Cyp1b1 deletion and retinol deficiency coordinately suppress mouse liver lipogenic genes and hepcidin expression during post-natal development

Abstract

Cyp1b1 deletion and gestational vitamin A deficiency (GVAD) redirect adult liver gene expression. A matched sufficient pre- and post-natal diet, which has high carbohydrate and normal iron content (LF12), increased inflammatory gene expression markers in adult livers that were suppressed by GVAD and Cyp1b1 deletion. At birth on the LF12 diet, Cyp1b1 deletion and GVAD each suppress liver expression of the iron suppressor, hepcidin (Hepc), while increasing stellate cell activation markers and suppressing post-natal increases in lipogenesis. Hepc was less suppressed in Cyp1b1-/- pups with a standard breeder diet, but was restored by iron supplementation of the LF12 diet.

Conclusions: The LF12 diet delivered low post-natal iron and attenuated Hepc. Hepc decreases in Cyp1b1-/- and GVAD mice resulted in stellate activation and lipogenesis suppression. Endothelial BMP6, a Hepc stimulant, is a potential coordinator and Cyp1b1 target. These neonatal changes in Cyp1b1-/- mice link to diminished adult obesity and liver inflammation.

Keywords: Cytochrome P450 1b1; Hepcidin; Lipogenesis; Vitamin A; Vitamin A deficiency.

Figure 1. GVAD and Cyp1b1 deletion each suppress diet-induced obesity, but R-Cyp1b1−/− mice do not

(A) Dietary administration for each group is shown. Body weight and epididymal fat pat pad weight in male offspring comparing obesity response on a control BD-HFD to (B) Cyp1b1−/− HFD and GVAD-HFD (C) R-Cyp1b1−/− HFD and (D) LF12 maternal diet (LF12-HFD). *p-value≤0.05, **p-value≤0.01

Figure 2. GVAD and Cyp1b1 deletion affect weanling pup physiological parameters

(A) Experimental design for C57Bl/6J (WT) and Cyp1b1−/− mice, each with GVAD examining offspring at birth (PN0), weaning (PN21), and 14weeks of age. At weaning, (B) Body weight, (C) nose to rump length, (D) epididymal fat pad, (E) liver weight, and (F) serum retinol, liver retinol, and liver retinyl ester content. *p-value≤0.05, **p-value≤0.01

Figure 3

Cyp1b1−/− and R-Cyp1b1−/− pups respond differently to GVAD. (A) Body weight, (B) epididymal fat pad, (C) nose to rump length, and (D) liver weight in Cyp1b1−/− and R-Cyp1b1−/− weanling pups (PN21) administered a gestational vitamin a sufficient (LF12) or deficient (GVAD) diet. (E) Serum retinol, liver retinol, and liver retinyl ester content. (F) Serum triglyceride measurements of WT, Cyp1b1−/−, and R-Cyp1b1−/− LF12 and GVAD pups at weaning. *p-value≤0.05, **p-value≤0.01

Figure 4. Developmental uncoupling of hepcidin genes, Hepc and Hamp2.

(A) Hepcidin (Hamp/Hepc) expression (Cy3) at birth (PN0) and at weaning (PN21). Fold change values are shown from EDGE3 processed data. (B) Raw relative expression (Cy3) microarray values of Hepc (solid line) and Hamp2 (dotted line) from birth through 14 weeks of age. (C) Body weight at weaning in WT and Cyp1b1−/− pups from dams fed the LF12 diet, LF12 + Iron diet or standard breeder diet (BD). (D) Hamp mRNA expression was measured by qPCR at weaning in WT and Cyp1b1−/− pups on the BD, LF12, and LF12 + Iron diet. Comparison between microarray and qPCR Hamp expression in WT and Cyp1b1−/− pups on the LF12 diet is highlighted. *p-value≤0.05, **p-value≤0.01

Figure 5. Pathways for synthesis and regulation of oleate and cholesterol from acetyl CoA and regulation

Pathways of synthesis (A) and regulation (B) highlight the role of the lipogenic genes shown in Table 5.

Figure 6. Major fatty acid metabolism gene expression responses by GVAD and Cyp1b1 deletion

Lipogenic liver mRNA was measured in C57Bl/6J (WT) and Cyp1b1−/− vitamin A sufficient (LF12) or deficient (GVAD) offspring by qPCR, which replicated array results. *p-value≤0.05, **p-value≤0.01

Figure 7. Proposed mechanism for vitamin A and CYP1B1 modulation of lipogenic genes through hepcidin expression

Hepcidin signaling is mediated by iron content, retinol, and CYP1B1 metabolism of estradiol, likely through GPR30/GPER/BMP6. Hepcidin acts to inhibit ferroportin, which releases iron into circulation from gut enterocytes or macrophage. Near depletion of hepcidin expression caused by GVAD, Cyp1b1 deletion, or the combination leads to increased iron concentrations and oxidative stress, which represses SREBP-mediated gene expression, including fatty acid and cholesterol synthesis genes.