Hepatic peroxisome proliferator-activated receptor γ coactivator 1α and hepcidin are coregulated in fasted/refed states in mice.

Hepcidin plays a central role in iron homeostasis and contributes to the pathogenesis of several disorders. Consequently, development of a clear understanding of hepcidin regulation in various pathophysiological conditions has been the subject of intensive research. Technical limitations, however, have limited the wide availability of assays for measuring this iron-regulatory hormone in human serum. Recently, Troutt et al. developed a specific and robust sandwich immunoassay for hepcidin-25 and measured it in the serum of healthy volunteers. They reported diurnal variation in its concentrations in the circulation (1). In particular, the authors demonstrated that hepcidin-25 concentrations were significantly increased after 3 days of fasting, an intriguing result considering the hyposideremic effect of hepcidin. The authors hypothesized that this increase in hepcidin could be caused by suppressed erythropoiesis to maintain tissue iron concentrations. To investigate the molecular mechanisms responsible for hepcidin regulation by fasting, we fasted C57BL/6J mice for 24 h and then separated them into 2 groups. The first group was fasted for an additional 12-h period (“fasted” conditions); the second group was refed during the 12-h period with a highcarbohydrate diet (“refed” conditions). Interestingly, mouse hepcidin antimicrobial peptide 1 (hepcidin1) mRNA concentrations were increased 4.2-fold with fasting [mean (SD) relative concentration, 1 (0.1) for the refed mice (n 6) vs 4.2 (0.1) for the fasted mice (n 6); P 0.0001], with a significant 25% decrease of plasma iron. These findings confirm the results of Troutt et al. and suggest that an increase in hepcidin-25 in human serum most likely reflects the direct upregulation of HAMP (hepcidin antimicrobial peptide) gene expression in response to fasting. To determine whether this regulation could be explained by systemic and/or cell autonomous pathways, we studied mouse Hamp1 (hepcidin antimicrobial peptide 1) gene expression in isolated hepatocytes maintained under culture conditions that mimic the fasting/ gluconeogenic state. To achieve these conditions, we deprived isolated mouse hepatocytes of serum for 16 h and then treated them for 8 h with dibutyryl-cAMP (to mimic the effect of increased glucagon when blood glucose concentrations are low). These gluconeogenic conditions per se induced Hamp1 gene expression 2-fold [mean relative concentration, 1 (0.1) for controls (n 3) vs 2.1 (0.1) in dibutyryl-cAMP–treated hepatocytes (n 3); P 0.0001]. Given the ability of peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ) to activate components of the fasting response in the liver, we investigated the role of this transcriptional factor by infecting mouse primary hepatocytes for 24 h at a multiplicity of infection of 25 with adenovirus producing either PGC-1 or green fluorescent protein as a control (2 ). Forced production of PGC-1 increased Hamp1 gene expression 2-fold [mean relative concentration, 1 (0.1) in control hepatocytes infected with green fluorescent protein–producing adenovirus (n 3) vs 2.0 (0.1) in hepatocytes infected with PGC1 –producing adenovirus (n 3); P 0.0001]. Also increased were glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, 2 well-known PGC-1 targets involved in gluconeogenesis. In addition, we found a significant correlation between relative concentrations of PGC-1 and hepcidin1 in the livers of fasted/refed animals (Fig. 1). 1 Nonstandard abbreviations: hepcidin1, mouse hepcidin antimicrobial peptide 1; PGC-1 , peroxisome proliferator-activated receptor coactivator 1 ; HNF4 , hepatocyte nuclear factor 4 ; FOXO1, forkhead box O1 protein. 2 Genes: HAMP, hepcidin antimicrobial peptide; Mus musculus gene Hamp1, hepcidin antimicrobial peptide 1. Hepcidin1 PG C -1 α y = 2.1546x + 0.0471 r 2 = 0.6303