Thus, we achieved a condition of increasing LIC in the face of stable (albeit high) circulating iron levels. In the chronic iron treatment setting, hepatic Hamp mRNA expression significantly and progressively increased between baseline and 48 hours,

and then plateaued for the remaining 3 weeks (Fig. 1D). Although both LIC and Tf sat positively correlated learn more with Hamp mRNA levels (r = 0.456, P = 0.002; r = 0.658, P < 0.001, respectively) and significantly influenced Hamp mRNA expression by simple linear regression analysis (R2 = 0.21, β = 0.456, P = 0.002; R2 = 0.43, β = 0.658, P < 0.001, respectively) by multivariate analysis, Tf sat was the only independent predictor of hepatic Hamp mRNA levels (R2 = 0.46, β = 0.57, P < 0.001) in this setting. Although the influence of LIC on Hamp mRNA levels was difficult to detect in the chronic iron treatment setting where both LIC and Tf sat were elevated, mice switched to a low iron diet MEK inhibitor after

receiving a high iron diet for 1 week maintained a high LIC for up 8 days (Fig. 2C), whereas serum iron and Tf sat decreased back to baseline levels by 24-48 hours (Fig. 2A,B), allowing us to examine the effects of an isolated elevated LIC with normal circulating iron levels. The low iron diet significantly decreased hepatic Hamp mRNA levels from those achieved by 1 week of a high iron diet within 24 hours and for up to 8 days (Fig. 2D), reflecting the decrease in serum iron and Tf sat, and consistent with a role for circulating iron in regulating hepcidin expression. Notably, Hamp mRNA levels remained significantly elevated above baseline for up to 8 days in these mice, suggesting

an independent role for LIC in inducing hepcidin expression. Indeed, by multivariate analysis, both Tf sat and LIC were independent predictors of hepatic Hamp mRNA levels in this model (R2 = 0.856; β = 0.004, P < 0.001 for Tf sat; β = 0.0004, Coproporphyrinogen III oxidase P < 0.001 for LIC). In the acute iron treatment experiment, both serum iron (Fig. 3A) and Tf sat (Fig. 3B) were significantly increased by a single dose of 2 mg/kg iron at 1 and 4 hours after oral gavage (black bars) compared with untreated animals (Baseline) or with mock gavage (gray bars), with a return to baseline by 8-24 hours. In contrast, LIC was unchanged at all timepoints in comparison to baseline and the respective mock groups (Fig. 3C). In the acute iron treatment experiment, hepatic Hamp mRNA showed a progressive temporal increase, and was significantly increased at 4 and 8 hours after iron gavage in comparison to baseline and the corresponding mock groups, with a return to baseline levels by 24 hours (Fig. 3D, black bars). The mock group did not manifest significant differences in Hamp mRNA compared to baseline, although there was a trend toward a higher value at 4 hours after mock gavage, suggesting a possible effect of the gavage procedure itself in a few animals (Fig. 3D, gray bars). In the iron group, hepatic Hamp mRNA was correlated with Tf sat (r = 0.455, P = 0.