Omega-3 Polyunsaturated Fatty Acids for Treatment of Nonalcoholic Fatty Liver Disease: A Possible Case for Personalized Therapy

In a recent issue of PLOSone Depner et al. [1] reported an excellent study on omega-3 fatty acid-induced changes in lipid metabolomics that may be involved in attenuating western diet-induced nonalcoholic steatohepatitis (NASH). A western diet, which is typically rich in saturated and trans fats, that leads to fat accumulation in the liver. This condition resembles alcoholic fatty liver disease; however, it progresses in the absence of any alcohol intake and is known as nonalcoholic fatty liver disease (NAFLD) [2]. In most individuals, the liver is devoid of any symptoms or problems that will only culminate in mild steatosis. However, in some cases, especially individuals with obesity and metabolic syndrome, the fat accumulation is accompanied with inflammation and causes NASH. If untreated, NASH worsens and causes fibrosis resulting in scarring of the liver. The pathophysiologic pathway of NAFLD is not clearly elucidated. Day and James [3] in 1998 proposed the “two-hit phenomenon,” where the “first hit” consists of triglyceride (TG) depositions in hepatocytes causing cellular dysfunction and death (lipotoxicity). The hyperinsulinemia and insulin resistance play a role in the first hit causing an alteration in uptake, synthesis, degradation, and secretion of free fatty acids leading to an accumulation of fat in hepatocytes, or steatosis [4,5]. Steatosis by itself is not detrimental, but the steatotic liver becomes more vulnerable to “second hits” by inflammatory cytokines, endotoxins, and oxidative stress causing mitochondrial dysfunction, which ultimately results in hepatocyte injury and fibrosis [6]. NAFLD is also associated with an increased risk of cardiovascular disease (CVD). To date there is no standardized optimal treatment, although several approaches to treat NAFLD have been adopted, including weight reduction, limiting dietary carbohydrate intake, inhibiting fat absorption by sibtramine and orlistat, lowering lipid profile by statins, improving insulin sensitivity by Metformin and thioglitazone and reducing oxidative stress by vitamin E [7-9].


Omega-3 Polyunsaturated Fatty Acids for Treatment of Nonalcoholic
In a recent issue of PLOSone Depner et al. [1] reported an excellent study on omega-3 fatty acid-induced changes in lipid metabolomics that may be involved in attenuating western diet-induced nonalcoholic steatohepatitis (NASH). A western diet, which is typically rich in saturated and trans fats, that leads to fat accumulation in the liver. This condition resembles alcoholic fatty liver disease; however, it progresses in the absence of any alcohol intake and is known as nonalcoholic fatty liver disease (NAFLD) [2]. In most individuals, the liver is devoid of any symptoms or problems that will only culminate in mild steatosis. However, in some cases, especially individuals with obesity and metabolic syndrome, the fat accumulation is accompanied with inflammation and causes NASH. If untreated, NASH worsens and causes fibrosis resulting in scarring of the liver. The pathophysiologic pathway of NAFLD is not clearly elucidated. Day and James [3] in 1998 proposed the "two-hit phenomenon," where the "first hit" consists of triglyceride (TG) depositions in hepatocytes causing cellular dysfunction and death (lipotoxicity). The hyperinsulinemia and insulin resistance play a role in the first hit causing an alteration in uptake, synthesis, degradation, and secretion of free fatty acids leading to an accumulation of fat in hepatocytes, or steatosis [4,5]. Steatosis by itself is not detrimental, but the steatotic liver becomes more vulnerable to "second hits" by inflammatory cytokines, endotoxins, and oxidative stress causing mitochondrial dysfunction, which ultimately results in hepatocyte injury and fibrosis [6]. NAFLD is also associated with an increased risk of cardiovascular disease (CVD). To date there is no standardized optimal treatment, although several approaches to treat NAFLD have been adopted, including weight reduction, limiting dietary carbohydrate intake, inhibiting fat absorption by sibtramine and orlistat, lowering lipid profile by statins, improving insulin sensitivity by Metformin and thioglitazone and reducing oxidative stress by vitamin E [7][8][9].
