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HEP_24359_sm_suppinfotable1.doc25KSupporting Information
HEP_24359_sm_suppinfofig1.tif8245KSupporting Figure 1. Thirteen days of a low iron diet leads to suppression of hepcidin expression without hypoferremia. The standard rodent diet is relatively high in iron (380 ppm in our rodent facility) compared with estimated minimal daily requirement, and prior studies have reported that baseline hepcidin levels in C57Bl/6 on a similar standard rodent diet were high and not significantly inducible by parenteral iron (Nemeth E et al. J Clin Invest. 2004;113:1271-6). Prior studies have reported that 10-15 days of a low iron diet (2-6 ppm iron) leads to suppression of endogenous hepcidin without hypoferremia, thereby enabling detection of hepcidin induction by iron administration (Nemeth E et al. J Clin Invest. 2004;113:1271-6; Rivera S et al. Blood. 2005;106:2196-9). We therefore chose to place mice on a low iron diet for 12-14 days prior to acute or chronic iron administration experiments in our study. To determine the effects of this 12-14 day low iron diet pretreatment in our hands, eight-week-old male C57BL/6 male mice on a control diet (380 ppm iron Prolab RMH 3000 diet) or a low iron diet (2-6 ppm iron TD.80396, Harlan Teklad) for 13 days prior to sacrifice (N = 5 per group) were analyzed for serum iron (A), transferrin saturation (Tf sat, B), and hepatic hepcidin (Hamp) relative to Rpl19 mRNA by quantitative real-time RT-PCR (C). Results are reported as the mean ± s.d. Exact P values are shown where significant or reported as NS where not significant. Similar to reports from previously published studies (Nemeth E et al. J Clin Invest. 2004;113:1271-6; Rivera S et al. Blood. 2005;106:2196-9), mice on a low iron diet for 13 days did not develop hypoferremia, maintaining serum iron of 162 μg/dL and Tf sat of 61%, which trended slightly lower but were not significantly changed from mice on a standard rodent diet. Hamp relative to Rpl19 mRNA levels were significantly lower in mice maintained on a low iron diet for 13 days compared with mice on a standard rodent diet. Thus, our results confirm previously reported studies (Nemeth E et al. J Clin Invest. 2004;113:1271-6; Rivera S et al. Blood. 2005;106:2196-9) that a low iron diet for 12-14 days leads to suppression of hepcidin expression without hypoferremia.
HEP_24359_sm_suppinfofig2.tif8245KSupporting Figure 2. Bmp6 mRNA expression in the duodenum is not affected by acute or chronic enteral iron administration, or by genetic iron overload. Although one publication suggests that the small intestine might also be a source of Bmp6 in response to iron administration (Arndt S et al. Gastroenterology. 2010;138:372-82), this has not been confirmed in other studies (Zhang AS et al. J Biol Chem. 2010;285:16416-23; Kautz et al. Haematologica. ePub 2010 Oct 15, doi:10.3324/haematol.2010.031963). To further clarify whether the liver is the main site of Bmp6 mRNA induction by iron, we analyzed the effects of chronic and acute iron administration and genetic iron overload on Bmp6 mRNA levels in the duodenum. A-B) The animals that underwent chronic iron administration from Figure 1 (panel A) and acute iron administration from Figure 2 (panel B) were analyzed for duodenal Bmp6 relative to Rpl19 mRNA expression by quantitative real-time RT-PCR (N = 6 per group). Results are expressed as the mean ± s.d. for the fold change compared to the Baseline. For each iron treated group, significant changes are shown as exact P values for the comparisons with the baseline (*), with the previous group (&), and with the corresponding mock group (#) (one way-ANOVA for panel A, and two-way ANOVA for panels B; Holm-Sidak or the Dunnett's post hoc tests (when appropriate) were used for pair-wise multiple comparisons). We did not find differences in duodenal Bmp6 mRNA expression after 48 hours or 3 weeks of chronic iron treatment compared with baseline animals (Panel A). After acute iron administration, there was a trend toward a temporal increase in duodenal Bmp6 mRNA for both mock and iron treated groups, without significant differences between the treatments (Panel B). C) As previously published (Corradini et al. Gastroenterology. 2009;137:1489-1497), male and female 129S6/SvEvTac WT mice (WT) and age, gender and background Hfe knock-out mice (Hfe-/-) were maintained on a standard rodent diet (Harlan, Tekland, Madison, WI) until 12 weeks of age. Dietary iron loading was achieved by feeding age, gender and background matched WT mice the same standard diet supplemented with 2% carbonyl iron for 4 weeks (WT IE diet). Age, gender and background matched WT mice were fed with a diet with no iron content (Harlan, Tekland, Madison, WI) for a period of 5 weeks (WT ID diet). All mice were sacrificed at 12 weeks and analyzed for duodenal Bmp6 relative to Rpl19 mRNA expression by quantitative real-time RT-PCR (N = 5-6 per group), Results are expressed as the mean ± s.d. for the fold change compared to the baseline. No significant differences were found between groups (two-way ANOVA).
