non - alcoholic fatty liver disease ( nafld ) is characterized by pathological fat accumulation in hepatocytes in the absence of excessive alcohol consumption , which comprises a wide spectrum of liver disease ranging from steatosis to steatohepatitis , cirrhosis and even hepatocellular carcinoma .
it is clear that nafld is strongly associated with insulin resistance , obesity , dyslipidemia and diabetes but its pathogenesis is not fully understood .
according to the  two hit  hypothesis , hepatic steatosis and insulin resistance occur first because of an increased uptake of lipids and/or decreased fatty acids oxidation , inadequate de novo synthesis of triglycerides , and/or decreased synthesis or secretion of very low density lipoproteins ( vldls ) in liver .
this induces a chronic inflammatory condition characterized by the release of pro - inflammatory cytokines and oxidative stress .
both of which are responsible of the  second hit  , which induces the progression from steatosis to more advanced stages of liver damage .
this theory , however , should be revised since the increased ratio of saturated - to - un - saturated fatty acids within the liver directly induce hepatic inflammation and insulin resistance , which may worsen nafld progression . currently , the first - line treatment of nafld is based on diet and lifestyle modification
. studies of the dietary habits of nafld patients showed low intake of n-3 poly - unsaturated fatty acids ( pufas ) and/or a lower relative concentration of n-3 pufas in tissues and blood [ 46 ] .
indeed , insufficient intake of n-3 pufas is associated with increased risk of nafld independent of traditional risk factors and data strongly support the detrimental role of high n-6/n-3 ratio on metabolic parameters .
deficiency of n-3 pufas leads to hepatic steatosis and insulin resistance , whereas dietary supplementation of n-3 pufas appears to safely reduce nutritional hepatic steatosis in adults .
the present review discusses the potential mechanisms through which n-3 pufas may show its benefits in nafld , and the current data supporting its use .
fatty acids , essential components of all cell membranes , play a number of key roles in metabolism such as being a major metabolic fuel ( storage and transport energy ) , and the ligands for transcription factors . although animals can synthesize saturated fatty acids and some monounsaturated fatty acids from carbon groups in carbohydrates and proteins , they lack the enzymes , desaturases , which are necessary to insert a cis double bond at the n-6 or the n-3 position of a fatty acid .
consequently , linoleic acid ( la , c18:2 ; n-6 ) and alpha - linolenic acid ( ala , c18:3 ; n-3 ) are essential nutrients that must be taken from food .
human can synthesize long - chain n-6 pufas , such as arachidonic acid ( aa ; c20:4;n-6 ) from la and long - chain n-3 pufas , such as eicosapentaenoic acid ( epa ; c20:5 ; n-3 ) and docosahexaenoic acid ( dha ; c22:6 ; n-3 ) from ala ( fig .
1 ) . however , epa and dha have to be obtained from diet because mammals inefficiently convert ala to epa and dha .
n-3 pufas are enriched in fish oil , flaxseed and some nuts whereas n-6 pufas are found predominantly in grain and vegetable oil .
it has been estimated that the ratio of n-6 to n-3 pufas in the diet of early humans was 1:1 , but the ratio in the typical western diet is now almost 10:1 due to increased use of vegetable oils rich in la as well as reduced fish consumption .
a lower intake in dietary sources of n-3 pufas seems to be associated with nafld .
in fact , patients with hepatic steatosis present a lower n-3/n-6 pufa ratio in liver tissue biopsies , namely in phospholipids subfractions , and in red blood cells .
health benefits of dietary n-3 pufas include decrease of triglyceridemia and hepatic steatosis in human and rodent models of obesity .
indeed , n-3 pufas supplementation lessened the hepatic steatosis in ob / ob mice [ 1316 ] and in rats fed a high - fat diet ( hfd ) .
in addition to improvement in hepatic steatosis , these rodent models had lowered plasma alanine aminotransferase ( alt ) and aspartate aminotransferase ( ast ) levels .
the lipid - lowering effect of n-3 pufas in rats fed high fat / cholesterol has been associated with the downregulation of de novo lipogenesis and upregulation of lipid oxidation .