Tissue biopsies from the liver of NAFLD/NASH patients show an accumulation of saturated and monounsaturated fatty acids (MUFAs) and a depletion of polyunsaturated fatty acids (PUFA) in TG and phospholipids (PL) [10]. The depletion of n-3 PUFA is typically more severe than for n-6 PUFA due to decreased elongation and desaturation of alpha-linoleic acid, the essential precursor fatty acids and because of decreased dietary intake [11]. In animal models, an association between n-3 PUFA deficiency and the development of hepatic steatosis was reported [12][13][14]. Several cross-sectional and case-control studies have also implicated the reduced dietary n-3 PUFA intake as a cause for NAFLD development [15][16][17][18]. Recent compelling evidence in the literature suggests that n-3 PUFA, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can be more beneficial than existing pharmaceutical agents for the treatment of NAFLD/NASH [17,[19][20][21]. Studies have shown that n-3 PUFA are involved in the control of glucose homeostasis and specifically affect the development of IR and NAFLD progression [22]. Dietary n-3 PUFA affects hepatic lipid homeostasis through imparting both lipid synthesis and lipid oxidation. n-3 PUFA have been shown to suppress lipogenesis by downregulating the activity of SREBP-1c and of carbohydrateresponsive element-binding protein (ChREBP) [23,24], a glucoseresponsive transcription factor that is linked to IR and hepatic steatosis. In addition, n-3 PUFA are natural ligands of PPAR-α receptors that increases hepatic mitochondrial β oxidation, and suppress de novo lipogenesis by increasing the transcription of fatty acid degradation genes [25]. Furthermore, EPA gives rise to E-resolvins and DHA gives rise to D-resolvins and protectins, which are inflammation-resolving cytokines and therefore play a critical role in suppressing oxidative stress and inflammation [26]. The suppressive effects of n-3 PUFA on inflammation, oxidative stress, and TG accumulation in the liver make them possible effective candidates for NAFLD/NASH. However, the molecular mechanism/s for n-3 PUFA to ameliorate NAFLD/ NASH is still obscure. Although data presented by Depner et al. [1] do not establish a cause and effect relationship, the lipidomic data further establishes the potential of n-3 PUFA as a therapeutic agent for NAFLD/NASH.
Depner et al. [1] performed experiments on LDLR-/-mice and fed them western diet with olive oil (WD + O), EPA (WD + E), DHA (WD + D) or both (WD + E + D) and measured changes in metabolomics. The WD + O diet induced classical NASH markers of hepatic damage, including inflammation, oxidative stress, and fibrosis. This analysis identified 524 total metabolites; 320 known and 204 unknown identities. The known metabolites belonged to 8 major pathways including amino acid, carbohydrate, energy, lipid, nucleotide, peptide, vitamins and cofactors, and xenobiotics. The metabolites associated with lipid and amino acid pathways were most affected by diet. Dietary supplementation with EPA and/or DHA attenuated many of the WD + O-induced effects; however, WD + D was found to be better than WD + E at reversing WD + O effects on the liver, Feeding mice the WD + O led to an accumulation of palmitoyl-sphingomyelin, sphinganine, sphingosine, MUFA (18:1,n-9 and 18:1,n-7), n-6 PUFA (20:4, n-6), and alpha-tocopherol (vitamin E), whereas this diet reduced hepatic n-3 PUFAs (EPA, DHA) and oxidized lipids derived from n-3 PUFA (18-hydroxyeicosapentaenoic acid [18-HEPE] and 17,18-dihydroxyeicosatetraenoic acid [17,). WD + O diet was also associated with the loss of S-lactoylglutathione, a detoxification product of methylglyoxal (MG); MG is involved in forming advanced glycation end products (AGEP) [27] and promotes NASH [28]. The authors reported that these changes in hepatic metabolites were associated with hepatic damage (ATL and AST) and the induction of multiple gene expression markers of NASH, including MUFA synthesis (SCD1), inflammation (MCP1, CD68, and TLR4), oxidative stress (HMOX-1 & NOX2) and fibrosis (proCOL1A1). In contrast, WD + D feeding decreased hepatic MUFA, n-6 PUFA, n-6 PUFA derived oxidized lipids, palmitoyl-sphingomyelin and α-Tocopherol. Changes in hepatic content of these metabolites were associated with a corresponding decline in the NASH gene expression markers (i.e., MCP1, CD68, ProCOL1A1, NOX2, SCD1 and TLR4). The authors conclude that DHA likely attenuated MUFA synthesis by decreasing fatty acid synthase (FASN), ATP-citrate lyase (ACL) and SCD1 expression. Their results suggest that DHA-containing diets regulate hepatic 16:0, 18:1,n-7, and 18:1,n-9 content by controlling multiple genes involved in DNL and MUFA synthesis as well as the availability of substrates required for DNL.
