High levels of n-6 PUFA in the Western diet could potentiate inflammatory response and may induce NASH. In the present study by mimicking typical Western diet, we investigated the impact of substitution of n-6 PUFA with n-3 PUFA (varying n-6:n-3 ratio) on the development of NASH. The n-6:n-3 ratios were chosen on the basis of our previous studies, wherein we have investigated the impact of varying n-6:n-3 ratios of 200, 50, 10, 5 and 2, representing wide range of n-6 and n-3 PUFA in the present human diet, on sucrose induced insulin resistance29,30. The results of the aforementioned studies demonstrated that compared to n-6:n-3 ratios of 200, 50 and 10, substitution of LA with ALA (n-6:n-3 ratio of 2) or LC n-3 PUFA (n-6:n-3 ratio of 5) prevented sucrose induced insulin resistance and dyslipidemia. Hence, n-6:n-3 ratios of 200 (representing high n-6 PUFA and n-3 PUFA deficient), 2 and 5 were selected. The results of the study, for the first time, demonstrated that partial replacement of LA with ALA (n-6: n-3 ratio of 2) or LC n-3 PUFA (n-6:n-3 ratio of 5) attenuated Western diet induced NASH. The protective effect of n-3 PUFA supplementation include attenuation of insulin resistance and glucose intolerance, optimization of plasma and liver lipid levels, mitigation of hepatic oxidative stress and inflammation, and improvements of aminotransferase activities and histological score.

It is established that NAFLD is the hepatic manifestation of metabolic syndrome and insulin resistance may play a key role in the development and progression of NAFLD43. However, whether insulin resistance is the cause or consequence of NAFLD is yet to be established44. Elevated levels of aminotransferase, which is the marker of liver injury has been reported in NASH subjects with insulin resistance, suggesting that insulin resistance may be involved in the progression of NAFLD45,46. Increased visceral adiposity by facilitating adipose tissue lipolysis and ectopic fat accumulation induces insulin resistance. Although obesity is the major risk factor for NAFLD, it is not rare in individuals with normal BMI particularly in Asian population47. Several animal studies demonstrated that both LC n-3 PUFA and ALA prevented high fat or high sucrose diet induced insulin resistance by reducing visceral adiposity48,49. Our earlier studies also showed that substitution of linoleic acid with ALA (n-6:n-3 ratio of 2) or LC n-3 PUFA (n-6:n-3 ratio of 5) prevented sucrose induced insulin resistance by increasing peripheral insulin sensitivity29,30. In the present study, rats fed HFHF diet increased visceral adiposity without altering the body weight. Also, substitution of n-6 PUFA with n-3 PUFA in HFHF diet effectively prevented the insulin resistance and glucose intolerance. The protective effect of n-3 PUFA supplementation was associated with decrease in visceral adiposity.

High level of fructose and n-3 PUFA deficiency in the Western diet has been implicated in the development of NAFLD50. Hepatic lipogenesis is modulated by SREBP-1c, which is the key transcription factor of de novo lipogenesis and regulates downstream genes such as SCD-1, ACC and FAS. SREBP-1c plays a crucial role in the development of NAFLD51,52. Indeed, Yamada et al. demonstrated that in patients with NASH, the gene expression of hepatic SREBP-1, SCD-1, FAS and PPARγ were enhanced53. In addition, C18:1/C18:0 ratio was associated with the steatosis score whereas C16:1/C16:0 ratio was associated with lobular inflammation score53. Several studies have shown that the fructose component of the Western diet is responsible for the de novo lipogenesis by up regulating SREBP-1c54. It is well documented that dietary n-3 PUFA prevents hepatic steatosis by down regulating SREBP-1c and up regulating PPAR-α which regulates genes involved in fatty acid oxidation55,56. In the present study, HFHF feeding induced hepatic steatosis by up regulating the hepatic mRNA expression of SREBP-1c and SCD-1 without altering PPAR-α expression. Additionally, the desaturation index C16:1/C16:0 and C18:1/C18:0, which is used to estimate the SCD-1 activity was significantly increased which further supports the involvement of SCD-1 in hepatic steatosis. Substitution of n-6 PUFA with n-3 PUFA (ALA or LC n-3 PUFA) in HFHF diet prevented hepatic steatosis by down regulating the expression of SREBP-1c and SCD-1. Decreased C18:1/C18:0 and C16:1/C16:0 ratios in liver by n-3 PUFA supplementation further confirms the anti-steatotic and anti-inflammatory role of n-3 PUFA. Studies have shown that among the LC n-3 PUFA, DHA is the potent regulator of SREBP-1c, whereas EPA is PPAR- α activator55. The observed anti-steatotic effect of ALA supplementation could be due to its conversion to DHA and EPA. Recently, Monteiro et al. demonstrated that using delta-5 and delta-6 knockout mice, ALA supplementation prevents hepatic lipogenesis and the development of steatosis57, suggesting that ALA could bring about its anti-steatotic effect which is independent of its conversion to LC n-3 PUFA.

Besides significant reduction in hepatic steatosis, n-3 PUFA supplementation also prevents HFHF induced dyslipidemia. Several animal and human studies suggest that dietary n-3 PUFA (ALA and LC n-3 PUFA) decrease the risk factors associated with metabolic syndrome including dyslipidemia58,59. It is worthy to mention that both ALA and LC n-3 PUFA are equally effective in preventing HFHF induced dyslipidemia.