lipogenesis is thought to be regulated via the transcription factors carbohydrate response element binding protein ( chrebp ) and sterol regulatory element binding protein-1c ( srebp-1c ) , which are activated by glucose and insulin , respectively . on the contrary , peroxisome proliferator activated receptor  ( ppar ) is a transcription factor known to decrease plasma lipids and induce enzymes and other proteins involved in lipid oxidation within the mitochondria , microsomes and peroxisomes .
the increased hepatic srebp-1c / ppar ratio and steatosis are strongly correlated with insulin resistance and n-3 pufa depletion in obese patients .
administration of n-3 pufas to obese patients and nafld - prone rodents decreased the nuclear abundance of chrebp / srebp-1c while induced ppar in the liver .
furthermore , the expression of chrebp / srebp-1c - dependent , glycolytic / lipogenic enzymes were reduced , while that of ppar-dependent , oxidative enzymes were increased by n-3 feeding .
fish oil feeding is also associated with elevated concentration of adiponectin in the circulation and visceral adipose tissue .
adiponectin , one of the insulin - sensitizing adipokines , has been shown to reduce hepatic lipids by activating fatty acid oxidation and inhibiting hepatic lipogenesis in liver .
specifically , nafld patients have lower levels of adiponectin whereas fish oil supplementation to the obese increases adiponectin in most [ 2931 ] , but not all studies . besides the function on metabolic homeostasis
, there is evidence from several studies that n-3 pufas have also shown the anti - inflammatory and anti - oxidant effects to protect the cells from ros - induced damage .
overall , these results suggest that n-3 pufas reduce hepatic lipids , markers of inflammation , and improve insulin sensitivity , all major events in nafld development and/or progression .
although increasing the ratio of dietary n-3 to n-6 pufas might be beneficial in the prevention and treatment of dyslipidemia , obesity , insulin resistance , nafld and diabetes , it is difficult to ascertain the contribution of certain n-3 pufas per se , without the potential confounding effects of other dietary components . since no pure n-3 and n-6 fatty acids are available , the major sources of the required n-3 and n-6 pufas are from fish oils and plant seed / vegetable oils , respectively .
these two kinds of oils contain different bioactive compounds , such as saturated fatty acids , monounsaturated fatty acids , cholesterol , anti - oxidants , contaminants and other unidentified substances , that affect the study outcomes .
indeed , while it is generally accepted that fish oil improves the serum lipid profile , it is controversial whether dietary flaxseed oil has a similar effect . studying the effects of dietary n-3 pufas in the context of high fat feeding has proven to be rather complicated because incorporation of n-3 pufas into rodent diets often prevents weight gain .
therefore it is ideal to develop a transgenic mouse capable of converting n-6 to n-3 pufas so that the results obtained in such an animal model will be more reliable and easy to interpret in terms of the effects of n-3 and n-6 pufas .
generated fat-1 transgenic ( fat-1 ) mice expressing the caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase that converts n-6 pufas to n-3 pufas ( fig .
1 ) . this allows production of two different fa profiles ( low vs high n-6/n-3 ratios ) in the fat-1 mice and wild - type littermates by using just a single diet , thus eliminating the potential diet variations .
hence , the fat-1 mice are valuable in vivo system for elucidating the role of n-3 pufas and the n-6/n-3 ratio in nafld development and progression .
studies on fat-1 mice yielded interesting results including reduction of inflammation , bone loss , colitis , colon cancer , melanoma growth , invasiveness of lung cancer cells , and seizure susceptibility [ 3742 ] .
white et al .   reported that transgenic restoration of n-3 pufas in insulin target tissues improved resolution capacity and alleviated obesity - linked inflammation and insulin resistance in hfd - fed mice .
the authors proposed that the endogenous n-3 pufas exerted their protective effects through their lipid oxygenation products , which reduced macrophage infiltration and inflammation in the expanding adipose tissue of obese mice .
however , the hepatic lipid accumulation was not changed in both hfd - fed wild - type and fat-1 mice , showing different results from dietary n-3 pufas supplementation reversed hepatic steatosis in ob / ob mice .
we have recently reported that endogenously synthesized n-3 pufas could ameliorate hfd - induced fatty liver and hyperlipidemia in fat-1 mice , leading to significant attenuation of nafld and hepatic injury , as reflected by significant decreases in either triglyceride ( tg ) or liver enzymes , including ast and alt .
