High Iron Exposure from the Fetal Stage to Adulthood in Mice Alters Lipid Metabolism

Abstract

Iron supplementation is recommended during pregnancy and fetal growth. However, excess iron exposure may increase the risk of abnormal fetal development. We investigated the potential side effects of high iron levels in fetuses and through their adult life. C57BL/6J pregnant mice from 2 weeks of gestation and their offspring until 30 weeks were fed a control (CTRL, FeSO₄ 0 g/1 kg) or high iron (HFe, FeSO₄ 9.9 g/1 kg) diets. HFe group showed higher iron accumulation in the liver with increased hepcidin, reduced TfR1/2 mRNAs, and lowered ferritin heavy chain (FTH) proteins in both liver and adipose tissues despite iron loading. HFe decreased body weight, fat weight, adipocyte size, and triglyceride levels in the blood and fat, along with downregulation of lipogenesis genes, including PPARγ, C/EBPα, SREBP1c, FASN, and SCD1, and fatty acid uptake and oxidation genes, such as CD36 and PPARα. UCP2, adiponectin, and mRNA levels of antioxidant genes such as GPX4, HO-1, and NQO1 were increased in the HFe group, while total glutathione was reduced. We conclude that prolonged exposure to high iron from the fetal stage to adulthood may decrease fat accumulation by altering ferritin expression, adipocyte differentiation, and triglyceride metabolism, resulting in an alteration in normal growth.

Keywords: dietary iron intake; ferritin heavy chain; high dietary iron; high iron exposure; iron homeostasis; lipid metabolism; maternal iron status.

Figure 1

Excessive dietary iron intake decreased body weight and fat mass. Diagram for feeding schedule (a) and amount of iron in liver and fat as measured by (b) ferrozine assay, plasma ferritin level (c), Prussian Blue Staining in the (d) liver and (e) mesenteric fat. (f) Cumulative food intake, (g) body weight, (h) food efficiency ratio (FER), (i) fat weight, and (j) organ weight at final week (week 30) of each group (CTRL, HFe). Data are expressed as means  ±  SE; Student’s t test was used for the test of difference. * p < 0.05, ** p < 0.01, *** p < 0.001. All sections were paraffin embedded. Scale bar, 100 µm; magnification, ×10. Black arrows show blue stain indicating iron accumulation in each tissue.

Figure 2

Iron-related mRNA and protein expression was affected by HFe diet. Amount of mRNA in the (a) liver, (b) fat, and (c) small intestine as measured by RT-PCR are presented. (d) Effects of iron overload on FTH protein expression levels of mice pups. Results are expressed as mean  ±  SE; Student’s t test was used for the test of difference. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

The change in FTH expression was not affected by DNA methylation in CGIs. (a) Schematic showing the tested regions for MSP at the FTH1 locus. Target regions were chosen using MethFinder (http://www.urogene.org/methprimer accessed on 19 April 2022) on regions upstream (−5 kb) and downstream from the FTH1 transcription start site. Agarose gel electrophoresis of MSP products are shown on (b) regions upstream (−5 kb) and (c) downstream (+3 kb) from CTRL- and HFe-fed offspring TFs in the FTH1 gene promoter region and were predicted using (d) PROMO ALGGEN, TRANSFAC and TFBIND software programs. (e) The binding sites of the TF detected in 3 databases. The spacing of the sites in the diagram reflects the typical spacing of the actual sequence. Solid arrows indicate transcription start sites. (f) Amount of protein levels of C/EBPα and Nrf2 and (g) mRNA levels of C/EBPβ and GATA2 in fat are presented. Data are expressed as means  ±  SE; Student’s t test was used to test differences between means. * p < 0.05. M, product amplified with methylated-specific primer; U, product amplified with unmethylated-specific primer; DW, double distilled water control; AP1, activating protein-1; GATA1, GATA-binding protein 1; SRF, serum response factor; YY1, Yin Yang 1.

Figure 4

HFe decreased the adipocyte size and TG levels in the blood and fat tissues. Representative images of hematoxylin and eosin (H&E) staining. (a) Decreased adipocyte size in HFe mice. (b) Plasma biochemistry related to lipid profiles and (c) FFA were measured at week 30. (d) TG and (e) TCHO levels in liver and fat of each group. (f) The results of OGTT, (g) AUC of OGTT, and (h) plasma fasting glucose at week 30 in mice fed CTRL and HFe diet. Data are expressed as means  ±  SE; Student’s t test was used to test differences between means. * p < 0.05, *** p < 0.001. TG, triglycerides; TCHO, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; FFA, free fatty acids.

Figure 5

Ann excessive iron diet affected FA biosynthesis and differentiation of adipocytes more than FA β-oxidation. (a) Amount of mRNA level in differentiation of adipocyte (b) de novo lipogenesis, lipid droplet FA uptake, and FA oxidation (c) and in mesenteric fat as measured by RT-PCR. Effects of HFe on protein levels in mesenteric fat related to (d) FA synthesis and (e) FA oxidation. (f) Hepatic mRNA levels of insulin sensitivity and glucose uptake. (g) Proposed schematic mechanism of the alteration on lipid metabolism by HFe. Data are expressed as means  ±  SE; Student’s t test was used to test differences between means. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

The HFe enhanced Nrf2-related antioxidant expression. (a) Estimation of MDA in mesenteric fat of each group (CTRL, HFe). (b) mRNA expression related to antioxidant enzymes. (c) Contents of tGSH and (d) RT-PCR analysis were performed to detect mRNA expression of GSH-Trx and production system in mice. All data are expressed as the mean ± S.E. Student’s t test was used to test differences between means. * p < 0.05. GPX4, glutathione peroxidase 4; HO-1, heme oxygenase-1; NQO1, quinone oxidoreductase 1; GR, glutathione disulfide reductase; GRX1, glutaredoxin 1; GST, glutathione S-transferase; GCLC, glutamate cysteine ligase catalytic; GCLM, glutamate cysteine ligase modifier.

Figure 7

Summarized scheme of the Heft diet affecting iron and lipid metabolism in mice in the present study.