According to multiple hit hypothesis of NAFLD pathogenesis, oxidative stress is considered as a key contributor to the transition from simple steatosis to NASH and fibrosis60. The excessive production of reactive oxygen species due to mitochondrial dysfunction causes lipid peroxidation which in turn induces inflammation and activation of stellate cells leading to fibrogenesis. Indeed, elevated levels of markers of oxidative stress and lipid peroxidation have been reported in patients with NASH60. In the present study, HFHF feeding induced oxidative stress as evidenced by increase in liver TBARS and decrease in antioxidant enzyme activity (catalase and SOD) and GSH. Due to high degree of unsaturation, there is a concern that n-3 PUFA particularly LC n-3 PUFA may induce oxidative stress and lipid peroxidation. However, emerging evidence indicate that although high levels of LC n-3 PUFA induce oxidative stress, moderate/appropriate dose of n-3 PUFA exerts antioxidant effect61. In fact, a recent study showed that LC n-3 PUFA supplementation decrease hepatic oxidative stress and triglyceride content in high fat diet induced fatty liver25. In the present study, both ALA and LC n-3 PUFA supplementation prevented the HFHF induced hepatic oxidative stress by improving the antioxidant status through restoring the antioxidant enzyme activities and GSH level. HO-1 is an inducible antioxidant enzyme and plays an important role in the cytoprotection and tissue injury62. Interestingly, n-3 PUFA supplementation up regulated the mRNA expression of HO-1 although HFHF feeding per se did not alter its expression. Recent in vitro studies have shown that LC n-3 PUFA prevents oxidative stress by up regulating HO-1 through activation of nuclear factor erythroid 2 related factor (Nrf-2)63. Further studies are necessary to understand the molecular mechanism by which LC n-3 PUFA modulates the antioxidant enzymes particularly in NAFLD. Oxidative stress triggers inflammatory process by activating redox - sensitive transcriptional factor, NF-κB thereby causing necroinflammation leading to NASH. Rats fed HFHF diet showed up regulation of proinflammatory cytokines with the development of NASH and n-3 PUFA supplementation has been shown to prevent NASH by suppressing the inflammatory response as evidenced by down regulation of proinflammatory cytokines. Tapia et al. showed the LC n-3 PUFA supplementation prevents high fat induced hepatic steatosis and inflammation by down regulating NF-κB23. Furthermore, a recent randomized clinical trial demonstrated that combined ALA and LC n-3 PUFA supplementation in NASH patients decreased plasma lipids with improvements in liver histology64. In addition to down regulating the proinflammatory cytokines, the anti-inflammatory effect of n-3 PUFA supplementation may also be mediated by resolvins and protectins derived from EPA and DHA which are known to resolve the inflammation65.

Dysregulation of adipocytokines by orchestrating proinflammatory and insulin resistance state may contribute to the development and progression of NAFLD. Leptin is known to induce insulin resistance, hepatic steatosis and has proinflammatory role and also contributes to fibrosis, whereas, adiponectin has anti-inflammatory and insulin sensitizing effect. Several clinical studies demonstrated that circulating leptin, TNF-α and IL-6 were significantly higher in patients with NAFLD/NASH, conversely adiponectin levels were significantly reduced in NAFLD/NASH patients66. However, the role of resistin which is proinflammatory in NAFLD is inconclusive. Lemoite et al. showed that in patients with NAFLD the adiponectin:leptin ratio is inversely related to the severity of NAFLD and proposed as predictive factor of NASH67. There is evidence that LC n-3 PUFA modulates the adipocytokines by increasing circulatory adiponectin and decreasing leptin levels68. Our study showed that both ALA and LC n-3 PUFA supplementation decreased plasma leptin and resistin levels and increased adiponectin:leptin ratio, suggesting that by correcting adipocytokine imbalance, n-3 PUFA ameliorates Western diet induced NASH.

ALA undergoes series of complicated desaturation and chain elongation pathway and gets converted into biologically active LC n-3 PUFA. High intake of LA and hence high LA:ALA ratio could inhibit the conversion of ALA. Studies have suggested that substituting LA with ALA would be the most appropriate way of optimizing the conversion of ALA to LC n-3 PUFA69. In the present study, substituting LA with ALA (n-6: n-3 ratio of 2) has been shown to increase the incorporation of LC n-3 PUFA at the expense of LC n-6 PUFA in liver phospholipids, suggesting the competitive interaction and inhibition of the desaturation and elongation of LA to LC n-6 PUFA and preferential conversion of ALA to LC n-3 PUFA.

In conclusion, the results of the present study demonstrated that substitution of dietary linoleic acid with α-linolenic acid (n-6:n-3 ratio of 2) or LC n-3 PUFA (n-6:n-3 ratio of 5) protects against the development of Western diet induced NASH as evidenced by improved liver histology, decreased liver and plasma lipids and reduced plasma aminotransferase levels. The protective effect of n-3 PUFA supplementation was attributed to the marked reduction in hepatic oxidative stress and proinflammatory cytokines. The present study also highlights the importance of balancing the n-6 and n-3 PUFA in the diet through suitable blending of vegetable oils for the prevention and management of diet related chronic diseases including NAFLD.