Lipidomic analysis of liver injury caused by a ω6‐PUFA‐enriched diet and ethanol exposure
Authors: Warner Dennis , Miller Matthew , Yin Xinmin , Wei Xiaoli , Prodhan Aminul , Zhang Xiang , Feldstein Ariel , McClain Craig , Kirpich Irina 
Published: November 2023

Introduction/AimAlcoholic liver disease (ALD) is one of the major medical problems in the US and worldwide. ALD progresses through the course of several pathologies including steatosis, steatohepatitis, and cirrhosis. Dietary factors play critical roles in ALD pathogenesis. It has been shown that dietary ω6 polyunsaturated fatty acids (ω6‐PUFAs), specifically linoleic acid (LA), exacerbated experimental ALD in mice; however the molecular mechanism(s) underlying this effect are not fully understood. In the present study we evaluated liver and blood lipidomic profiles; and tested the hypothesis that oxidized LA metabolites (OXLAMs) exacerbate EtOH‐induced liver injury via OXLAM‐mediated induction of a pro‐inflammatory response in a mouse model of ALD.Materials and MethodsC57BL/6 male mice were fed 5% (v/v) EtOH in a liquid diet for 10 days followed by a single EtOH gavage (5 g/kg) on day 11. Two different diets were administered: corn oil (unsaturated fat, USF), and a combination of medium chain triglyceride oil and beef tallow (saturated fat, SF) enriched diets. Liver injury and steatosis were evaluated. Expression of genes involved in inflammation and lipid metabolism was examined. Lipidomic analysis was performed on the liver and plasma samples. For the in vitro study, RAW264.7 cells, a mouse macrophage cell line, were used to directly test the effect of LA metabolites on production of pro‐inflammatory cytokines.ResultsMice fed USF+EtOH had significantly elevated plasma alanine aminotransferase levels (a marker of liver injury) and hepatic fat accumulation compared to mice fed SF+EtOH. Expression of the pro‐inflammatory cytokines, tumor necrosis factor α (Tnf‐α) and Interleukin‐1β (Il‐1b), was significantly higher in the USF+EtOH group. Hepatic carnitine palmitoyltrasferase, the rate‐limiting enzyme in fatty acid β‐oxidation, was down‐regulated in response to EtOH in both SF and USF dietary groups, while fatty acid synthase, an enzyme involved in fatty acid synthesis, was up‐regulated predominantly by USF+EtOH. Lipidomic analysis revealed low levels of hepatic medium chain (octanoic, decanoic, and dodecanoic), as well as ω3‐PUFAs (eicosapentaenoic and docosahexaenoic, [EPA and DHA]) fatty acids in USF+EtOH compared to SF+EtOH‐fed mice. Serum levels of LA and OXLAMs (9‐ and 13‐hydroxyoctadecadienoic acids [9‐HODE and 13‐HODE]) were significantly higher in USF+EtOH compared to SF+EtOH group. In RAW264.7 macrophages 9‐HODE alone induced Tnf‐α, and Il‐1b expression and enhanced lipopolysaccharide‐mediated induction of these cytokines.ConclusionCompared to SF, dietary USF exacerbated liver steatosis, inflammation and injury caused by EtOH. The greater degree of steatosis was, in part, the result of elevated lipid synthesis, rather than differences in fatty acid oxidation, and possibly, a decrease in EPA and DHA levels. OXLAMs may contribute to EtOH‐induced liver injury, most likely via induction of pro‐inflammatory response in hepatic macrophages.Support or Funding InformationSupported by grants by the NIH and Department of Veterans Affairs