ω3‐PUFA Supplementation Reduced ALT Levels In Mice Exposed To A Low Linoleic Acid Diet With Or Without Ethanol In In A Drinking In The Dark Binge Model
Authors: Warner Dennis , Chen Chuck , Hibbeln Joseph , McClain Craig , Kirpich Irina 
Published: November 2023

Introduction/AimThe Western type diet is low in ω‐3 polyunsaturated fatty acids (PUFAs) and rich in ω‐6 PUFAs, which results in an increased ω‐6/ω‐3 PUFA intake ratio, typically ~10:1, whereas it has been estimated that in a Paleolithic diet, the ratio was between 2:1 – 3:1. This dietary imbalance between ω‐6 and ω‐3 PUFAs leads to multiple adverse health effects, including fatty liver disease. Thus, a reduction in dietary ω‐6 PUFAs to Paleolithic levels may have beneficial effects. Chronic and binge ethanol (EtOH) consumption leads to liver steatosis, inflammation, and injury. The aim of the present study was to test the hypothesis that lowering the dietary ratio of ω‐6:ω3 can protect mice from liver damage in a Drinking in the Dark (DID) EtOH binge exposure animal model.Materials and MethodsC57BL/6 male mice (n=7–9 animals/group) were randomized into four custom diets with varying levels of ω6 and ω3 PUFAs. Specifically, the mice were fed diets providing high or low levels of the ω6 PUFA, linoleic acid (LA, 8%E or 1%E, respectively [Hω6 or Lω6]), with or without ω3 PUFA (eicosapentaenoic and docosahexaenoic [EPA and DHA] acids) supplementation. There were 4 experimental dietary groups: Hω6:Lω3 (15:1), Hω6:Hω3 (9:1), Lω6:Lω3 (2:1), Lω6:Hω3 (1:1). After 8 weeks of dietary intervention, the mice were exposed to ethanol (20% v/v) for 6 weeks according to DID protocol: 4 hours of access to EtOH in their water during the peak drinking hours at night. At the end of the experiment, plasma alanine aminotransferase (ALT) levels (as a marker of liver injury) and fatty acid profile were evaluated. Liver RNA was extracted, and expression of genes involved in hepatic pro‐inflammatory response was examined by qRT‐PCR.ResultsThe mice fed either high or low LA diets revealed relatively similar levels of ALT activity, with the notable exception of the Lω6:Hω3 group. This low dietary LA group supplemented with EPA and DHA had a lower ALT activity with no effect of EtOH on the ALT levels. The decrease in ALT activity was correlated with a reduction in plasma linoleic and arachidonic acids and a significant increase in plasma EPA levels, independently of EtOH administration. We assessed the expression of hepatic pro‐inflammatory cytokines in order to determine a potential underlying cause for the decreased ALT in the Lω6:Hω3 group. There were no significant changes in the expression of hepatic pro‐inflammatory cytokines, including tumor necrosis factor alpha, macrophage inflammatory protein 2‐alpha, and monocyte chemoattractant protein‐1, in the absence or the presence of EtOH.ConclusionsThe data demonstrated that dietary ω3 supplementation was able to reduce ALT levels in mice fed low but not high LA diet regardless the presence or the absence of ethanol exposure, suggesting that high LA diet is more damaging to the liver health. The mechanism(s) underlying this effect remain to be determined.Support or Funding InformationSupported by the NIH and the Veterans Administration.