One interesting finding was the observed changes in lysophospholipids (lyso-PLs), revealing significant changes in oleic, linoleic, and arachidonic acid but not DHA-containing lyso-PLs. The metabolomic analysis identified a total of 42 lysophospholipids with 26 lyso-PLS with acyl chains in the sn-1 position, and 16 identified lyso-PLs with acyl chains in the sn-2 position. 1oleylphosphoethanolamine, 1-arachidonylphosphocholine, and 1-arachidonylphosphoethanolamine were increased significantly by the WD + O diet, while diets containing EPA or DHA decreased these lysophospholipids. Hepatic 2-oleoylglyceroethanolamine was increased significantly while levels of 2-linolenoylphosphoethanolamine were suppressed by WD + O. DHA or EPA diet has no effect on these lyso-PLs. Levels of 2-arachidonylphosphocholine and 2-arachidonylphosphoethanolamine, were suppressed by DHA and EPA diets. As suggested by the authors, changes in lysoPLs composition represent either de novo phospholipid synthesis or remodeling of membrane phospholipids.
As reported by others, this study also presents the protective effects of n-3 PUFAs by modulating oxidative stress. The metabolomic analysis identified several oxidized PUFAs generated from 18:2,n-6, 20:4,n-6 and 20:5,n-3. The WD + D diet was the most effective diet at suppressing WD-mediated accumulation of proinflammatory oxidized lipids derived from n-6 PUFA. Interestingly, two n-3 PUFA-derived anti-inflamamtory oxidized lipids, 18-HEPE and 17,18-DiHETE, were detected. The data is consistent with the protective effects of oxidized n-3 PUFA metabolities in liver. Other studies have shown that n-3 PUFAs change cardiolipin content in liver mitochondrial membranes, thus affecting the fatty acid composition and PL class content of the membranes and influencing the activities of some of the respiratory chain complex enzymes [29]. Furthermore, livers from WD + n-3 PUFA groups had reduced vitamin E levels. We have also found similar results of vitamin E depletion on fish oil based emulsion infusion in guinea pigs (unpublished data). This may be due to utilization of hepatic Vitamin E by the abundance of EPA and DHA.
Although the authors describe several limitations to their studies, the data demonstrates that n-3 PUFA consumption changes carbohydrate and lipid metabolism, which is clearly linked to reduced TG deposits and hepatic inflammation. This work further provides evidence for the potential use of n-3 PUFAs for treating NALD/NASH. n-3 PUFAs are widely accepted as beneficial nutritional supplements, especially for their cardioprotective effects. It is becoming more convincing that patients at risk of obesity, metabolic syndrome, diabetes, and cardiovascular disease will greatly benefit from the regular use of n-3 PUFAs. EPA and DHA can be obtained either through diet (e.g., fatty fish) or through fish oil supplements. The American Heart Association , American Diabetes Association, and The World Health Organization recommend an average of 500 mg/day of EPA/DHA intake or at least two servings of fatty fish weekly [30,31]. Although these suggestions are based on various epidemiological studies for disease prevention, there is not enough data to suggest an optimal dose and duration for n-3 PUFA for therapeutic purposes for NAFLD/NASH. The amount of n-3 PUFAs and their chemical form (TG, PL, and free FA) varies greatly among marine animals and marine oil-based supplements. A number of pharmaceutical companies are now marketing standardized commercial preparations of n-3 PUFA products, eliminating batchto-batch variation in n-3 PUFA content. Federal Drug Administration (FDA) has approved highly purified DHA and EPA ethyl ester (Lovaza) and EPA ethyl ester (vescepa) to reduce high TG levels in adults. In a recent study these n-3 PUFA preparations are compared; both n-3 PUFA products decreased TG but DHA-containing supplements have been associated with a significant increase in LDL-C [32]. However, these products do not show additional reduction in adverse cardiovascular events.
Often clinical studies are based on uniform doses. It is perhaps too early to recommend a uniform dose of n-3 PUFA to NAFLD/NASH patients. The effect of n-3 PUFAs may vary from individual to individual because of their metabolic and genetic variations. Furthermore, individuals with NAFLD/NASH may possibly have variable stages of disease and hepatic accumulation of TG. In addition, dietary habits also vary from person to person which can also influence the effect of n-3 PUFA. Some of these conditions are difficult to control in a clinical trial. It is possible that n-3 PUFA treatment in NAFLD patients can be individualized, depending on the clinical progression of disease. This low cost and relatively safer treatment may be an attractive solution to costly and toxic alternative that are currently available.