Ingestion of wheat amylase trypsin inhibitors promotes a proinflammatory liver and adipose tissue phenotype in diet induced non-alcoholic fatty liver disease in mice

Non-alcoholic fatty liver disease (NAFLD) is a liver disease with an escalating demand for liver transplantation over the next decade. Apart from life style changes and pharmacological strategies, intestinal microbiota and specific (micro-) nutrients may play an important role in NAFLD pathogenesis. A likely nutritional candidate is the family of what amylase trypsin inhibitors (ATIs) that represent 3 – 4% of wheat protein. ATIs are highly resistant to digestive proteolysis and activate intestinal innate immunity via toll like receptor 4 (TLR4) on monocytes, macrophages and dendritic cells (Junker Y et al, J Exp Med 2012, Zevallos VF et al, Gastroenterology 2016). We therefore assessed the effect of nutritional ATIs equivalent in quantity to human average wheat ingestion on the severity of diet induced NAFLD in mice.

Male mice received a carbohydrate and protein (zein from corn, 22.1. % of weight) defined low or high fat diet (HFD, 53 KJ% vs. 13 KJ% of calories as saturated fats), with or without 30% of the zein being isocalorically replaced by wheat gluten (containing 0.1 g ATIs/10 g gluten) or 0.7% of the zein replaced by purified ATIs for 8 weeks. Mice on ATIs containing diets gained significant obesity as well as a significantly higher serum triglycerides vs. HFD alone. HFD/ATI group were significantly insulin resistant and has significantly higher NAS score. Transcript levels for CD68 (total macrophages), IL-1β, IL-6 and TNF-α were increased significantly both in the liver and in adipose tissues whereas M2 macrophages anti-inflammatory transcripts for ARG1 and Ym-1 were decreased in the liver tissues of HFD/ATI fed mice. Histologically elevated CD68+ total liver macrophages and decreased numbers of YM-1⁺ M2 liver macrophages in the HFD/G+ATI and HFD/ATIs were witnessed. FACS analysis revealed an increased population of CD11b⁺F4/80⁺ cells in the livers of HFD/ATIs group. Finally, HFD/ATI fed mice show higher fibrosis related genes and a significantly more CD68⁺ Crown like structure, a marker of adipose tissue inflammation.

Our study clearly implicates dietary ATIs from wheat as proinflammatory nutritional drivers of NAFLD/NASH. This effect occurs at a daily intake that is comparable to average human consumption of wheat products irrespective of caloric value.