A Low Iron Diet Protects from Steatohepatitis in a Mouse Model

Figure 1. Metabolic phenotypes after 3–4 months on diet: The following were measured in mice on the FF/NC diets for 3–4 months (n = 3–5 per group). (A) Body weight. (B) Blood glucose after 6 h fasting. (C) Glucose tolerance (area under the glucose curve [AUC] after intra-peritoneal glucose tolerance testing). (D) Homeostasis model assessment of β-cell function (HOMA B). (E) Homeostasis model assessment of insulin resistance (HOMA IR). (F) Serum cholesterol and (G) Fasting serum triglycerides (TG). Shown are means ± SE. p values were calculated by two-way ANOVA with post hoc Tukey’s multiple comparisons test. * 0.01 ≤ p ≤ 0.05; **** p < 0.0001, ns: not significant, i.e., p ≥ 0.05.

Figure 2. Metabolic phenotypes after 5–6 months on diet: The following were measured in mice on the FF/NC diets for 5–6 months (n = 8–10 per group). (A) Body weight. (B) Blood glucose after 6h fasting. (C) Glucose tolerance (area under the glucose curve [AUC] after intra-peritoneal glucose tolerance testing). (D) Homeostasis model assessment of β-cell function (HOMA B). (E) Homeostasis model assessment of insulin resistance (HOMA IR). (F) Serum cholesterol and (G) Fasting serum triglycerides (TG). Shown are means ± SE. p values were calculated by two-way ANOVA with post hoc Tukey’s multiple comparisons test. * 0.01 ≤ p ≤ 0.05; ** 0.001 ≤ p ≤ 0.01; *** 0.0001 ≤ p ≤ 0.001; **** p < 0.0001. ns: not significant.

Figure 3. Effects of the FF diet on iron parameters and of dietary iron on hepatic lipid accumulation. (A,B) Serum ferritin at 3–4 and 5–6 months (n = 3–5 per group). (C,D) Hepatic iron content at 3–4 and 5–6 months (n = 3–6 per group). (E,F) Hepcidin mRNA (Hamp) normalized to 18s rRNA at 3–4 and 5–6 months (n = 3–5 per group). (G,H) Bone morphogenetic protein 6 (Bmp6) mRNA at 3–4 and 5–6 months (n = 3–5 per group). (I,J) Liver triglyceride content (TG) at 3–4 and 5–6 months (n = 3–10 per group). Shown are means ± SE. p values were calculated by two-way ANOVA with post hoc Tukey’s multiple comparisons test. ns: not significant, i.e., p ≥ 0.05; * 0.01 ≤ p ≤ 0.05; ** 0.001 ≤ p ≤ 0.01; *** 0.0001 ≤ p ≤ 0.001; **** p < 0.0001.

Figure 4. Effects of dietary iron and FF on NASH biomarkers: (A,B) Serum ALT at 3–4 and 5–6 months (n = 3–11 per group). (C,D) Tumor necrosis factor alpha (Tnfa) mRNA at 3–4 and 5–6 months (n = 3–5 per group). (E,F) Transforming growth factor beta (Tgfb) mRNA at 3–4 and 5–6 months (n = 3–5 per group). (G,H) of collagen1alpha1 (Col1a1) mRNA at 3–4 and 5–6 months (n = 3–5 per group). All genes were normalized to 18sRNA. Shown are means ± SE. p values were calculated with ordinary two-way ANOVA with post hoc Tukey’s multiple comparisons test. ns: not significant, i.e., p ≥ 0.05; * 0.01 ≤ p ≤ 0.05; ** 0.001 ≤ p ≤ 0.01; *** 0.0001 ≤ p ≤ 0.001; **** p < 0.0001.

Figure 5. Differential gene expression in the hepatic transcriptome induced by iron and FF: (A–C) Heat maps of DEG using cutoff log2FC >1 or <−1, p < 0.01 on DeSEq2 data. Each row represents a DEG and each column is a sample from an individual mouse fed the diet as indicated by the column label. (A) Effect of iron in the FF-fed mice at 3–4 months. First two columns: low iron (LI) FF; next two columns: normal iron (NI) FF; last two columns: high iron (HI) FF. (B) Effect of iron in the FF-fed mice at 5–6 months; three samples each for LIFF and NIFF and two samples for HIFF. (C) Effect of FF compared to normal chow (NC). First three columns: LINC; next three columns: LIFF. (D) Venn diagram of DEGs affected by iron and FF, showing only 1% of genes altered by both iron and FF. (E) Bar chart of top 3 canonical pathways enriched by IPA core analysis. (F) Stacked bar chart of the number of genes upregulated and downregulated in the top 6 canonical pathways enriched by IPA core analysis.

Figure 6. Hepatocyte-stellate cell interaction is necessary for iron- and fat-induced fibrogenesis: mRNA expression of smooth muscle actin (ACTA2), upon treatment with iron (FAC) and palmitate (Pal). n = 3 biological replicates. Error bars are shown as means ± SE. p values were calculated by two-way ANOVA with post hoc Tukey’s multiple comparisons test. ** 0.001 ≤ p ≤ 0.01; **** p < 0.0001.