Oils rich in linolenic acid independently protect against characteristics of fatty liver disease in the 6desaturase null mouse

Alpha linolenic acid (ALA) biological activity is poorly understood and primarily associated with its conversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Delta 6 desaturase (D6D) initiates the metabolism of linoleic acid (LA) and ALA to arachidonic acid, EPA, and DHA, respectively. In this study, D6D knock-out (D6KO) mice were used to evaluate the effects of ALA rich oils in preventing hepatic steatosis and inflammation. D6KO and wild type mice were fed 1 of 4 high-fat (14percent w/w) diets: (i) lard (LD, 0percent n3 PUFA), (ii) canola oil + ARASCO (CD, 8percent ALA), (iii) flax seed oil + ARASCO (FD, 55percent ALA), (iv) menhaden oil (MD, 30percent EPA/DHA) for 8 or 20 weeks. Livers of D6KO mice consuming CD and FD were depleted of EPA/DHA, and enriched in ALA. Markers of fat accumulation and inflammation were lowest in the MD fed mice, at 8 and 20 weeks, regardless of genotype. CD and FD fed D6KO groups were found to have lower liver lipid accumulation and lower hepatic inflammation relative to the LD-fed mice at 8 weeks. In conclusion, while MD was the most protective, this study shows that ALA can act independently on risk factors associated with the development of fatty liver disease. (Authors abstract)

In recent years, non-alcoholic fatty liver disease (NAFLD) has emerged as the leading liver disease in North America. NAFLD is defined by the accumulation of lipids in hepatocytes, and is prevalent in individuals with metabolic abnormalities associated with Metabolic Syndrome (MetS). Low intake of polyunsaturated fatty acid (PUFA) is associated with increased prevalence of NAFLD.  The recent generation of the 6 desaturase knock out (D6KO) mouse has made it possible to study the independent effects of ALA for the first time. The D6KO mouse cannot synthesize the D6D enzyme and so is incapable of converting ALA or LA to their usual downstream fatty acid metabolites: EPA, DHA, or AA.  In this study, the D6KO mouse model was utilized to determine the effects of ALA rich diets on the phospholipid (PL) fatty acid composition in the liver, the main organ involved in PUFA metabolism, and also to determine whether ALA can independently prevent hepatic steatosis and inflammation associated with NAFLD. To further characterize the metabolic changes associated with NAFLD, systemic inflammation and weight gain were also evaluated. It was hypothesized that, similar to EPA and DHA, ALA rich diets could prevent the development of risk factors associated with the development of NAFLD in a dose dependent manner.

In the present study, the metabolic outcomes of D6KO mice supplemented with canola oil + ARASCO or flaxseed oil + ARASCO, which contain 8percent ALA or 55percent ALA respectively, were compared with D6KO control animals supplemented with either lard, which contains negligible n3 PUFA (negative control), or menhaden oil, which is rich in EPA and DHA (positive control). Eight weeks after supplementation began, it was confirmed that the livers of D6KO mice fed canola or flaxseed oils contained significantly less EPA and DHA relative to WT animals.  At 8 weeks, liver lipid accumulation was significantly greater in LD and CD fed D6KO mice relative to their WT counterparts, while smaller differences in liver lipid mass relative to WT animals were observed in FD and MD fed D6KO mice . This finding supports the initial hypothesis that ALA is associated with lower liver lipid accumulation,  In agreement with liver lipid mass measurements, LD, CD, and FD fed D6KO animals had greater hepatic fat accumulation relative to WT animals. However, at both time-points, ALA rich oils were not associated with lower steatosis scores in either WT or KO animals relative to LD fed mice. Consumption of EPA and DHA provided the best protection, but nevertheless, marked differences were also observed in ALA fed mice compared with LD fed mice.  At 8 weeks, liver inflammation scores of the FD and CD fed D6KO mice were intermediate between the LD and MD fed D6KO mice. Overall, ALA rich oils were associated with reduced hepatic inflammation scores at an early stage of NAFLD; however, only EPAand DHA rich fish oil was associated with lower steatosis scores. Serum CRP values did not differ among the treatment groups. 

A diet rich in EPA and DHA provided the greatest protection against steatosis and hepatic inflammation. Protective effects were observed in animals provided with high dietary ALA, as FDfed D6KO mice consistently had lower liver lipid masses, and were slower to develop hepatic inflammation. While animals provided with the low-ALA canola diet also had reduced liver inflammation score at 8 weeks, they generally responded similarly to animals fed the n3 depleted lard diet. This suggests that the protective effects of ALA are more readily conferred at high levels of intake, but does not exclude the potential of beneficial effects from chronic long-term consumption of ALA at lower levels. Overall, this study provides evidence that ALA has independent biological activity that is not dependent upon its conversion to EPA and DHA. (Editors comments)