HEP_24359_sm_suppinfofig3.tif8246KSupporting Figure 3. Bmp6 mRNA expression in the spleen is not affected by acute or chronic enteral iron administration. To further clarify whether the liver is the main site of Bmp6 mRNA induction by iron, we analyzed the effects of chronic and acute iron administration on Bmp6 mRNA levels in the spleen, another organ that plays a key role in iron homeostasis. The animals that underwent chronic iron administration from Figure 1 (Panels A,C) and acute iron administration from Figure 3 (Panels B, D) were analyzed for spleen iron content (Panels A-B) or splenic Bmp6 relative to Rpl19 mRNA expression by quantitative real-time RT-PCR (Panels C-D), N = 6 per group. Results are expressed as the mean ± s.d. For each iron treated group, significant changes are shown as exact P values for the comparisons with the baseline (*), with the previous group (&), and with the corresponding mock group (#) (one way-ANOVA for Panels A-C, two-way ANOVA for panel D, with Holm-Sidak or the Dunnett's post hoc tests used for pair-wise multiple comparisons when appropriate). Chronic iron treatment significantly increased spleen iron content in a progressive fashion in comparison to the baseline group starting at one week (Panel A). Acute iron treatment did not significantly change spleen iron content in comparison to the baseline (Panel B). There were no significant changes in splenic Bmp6 mRNA expression in response to chronic (Panel C) or acute iron treatment (Panel D).
HEP_24359_sm_suppinfofig4.tif7763KSupporting Figure 4. Hepatic Il6 and Crp mRNA expression are not increased by chronic or acute enteral iron administration or by oral gavage. The animals that underwent chronic iron administration from Figure 1 (Panels A-B) and acute iron administration from Figure 3 (Panels C-D) were analyzed for hepatic Il6 (Panels A, C) and Crp (Panels B, D) relative to Rpl19 mRNA expression by quantitative real-time RT-PCR using previously described primers (Xia Y et al. J Immunol. 2011;186:1369-76; Corradini E, et al. Gastroenterology. 2010;139:1721-9). Results are expressed as the mean ± s.d for the fold change compared to the baseline, N = 6 per group. Neither mock gavage (Panels C-D, gray bars), acute iron gavage (Panels C-D, black bars), nor chronic iron administration (Panels A-B) significantly increased hepatic Il6 or Crp relative to Rpl19 mRNA as determined by one-way ANOVA (Panels A-B) or two-way ANOVA (Panels C-D). These data suggest that the inflammatory pathway is not activated in the liver by gavage, acute iron administration, or chronic iron administration.
