Reduced Expression of Ferroportin-1 Mediates Hyporesponsiveness of Suckling Rats to Stimuli That Reduce Iron Absorption  Deepak Darshan, Sarah J. Wilkins, David M. Frazer, Gregory J. Anderson  Gastroenterology  Volume 141, Issue 1, Pages 300-309 (July 2011) DOI: 10.1053/j.gastro.2011.04.012 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 59Fe absorption during suckling and after weaning. Rats at the postnatal ages indicated were gavaged with 3 μCi of 59Fe/animal and intestinal iron absorption determined. Iron absorption is presented as the percentage of radioactivity retained by the body after 5 days, relative to the initial dose. Data represent mean ± standard error of mean; n = 5–10 rats/group. Gastroenterology 2011 141, 300-309DOI: (10.1053/j.gastro.2011.04.012) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Comparison of iron homeostasis in iron deficient weaned rats and suckling animals. (A) Hepatic iron concentration, (B) hepcidin expression, and (C) 59Fe absorption were measured. Data represent mean ± standard error of mean. Bars with the same letters are significantly different (P ≤ .05); n = 5–6 rats/group. Gastroenterology 2011 141, 300-309DOI: (10.1053/j.gastro.2011.04.012) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Intestinal iron transporter gene expression in iron-deficient weaned rats and suckling animals. RNA was extracted from gut epithelial cells isolated from 25-day-old rats that had been weaned onto iron-deficient or control diets and 15-day-old suckling animals, and gene expression was determined by quantitative real-time polymerase chain reaction. Expression of (A) total Fpn1, (B) Dmt1, (C) duodenal cytochrome B (DcytB), and (D) Hephaestin are shown relative to basic transcription factor 3 (BTF3). Data represent mean ± standard error of mean. Bars with the same letters are significantly different (P ≤ .05); n = 6 rats/group. Gastroenterology 2011 141, 300-309DOI: (10.1053/j.gastro.2011.04.012) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Regulation of iron absorption and homeostasis in response to iron loading and inflammation in suckling and weaned animals. Suckling animals (15 days old) and 25-day-old weaned animals were treated with either iron dextran (0.3 mg/g) or LPS (0.1 mg/kg) and (A) transferrin saturation, (B) hepatic iron concentration, (C) hepatic Hamp messenger RNA levels, and (D) intestinal 59Fe absorption were determined as described in the Materials and Methods section. Data represent mean ± standard error of mean. Bars with the same letters are significantly different (P ≤ .05); n = 4–6 rats/group. Gray, neonate; black, adult animals. Gastroenterology 2011 141, 300-309DOI: (10.1053/j.gastro.2011.04.012) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Expression and regulation of Fpn1 in suckling animals. (A) RNA was extracted from duodenal enterocytes isolated from suckling or weaned rats following iron dextran or LPS administration, and Fpn1 messenger RNA levels were determined by quantitative real-time polymerase chain reaction. Protein was extracted from duodenal enterocytes obtained from animals of varying ages, and the level of (B) Fpn1 and (C) ferritin protein was determined by Western blotting. The lower panels show quantitation of protein levels normalized to the loading control actin. Data represent mean ± standard error of mean. Bars with the same letters are significantly different (P ≤ .05); n = 5–6 rats/group. Gray, neonate; black, adult animals. Gastroenterology 2011 141, 300-309DOI: (10.1053/j.gastro.2011.04.012) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Relative abundance of Fpn1 splice variants throughout suckling and weaning. RNA was extracted from the intestine of rats of varying ages and Fpn1A(+IRE) and Fpn1B(−IRE) transcript levels were measured by quantitative real-time polymerase chain reaction. Data represent mean ± standard error of mean. n = 5–12 rats/group. Gray, Fpn1A(+IRE); black, Fpn1B(−IRE) transcripts. Gastroenterology 2011 141, 300-309DOI: (10.1053/j.gastro.2011.04.012) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Regulation of Fpn1 and iron absorption by corticosteroids. Animals were treated with dexamethasone (0.4 μg/g body weight) at the ages indicated, and the expression of (A) sucrose-isomaltase, (B) Fpn1A(+IRE) (black bars), and Fpn1B(−IRE) (gray bars) was determined by quantitative real-time polymerase chain reaction and (C) Fpn1 protein levels by Western blotting. (D) Absorption of 59Fe was also determined. Data represent mean ± standard error of mean. Bars with the same letters are significantly different (P ≤ .05); n = 3–8 rats/group. Gastroenterology 2011 141, 300-309DOI: (10.1053/j.gastro.2011.04.012) Copyright © 2011 AGA Institute Terms and Conditions