To our knowledge, this is the first study that reported the protective effect of a diet rich in FO in relation to a diet rich in SF against LPS-induced pathologies in mice with functional TLR-4. SF-fed mice that were challenged with LPS got sicker and had higher mortality rate compared to FO-fed mice. After LPS challenge, SF-fed mice lost more weight and had greater drop in blood glucose concentrations compared to FO-fed mice. These coincided with higher serum IL-1β concentrations in SF-fed mice challenged with LPS compared to their counterparts. Additionally, net body weight gain, abdominal fat weight, serum cholesterol, and blood glucose were significantly higher in SF-fed mice compared to FO-fed mice.
Although the food intake during the 60 day feeding was not significantly different between FO- and SF-fed mice, the net body weight gain and epididymal fat weight were significantly increased in SF-fed mice suggesting that the weight gain observed in SF-fed mice was due to type of fat administered and not due to hyperphagia. Generally, increased adiposity is a cause for elevated inflammation and further fat intake/infusion is likely to lead elevated free fatty acids which further leads to elevated inflammation [24–26]. Intake of n-3 fatty acids from marine sources reduces adiposity by inhibiting the sterol regulatory element binding protein-1 and thereby decreasing the expression of lipogenic genes (acetyl CoA carboxylase and fatty acid synthase), increasing the gene expression of lipolytic genes (hormone sensitive lipase and serum amyloid A) , and decreasing expression of perilipin, a lipid droplet-protective protein . Similar to our findings, Samane et al  found that FO decreased visceral adiposity compared to a diet rich in lard. They also reported no difference in weight gain between mice fed a high fat diet with 6% of fat replaced by fish oil or argan oil (mainly oleic acid) for 4 weeks, suggesting the effect of fish oil on body weight is affected by the duration and amount of fish oil intake.
Another beneficial effect of FO diet in relation to SF diet is decreased circulating cholesterol. After 60-day feeding, on average, mice fed FO diet had 87.5% lower serum cholesterol compared to mice fed SF diet. Depressed circulating cholesterol is a well established cardioprotective factor . Generally, decreased blood cholesterol is due to lowered LDL cholesterol. It has been well documented diets rich in FO are cardioprotective [31, 32]. However, effects of FO on blood lipids have not been uniform . Chang et al  reported that the cardioprotective effect of FO in comparison to SF diet was not related to blood cholesterol. In their study, SF and FO diets had similar increase in blood cholesterol but free fatty acids and triglycerdes were lower with FO diet in relation to SF diet. Increased blood cholesterol was likely due to higher cholesterol content of SF and FO diets . Additionally, cardioprotective effect of n-3 fatty acids compared to SF was due to reduced total LDL uptake and cholesterol ester deposition in arterial walls [32, 33].
Studies have shown that diets high in fat (other than FO) and specifically high in SF induced metabolic endotoxemia and adipose tissue inflammation by altering gut microbiota and permeability to LPS [34–36] and by stimulating TLR-4 expression . Antibiotic treatment reversed these effects significantly . Thus metabolic endotoxemia (increased circulating LPS) may partly explain the increased inflammation with SF diet. In our study, the SF diet group had a significant decrease in colon weight compared to FO diet group (P < 0.013). This decrease in colon weight observed in the SF group may be a result of atrophy due to a change in the intestinal microbiota or increased colon weight in FO diet group due to protective effect of FO on the gut. More research investigating the effect of saturated fat and n-3 fatty acids on intestinal microbiota and pathobiology of gut is needed to verify this observation.
After administration of LPS, mice were observed for changes in behavior. In the post-500 ng LPS phase, 3 mice died in the SF group out of 9 (33.3% mortality rate) and 1 mouse died out of 10 (10% mortality rate) in the FO group suggesting that FO offered protection against LPS-induced sickness and mortality. Furthermore, after LPS-challenge, SF-fed mice had lost more body weight and experienced a greater decline in blood glucose (decrease was 42.8% with SF and 19.7% with FO) than FO-fed mice suggesting that the SF-fed mice were in a more catabolic state. Following the 60 day period, we noticed no overt in-between group differences in the physical appearance of mice. However, following 100 ng LPS-challenge, the SF diet group mice became reclusive and shortly fell asleep. In contrast, the FO-fed mice exhibited no abnormal behavior. After the 500 ng LPS-challenge, both groups of mice were moribund. However, mice in FO diet group recovered quicker (within 30 minutes) than the mice in SF diet group. These observations illustrate an over-all protective effect of fish oil from LPS-induced ill health.
After 60 days of feeding, FO diet group had a 17.7% decrease in fasting blood glucose compared to SF diet group (P < 0.01) suggesting overall protection of FO from hyperglycemia. Flachs P et al  reported a ≈4% decrease in blood glucose in mice fed a FO diet. Rossmeisl et al  reported a blood glucose decrease of ≈19.2% in mice fed DHA diet compared to mice fed a corn oil diet for 4 months. In contrast, Samane et al  reported no significant decrease in fasting blood glucose. It is likely that the differences in results in between these studies may be due to differences in duration of feeding and amount of FO present in experimental diets.
Although insulin concentration did not reach statistical significance, there was higher insulin concentration in SF group compared to FO group after mice were challenged with LPS (P < 0.06). FO diet group compared to SF diet group had lower HOMA-IR values after 60 day feeding and after LPS challenge. The observation of improved insulin sensitivity in rodents fed FO diet compared to rodents fed SF diet has been previously reported . Differential effects of saturated and n-3 fatty acids on TLR-4 signaling may explain the variation in insulin resistance. Saturated fatty acids and LPS induce dimerization and recruitment of TLR-4 to the membrane which are essential for TLR-4 signaling. In contrast, DHA of FO inhibits the dimerization and recruitment of TLR-4 into the membrane  leading to attenuated TLR-4 signaling. In vivo studies have reported that FO is incorporated into the plasma membrane where it inhibits downstream signaling of TLR-4 [41, 42] leading to decreased insulin resistance [43–45].
Obesity and DM2 are characterized by elevated circulating concentrations of inflammatory markers . These inflammatory markers originate from adipose tissue . In diet-induced obesity, macrophages infiltrate into white adipose tissue and these infiltrated macrophages produce inflammatory markers such as IL-1β [48, 49]. We found a protective effect of FO against IL-1β, a potent inflammatory marker, in LPS-challenged mice. Several studies have demonstrated that consumption of FO decreased production of IL-1β from mononuclear cells [50–52]. Eevated IL-1β promotes insulin resistance . IL-1β is produced from large pro-inflammatory protein complexes called inflammasomes . These are part of innate immune system activated by various microbial stimuli. Other mechanisms through which FO exerts anti-inflammatory properties include displacement of arachidonic acid in the plasma membrane leading to the synthesis of anti-inflammatory PGE3 and LTB5 instead of PGE2 and LTB4, respectively . Recent studies revealed that n-3 fatty acids produce E- and D-series resolvins [56–58] which are found to be potently anti-inflammatory .
There is a possibility that the overall beneficial effect of FO against LPS-induced ill health that we found in our study may also be due to mechanisms unrelated to TLR-4 and NRLs. Although n-3 fatty acids from FO are known to antagonize the effects of LPS-induced TLR-4 signaling, the protection of FO diet could potentially be due to increased production of anti-inflammatory components of n-3 fatty acid-derived eicosanoids. Based on the findings from this study, consumption of a diet rich in n-3 fatty acids from marine sources is beneficial against the deleterious effects of Gram negative bacterial infection.