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, 9 (1), 17463

Dietary Wheat Amylase Trypsin Inhibitors Promote Features of Murine Non-Alcoholic Fatty Liver Disease

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Dietary Wheat Amylase Trypsin Inhibitors Promote Features of Murine Non-Alcoholic Fatty Liver Disease

Muhammad Ashfaq-Khan et al. Sci Rep.

Abstract

We previously demonstrated that a common dietary protein component, wheat amylase trypsin inhibitors (ATI), stimulate intestinal macrophages and dendritic cells via toll like receptor 4. Activation of these intestinal myeloid cells elicits an inflammatory signal that is propagated to mesenteric lymph nodes, and that can facilitate extraintestinal inflammation. Mice were fed a well-defined high fat diet, with (HFD/ATI) or without (HFD) nutritionally irrelevant amounts of ATI. Mice on HFD/ATI developed only mild signs of intestinal inflammation and myeloid cell activation but displayed significantly higher serum triglycerides and transaminases compared to mice on HFD alone. Moreover, they showed increased visceral and liver fat, and a higher insulin resistance. ATI feeding promoted liver and adipose tissue inflammation, with M1-type macrophage polarization and infiltration, and enhanced liver fibrogenesis. Gluten, the major protein component of wheat, did not induce these pathologies. Therefore, wheat ATI ingestion in minute quantities comparable to human daily wheat consumption exacerbated features of the metabolic syndrome and non-alcoholic steatohepatitis, despite its irrelevant caloric value.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Enhanced insulin resistance, higher serum transaminases and increased lipogenic gene expression in mice fed ATI. Age-matched, wildtype mice were fed four different diets for 8 weeks. A low fat diet (LFD), a high fat diet (HFD), a HFD/G/ATI diet (G, 30% of protein as gluten, and 0.45% as ATI) and a HFD/ATI diet (0.7% of protein as ATI). (A) Average food consumption per week (B) Serum ALT (C) Serum triglycerides. (D) Average AUC (area under the curve) of the aforementioned groups (E) Intraperitoneal glucose tolerance test (IPGTT), in the same dietary groups as shown in A. (F) Hepatic transcript levels of srebp1c, acc, fas. Comparisons by ANOVA; data are means ± SEM for 7–10 mice per group; *p < 0.05, **p < 0.01; $p < 0.05, $$p < 0.01.
Figure 2
Figure 2
ATI feeding promotes hepatic steatosis and inflammation. (A) Representative images of H&E stained liver sections (original magnifiction 20x and 40x). (B,C) Grading of steatosis, lobular inflammation and hepatocyte ballooning (arrows), according to the NAFLD Activity Score (NAS). (D) Frozen liver sections stained with Sudan III (original magnification 20x). (E) Quantification of % of Sudan III stained area. Comparisons by ANOVA; data are means ± SEM for 5 representative sections per mouse and 7–10 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
ATI feeding increases liver macrophage numbers and their M1- vs M2-type polarization. (AC) Immunohistochemistry and quantitative morphometry for CD68 and YM-1 positive cells (original magnification 40x). (D) Ratio of total (CD68+) vs M2-type (Ym-1+) macrophages. (E) CD11b+ F4/80+ macrophage subset (% of CD45 positive total immune cells) as determined by FACS analysis. Comparisons by ANOVA; data are means ± SEM for 10 representative sections per mouse and 7–10 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
ATI feeding increases hepatic pro-inflammatory and macrophage M1- vs M2-type gene expression. (AF) Hepatic transcript levels of cd68, tnfa, il1b, il6, arg1 and ym1. Comparisons by ANOVA; data are expressed as means ± SEM for 7–10 mice per group; *p < 0.05, **p < 0.01.
Figure 5
Figure 5
ATI feeding promotes hepatic fibrogenesis. (A) Sirius Red and (B) α–SMA immunohistochemistry and (C,D) quantitative morphometry (original magnification 20x). Hepatic transcript levels of tgfbeta, mmp2, mmp9, mmp13, col1a1, timp1. Comparisons by ANOVA; data are expressed as means ± SEM of 5 representative sections per mouse and 7–10 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6
Nutritional ATI promote central adipose tissue inflammation. (A) Crown like structures (CLS = accumulation of macrophages) in CD68+ stained sections of epididymal adipose tissue in the 4 experimental groups (original magnification 40x), the number of CD68+ CLS as determined by morphometry, and epididymal fat as % of body weight. (B) fat weights, and (C) transcript levels of cd68, il6 and il1b. Comparisons by ANOVA; data are means ± SEM for 10 representative sections per mouse and 7–10 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7
Figure 7
ATI feeding increases intestinal macrophage and dendritic cell activation and maturation. (A–C) CD68, CD86 and MCH-II expressing cells in the terminal ileum; scale bar: 100 and 50 µm. (D) Morphometric quantification of CD68, CD86 and MHC-II positive cells. (E) Transcript levels of il1b, tnfα and il6. Comparisons by ANOVA; data are expressed as means ± SEM of 6 mice per group and 5 representative sections per mouse; *p < 0.05, **p < 0.01, ***p < 0.001.

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