Dietary fat is a lipid source in 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD)-elicited hepatic steatosis in C57BL/6 mice

Abstract

2,3,7,8-Tetrachlorodibenzo-ρ-dioxin (TCDD) increases fatty acid (FA) transport and FA levels resulting in hepatic steatosis in mice. Diet as a source of lipids was investigated using customized diets, stearoyl-CoA desaturase 1 (Scd1) null mice, and (14)C-oleate (18:1n9) uptake studies. C57BL/6 mice fed with 5, 10, or 15% fat or 50, 60 or 70% carbohydrate diets exhibited increased relative liver weight following gavage with 30 µg/kg TCDD for 168 h. Hepatic lipid extract analysis from mice fed with 5, 10, and 15% fat diets identified a dose-dependent increase in total FAs induced by TCDD. Mice fed with fat diet also exhibited a dose-dependent increase in the dietary essential linoleic (18:2n6) and α-linolenic (18:3n3) acids. No dose-dependent FA increase was detected on carbohydrate diets, suggesting dietary fat as a source of lipids in TCDD-induced steatosis as opposed to de novo lipogenesis. TCDD also induced oleate levels threefold in Scd1 null mice that are incapable of desaturating stearate (18:0). This is consistent with oleate representing > 90% of all monounsaturated FAs in rodent chow. Moreover, TCDD increased hepatic (14)C-oleate levels twofold in wild type and 2.4-fold in Scd1 null mice concurrent with the induction of intestinal and hepatic lipid transport genes (Slc27a, Fabp, Ldlr, Cd36, and Apob). In addition, computational scanning identified putative dioxin response elements and in vivo ChIP-chip analysis revealed regions of aryl hydrocarbon receptor (AhR) enrichment in lipid transport genes differentially regulated by TCDD. Collectively, these results suggest the AhR mediates increased uptake of dietary fats that contribute to TCDD-elicited hepatic steatosis.

FIG. 1.

Hepatic absolute and essential FA levels in mice fed with increasing fat or carbohydrate diets treated with sesame oil vehicle (V) or 30 µg/kg TCDD (T) for 168 h. (A–C) Mice fed with 5, 10, and 15% fat diet, (A) total fatty acids (TFA), (B) α-linoleic acid (18:2n6), and (C) α-linolenic acid (18:3n3) levels. *p < 0.05 for TCDD compared with vehicle within a diet, **p < 0.05 for TCDD 15% fat or 10% fat compared with TCDD 5% fat. (D–F) Mice fed with 50, 60, and 70% carbohydrate diet, (D) TFAs, (E) 18:2n6, and (F) 18:3n3. *p < 0.05 for TCDD compared with vehicle within a diet; **p < 0.05 for TCDD 70% carbohydrate compared with TCDD 50% carbohydrate. (A–F) Bars represent mean ± standard error of the mean (SEM), n = 5.

FIG. 2.

Hepatic ¹⁴C and lipid levels in Scd1 wild type and null mice 120 h postdose with 30 µg/kg TCDD. (A) ¹⁴C-levels were measured in lipid extracts by liquid scintillation counting after gavage with 2 µCi ¹⁴C-oleate 4 h prior to sacrifice. Gas chromatography mass spectrometry analysis of hepatic oleate (18:1n9) (B) and monounsaturated fatty acid (C) levels (expressed in nmol/g) in Scd1 wild type and null mice 168 h after oral gavage with 30 µg/kg TCDD (Angrish et al., 2011). *p < 0.05 for TCDD compared with vehicle, **p < 0.05 for Scd1 wild type TCDD compared with Scd1 null mice TCDD. Bars represent mean ± SEM, n = 5.

FIG. 3.

AhR-mediated increase in dietary lipid in TCDD-elicited hepatic steatosis. Steps 1–4: Fat absorption by the intestinal epithelium and export to the circulatory system. Steps 5–11: Enhanced hepatic fatty acid uptake and storage. Inhibition of efflux and β-oxidation-mediated degradation pathways. Steps 12–17: Hepatic glucose metabolism including glycogen synthesis, gluconeogenesis and fatty acid synthesis are inhibited. Lines with arrowheads indicate reaction/pathway direction. Lines with blunted ends indicate reactions/pathways that are inhibited. Red boxes indicate induced gene expression. Green boxes indicate repressed gene expression. A more detailed description is provided in the Discussion section.