and related inflammation . However, contrary to our results, the report by Laugerette et al.  states that rape seed (canola) and sunflower oil, with its high unsaturated fatty acid content, augmented plasma endotoxemia by 50-75%. Cani et al. , also observed a similar increase in endotoxemia in mice orally administered corn oil with or without LPS compared to water alone. However, we observed no change in serum postprandial endotoxin concentration or intestinal endotoxin transport due to dietary vegetable oil compared to the saline control (Figures 1 and 3). This discrepancy of these parameters between the studies might be partially explained by the use of different animal models and also the nature of the experimental design. Whereas Laugerette et al.  performed a chronic feeding study using mice for eight weeks using oils with different fatty acid composition; we used an acute meal bolus model with swine to examine endotoxin permeability. Therefore, the mice fatty acid profiles would have mimicked their diets and this tissue enrichment may also modify endotoxin permeability, signaling and acute inflammation. Further work is needed to explain the molecular aspects of these differences between the two studies with regard to acute versus chronic dietary fat signaling in the gastrointestinal tract. Additionally, we observed a significant increase in postprandial endotoxemia after a porridge meal mixed with coconut oil (Figure 1). Again, this contradicts data presented by Laugerette et al.  in which palm oil, high in saturated fatty acids, had no effect on plasma endotoxin concentrations in mice. However, these authors did report an increase in plasma LPS binding protein and argued that this protein is a better marker of endotoxemia due to the short half-life of circulating endotoxin. One issue is that LPS binding protein can be up regulated by inflammation and acute stress as well as both gram positive and negative infections. As the magnitude of LBP response goes down with multiple episodes of infection , this could be a result of the agonistic effects of saturated fatty acid on pro-inflammatory signaling and not circulating endotoxin.
Gram negative bacteria, particularly those found in the distal ileum and colon, might be one of the major sources for circulating endotoxin . It has been estimated that a single cell of Escherichia coli contains approximately 106 Lipid A or endotoxin molecules and a typical human intestinal tract could harbor approximately one gram of endotoxin [33–35]. Interestingly, the bacterial population in the intestine is not static. Multiple studies have shown that bacterial composition shifts to either gram positive majority or gram negative majority based on the composition of the diet consumed [30, 36, 37]. A majority of these studies show that consuming high saturated fat diet for a longer period results in higher gram negative bacterial populations and high fiber diets results in gram positive bacterial populations [30, 38]. Laugerette et al. , reaffirmed this and showed that fatty acid composition of different dietary oils can alter intestinal microbiota populations. Moreover, these authors demonstrated that feeding a diet high in palm oil which is rich in SFAs significantly increased the gram negative bacteria Escherichia coli groups, which can be significant source of endotoxin in the cecal content of mice compared to milk fat, rape seed and sunflower oil fed diets.
During intestinal stress, ischemia, inflammation and diseases, paracellular transport occurs through the tight junction, as known as “leaky gut” . Alternatively, transcellular or intracellular transport can occur, particularly in healthy individuals . Transcellular endotoxin transported across a cell membrane has been shown to occur via TLR4 and soluble GPI anchored receptor CD14 in a lipid raft mediated mechanism [41, 42]. Additionally, chylomicron associated LPS transport has also been suggested to play a key role in intestinal LPS transport from the intestinal epithelial cell [11, 43, 44]. Importantly, we observed no decrease in intestinal integrity which might enhance paracellular permeability as assessed by transepithelial resistance or FITC-dextran permeability due to treatment or short term raft destabilization (Figure 4B). These data suggest that under healthy intestinal epithelial conditions, endotoxin is most likely transported via lipid raft mediated endocytosis.
The signaling and transport process for endotoxin is initiated in specialized membrane micro domains called lipid rafts . Lipid rafts are membrane regions rich in cholesterol, glycolipids, sphingolipids and saturated fatty acids, which result in a ‘rigid’ membrane structure compared to the adjacent ‘fluid’ regions . In immune cells, endotoxin triggers the recruitment of TLR4 into the lipid raft, where it interacts with CD14 and other associated proteins such as MD-2 resulting in an inflammatory signaling cascade [46, 47]. Thus, the two major consequences of preventing endotoxin recognition by dissociating the lipid raft to attenuate TLR4 recruitment include reduced inflammatory signaling and attenuated endotoxin transport. We observed that if intestinal lipid rafts are dissociated ex vivo with MβCD, then endotoxin permeability is attenuated in the ileum (Figure 4). Interestingly, saturated fat induced endotoxin permeability is also significantly reduced. Stimulation of TLR4 receptor has been shown to result in the endotoxin transport across the intestinal epithelial cells . TLR4 is not only implicated in the transcellular transport of LPS but also for live bacteria . Since saturated fatty acids and n-3 PUFA can reciprocally modulate TLR4 signaling , the fatty acid composition of oil in the diet has the potential to increase or decrease endotoxin transport. Altogether, these data suggest that apical endotoxin transport in the intestines is arguably raft mediated in healthy individuals.
In vitro experiments show clearly that n-3 PUFA disrupt TLR4 signalling and the activation of NFκB by LPS in a murine monocytic cell line . Moreover, DHA modulates TLR4 signaling in vitro in RAW 264.7 macrophages and 293 T cells , human monocytes and dendritic cells  and adipose tissue. We have previously shown in pigs that dietary EPA and DHA are effective means of influencing the inflammatory status and pathways influenced by TLR4 signaling induced by LPS  and in altering intestinal function [24, 53]. Therefore, one could postulate that antagonizing TLR4 recruitment to lipid rafts and it’s signaling by DHA and EPA, or stimulating these processes with saturated fatty acids, would alter endotoxin transport and circulating postprandial endotoxin.
Another mechanism through which endotoxin can enter the circulation is through micelles. Since the endotoxin side chains are made up of fatty acids, endotoxins can be incorporated into the micelles and transported into the intestinal epithelial cell . In intestinal epithelial cells, chylomicrons transport the absorbed lipids into various parts of the body. High fat administration has been shown to proportionately increase the endotoxin content of the chylomicron indicating that high fat consumption indeed enhances higher endotoxin transport into the intestinal epithelial cell and incorporation into chylomicron [11, 28]. Furthermore, even though the mechanism is not clear, high intake of fat has been shown to cause internalization of tight junction proteins and increase in the paracellular permeability to macro molecules including endotoxin . Even though, this mode of endotoxin transport cannot be ruled out, we speculate that the rate of incorporation of fatty acids into micelles would not vary due to oil composition. Therefore, we propose that the difference in intestinal endotoxin transport we observed is primarily transcellular transport that involves lipid rafts and receptor mediated endocytosis .
In conclusion, these data suggest that dietary oils can differentially alter intestinal endotoxin transport. Oils rich in DHA and EPA attenuate endotoxin transport, while oils high in saturated fatty acids augment endotoxin transport. Furthermore, intestinal endotoxin transport in healthy subjects may be regulated through a lipid raft mediated mechanism. Saturated fatty acids may be stabilizing the lipid rafts allowing for greater endotoxin transport.