protection against hyperlipidemia in fat-1 mice was associated with significant upregulation of genes involved in cholesterol uptake ( ldlr ) , bile acid metabolism ( cyp7a1 ) or excretion ( abcg5 and abcg8 ) , and downregulation of apoa4 , a component of chylomicrons and hdl .
these results suggested that endogenously synthesized n-3 pufas ameliorated fatty liver and hypercholesterolemia through modulating cholesterol and bile acid metabolism .
recently , cholesterol accumulation is proposed to participate to the pathogenesis of nafld and non - alcoholic steatohepatitis .
the sole pathways for hepatic cholesterol elimination are bile acid synthesis and cholesterol exported into the bile .
cyp7a1 is the rate - limiting enzyme in the bile acid synthetic pathway that converts cholesterol into bile acids in the liver .
recent studies have shown that transgenic mice overexpressing cyp7a1 were protected against hfd - induced hypercholesterolemia , obesity , and insulin resistance . moreover ,
the hepatic cholesterol transporters abdg5 and abcg8 were significantly induced in cyp7a1-tg mice , as shown in fat-1 mice .
therefore , the findings regarding to the regulation of ldlr and cyp7a by endogenous n-3 pufas paralleled its effects on circulating cholesterol , tg , and ldl cholesterol levels during hfd treatment and provided mechanistic evidence for decreased cholesterol accumulation and increased hepatic uptake of circulating ldl cholesterol .
although simple steatosis is relatively benign and principally reversible , steatohepatitis is the progressive form of nafld and can develop cirrhosis , hepatic failure , and hepatocellular carcinoma .
the only approved management of nafld is lifestyle modification including increased physical activity , dietary behaviors , and weight reduction .
dietary n-3 pufas reduce hepatic steatosis , inflammation and oxidative stress and improve insulin sensitivity .
studies on fat-1 mice revealed that endogenously synthesized n-3 pufas ameliorated fatty liver and hypercholesterolemia resulting from enhanced hepatic cyp7a1 expression .
these findings underscore the importance of endogenous n-3 pufas to maintain whole - body bile acid homeostasis , which plays a key role in lipid , glucose , and energy homeostasis . to confirm the clinical relevance of the preventive effect of exogenous n-3 pufas on nafld , the physiological concentration of n-3 pufas required to produce similar effects on the fatty livers of fat-1 mice should be determined . for this purpose
it will be helpful to investigate the concentrations of n-3 pufas in the liver after administration of various doses exogenous n-3 fas in wt mice compared to fat-1 mice .
 non - alcoholic fatty liver disease ( nafld ) is one of the most common causes of chronic liver disease that affects one - third of adults in westernized countries . 
 nafld represents a wide spectrum of hepatic alterations , ranging from simple triglyceride accumulation in the liver to steatohepatitis . 
 several pharmaceutical approaches to nafld management have been examined , but no particular treatment has been considered both safe and highly effective . growing evidence 
 reveal that supplemental fish oil , seal oil and purified n-3 fatty acids can reduce hepatic lipid content in nafld through extensive regulation by inhibiting lipogenesis , promoting fatty acid oxidation and suppressing inflammatory responses . 
 recently , the fat-1 transgenic mice capable of converting n-6 to n-3 polyunsaturated fatty acids ( pufas ) have been used to examine the effects of endogenous n-3 pufas on nafld . 
 the increased n-3 pufas in fat-1 transgenic mice reduced diet - induced hyperlipidemia and fatty liver through induction of cyp7a1 expression and activation of cholesterol catabolism to bile acid . 
 this article introduces the n-3 pufas , and addresses the evidence and mechanisms by which endogenously synthesized n-3 pufas or increased dietary n-3 pufas may ameliorate nafld . 
INTRODUCTION
METABOLISM OF n-3 PUFAs
THE EFFECTS OF DIETARY n-3 PUFAs AND UNDERLYING MECHANISMS
THE 
ENDOGENOUSLY SYNTHESIZED n-3 PUFA EFFECTS ON NAFLD
CONCLUSION