HEP_24359_sm_suppinfofig5.tif8245KSupporting Figure 5. Effects of acute enteral iron administration on hepatic Hfe2 and Tmprss6 and mRNA expression. Hemojuvelin (encoded by HFE2, also known as HJV) is a co-receptor for the BMP-SMAD signaling pathway that enhances SMAD signaling and hepcidin induction in response to BMP ligand (Babitt JL et al. Nat Genet. 2006;38:531-9). TMPRSS6 is a serine protease that inhibits hepcidin expression through a mechanism that is thought to involve cleavage of hemojuvelin and thereby inhibition of BMP-SMAD signaling (Du X et al. Science. 2008;320:1088-92; Silvestri L et al. Cell Metab. 2008;8:502-11). To determine whether an increase in Hfe2 mRNA expression or a decrease in Tmprss6 mRNA expression could account for the increase in downstream Smad signaling and hepcidin induction by acute iron gavage, the animals that underwent acute iron administration from Figure 3 were analyzed for hepatic Hfe2 (Panel A) and Tmprss6 (Panel B) relative to Rpl19 mRNA expression by quantitative real-time RT-PCR using the following primers: Hfe2 forward 5′-CACGGCAGCCCTCCAACTCTAA-3′ , Hfe2 reverse 5′ -GACATACTCGGCATTGCAGCGG-3′ , Tmprss6 forward 5′ - TTGCTGGTCTTGGCTGCGCT-3′ , Tmprss6 reverse 5′ - AATGACGGTTGAGCACCCGGAG-3′ . Results are expressed as the mean ± s.d for the fold change in comparison to the baseline group. Statistical significance was determined by two-way ANOVA with Holm-Sidak or the Dunnett′ s post hoc tests for pair-wise multiple comparisons. (*) P < 0.05 for the comparison with the baseline, (#) P <0.05 for the comparison with the corresponding iron group. Panel A) Acute iron gavage had no significant effect on hepatic Hfe2 relative to Rpl19 mRNA in comparison to the baseline group (black bars). Mock-treated animals did show a significant decrease of Hfe2 mRNA expression in comparison to the baseline group at 1 and 8 hours (gray bars). Iron-treated animals showed a temporal trend similar to the mock group, but with higher levels than the respective mock time points at 1 and 24 hours. These data suggest a possible effect of the gavage itself and/or circadian fluctuations to decrease Hfe2 mRNA expression. The higher levels of Hfe2 mRNA in iron treated groups compared with mock treated groups at some time points could suggest a possible effect of iron on Hfe2 mRNA expression. However, the lack of increase in Hfe2 mRNA over baseline by acute iron gavage in contrast to the trend toward increased P-SMAD1/5/8 protein and significantly increased Id1 and Hamp mRNA over baseline by acute iron gavage (See Figs 3D, 5B, 6C) suggests that an increase in Hfe2 mRNA expression does not account for the increase in downstream Smad signaling and Hamp mRNA induction by acute iron administration under these experimental conditions. Additional experiments using different conditions that do not affect underlying Hfe2 mRNA expression may be helpful to more definitively rule out an effect of acute iron administration on Hfe2 mRNA expression. Panel B) Although Tmprss6 relative to Rpl19 mRNA expression was significantly decreased from baseline in the mock gavage group at 1 hour, and in both the iron and mock gavage groups at 4 hours, there was no significant difference between iron and mock gavage groups for all time points. These data suggest that although there might be an effect from the gavage itself and/or circadian fluctuation to decrease Tmprss6 mRNA expression, Tmprss6 mRNA is not significantly affected by acute iron administration under these experimental conditions.
HEP_24359_sm_suppinfofig6.tif204KSupporting Figure 6. BMP6 antibody administration inhibits hepcidin induction by a high iron diet and increases iron deposition in the liver. Seven-week-old C57Bl/6 male mice were placed on a low iron diet for 12 days (2-6 ppm iron) and then sacrificed (Control), or switched to a 2% carbonyl iron diet for 1 week either alone (High iron), or in combination with a neutralizing anti-BMP6 antibody administered IP once daily (BMP6 Ab) prior to sacrifice (N = 6 per group). Serum iron (Panel A), Tf sat (Panel B), hepatic Hamp relative to Rpl19 mRNA (Panel C), and LIC (Panel D) were measured as described in Figure 1. Results are reported as the mean ± s.d. Exact P values as determined by one-way ANOVA are shown. As expected, the high iron diet significantly increased serum iron, Tf sat, hepatic Hamp relative to Rpl19 mRNA expression, and LIC compared to Control animals (Compare bar 2 to 1, Panels A-D respectively). BMP6 antibody administration in conjunction with the high iron diet significantly impaired the ability of the high iron diet to induce hepcidin expression. Indeed, Hamp relative to Rpl19 mRNA levels in the BMP6 antibody group were significantly reduced from the High iron diet group and were not significantly changed from Control animals (Panel C, bar 3). The ability of BMP6 antibody administration to block hepcidin induction by the high iron diet did not significantly increase serum iron or Tf sat levels above those achieved in the High iron diet group, likely because these levels were already near maximum (>300 ± g/dL, 93% respectively, Panels A-B bar 3), but did significantly increase iron deposition in the liver to levels almost twice those in the High iron diet group (Panel D, bar 3). Thus, specific inhibition of BMP6 activity with a neutralizing BMP6 antibody inhibits hepcidin induction by chronic iron administration, leading to increased iron deposition in the liver. These data suggest that chronic iron administration induces hepatic hepcidin expression by a BMP6 dependent mechanism.

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