Gluconeogenic Signals Regulate Iron Homeostasis via Hepcidin in Mice.

Hepatic gluconeogenesis provides fuel during starvation, and is abnormally induced in obese individuals or those with diabetes. Common metabolic disorders associated with active gluconeogenesis and insulin resistance (obesity, metabolic syndrome, diabetes, and nonalcoholic fatty liver disease) have been associated with alterations in iron homeostasis that disrupt insulin sensitivity and promote disease progression. We investigated whether gluconeogenic signals directly control Hepcidin, an important regulator of iron homeostasis, in starving mice (a model of persistently activated gluconeogenesis and insulin resistance).|We investigated hepatic regulation of Hepcidin expression in C57BL/6Crl, 129S2/SvPas, BALB/c, and wild-type and Creb3l3-/- null mice. Mice were fed a standard, iron-balanced chow diet or an iron-deficient diet for 9 days before death, or for 7 days before a 24- to 48-hour starvation period; liver and spleen tissues then were collected and analyzed by quantitative reverse-transcription polymerase chain reaction and immunoblot analyses. Serum levels of iron, hemoglobin, Hepcidin, and glucose also were measured. We analyzed human hepatoma (HepG2) cells and mouse primary hepatocytes to study transcriptional control of Hamp (the gene that encodes Hepcidin) in response to gluconeogenic stimuli using small interfering RNA, luciferase promoter, and chromatin immunoprecipitation analyses.|Starvation led to increased transcription of encodes phosphoenolpyruvate carboxykinase 1 (a protein involved in gluconeogenesis) in livers of mice, increased levels of Hepcidin, and degradation of Ferroportin, compared with nonstarved mice. These changes resulted in hypoferremia and iron retention in liver tissue. Livers of starved mice also had increased levels of Ppargc1a messenger RNA and Creb3l3 messenger RNA, which encode a transcriptional co-activator involved in energy metabolism and a liverspecific transcription factor, respectively. Glucagon and a cyclic adenosine monophosphate analog increased promoter activity and transcription of Hamp in cultured liver cells; levels of Hamp were reduced after administration of small interfering RNAs against Ppargc1a and Creb3l3. PPARGC1A and CREB3L3 bound the Hamp promoter to activate its transcription in response to a cyclic adenosine monophosphate analog. Creb3l3-/- mice did not up-regulate Hamp or become hypoferremic during starvation.|We identified a link between glucose and iron homeostasis, showing that Hepcidin is a gluconeogenic sensor in mice during starvation. This response is involved in hepatic metabolic adaptation to increased energy demands; it preserves tissue iron for vital activities during food withdrawal, but can cause excessive iron retention and hypoferremia in disorders with persistently activated gluconeogenesis and insulin resistance

The SMAD pathway is required for hepcidin response during endoplasmic reticulum stress

Hepcidin, the iron hormone, is regulated by a number of stimulatory and inhibitory signals. The cAMP responsive element binding protein 3-like 3, CREB3L3, mediates hepcidin response to endoplasmic reticulum (ER) stress. In this study we asked whether hepcidin response to ER stress also requires the SMAD1/5/8 pathway that has a major role in hepcidin regulation in response to iron and other stimuli. We analyzed hepcidin mRNA expression and promoter activity in response to ER stressors in HepG2 cells in the presence of the BMP type I receptor inhibitor LDN-193189, mutated hepcidin promoter or siRNA against different SMAD proteins. We then used a similar approach in vivo in wild-type, Smad1/5 or Creb3l3 -/- animals undergoing ER stress. In vitro, LDN-193189 prevented hepcidin mRNA induction by different ER stressors. Seemingly, mutation of a BMP-responsive element in the hepcidin promoter prevented ER stress-mediated upregulation. Moreover, in vitro silencing of SMAD proteins by siRNA, in particular SMAD5, blunted hepcidin response to ER stress. On the contrary, hepcidin induction by ER stress was maintained when using antibodies against canonical BMP receptor ligands. In vivo, hepcidin was induced by ER stress and prevented by LDN-193189. In addition, in Smad1/5 knock-out mice, ER stress was unable to induce hepcidin expression. Finally, in Creb3l3 knock-out mice, in response to ER stress, SMAD1/5 were correctly phosphorylated and hepcidin induction was still appreciable, although to a lesser extent as compared to control mice. In conclusion, our study indicates that hepcidin induction by ER stress involves the central regulatory SMAD1/5 pathway

Huh-7: a human hemochromatotic cell line.

Hereditary hemochromatosis (HC) is commonly associated with homozygosity for the cysteine-to-tyrosine substitution at position 282 (C282Y) of the HFE protein. This mutation prevents HFE from binding beta(2)-microglobulin (beta(2)M) and reaching the cell surface. We have discovered that a widely used hepatoma cell line, Huh-7, carries a HFE mutation similar to that associated with human HC. By HFE gene sequencing of Huh-7 genomic DNA, we found a TAC nucleotide deletion (c. 691_693del) responsible for loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This mutation affects a conserved hydrophobic region in a loop connecting two beta strands that make up the alpha3 domain of HFE, not far from the 282 site. HIE was detected by western blot in HepG2 but not in Huh-7 cell membrane fractions. In WRL-68 cells expressing wild-type HIE, the HIE protein was largely found at the plasma membrane where it colocalizes with beta(2)M. On the contrary, the HFE-Y231del mutant, similarly to an exogenously expressed HFE-C282Y mutant, failed to reach the plasma membrane and did not colocalize with membrane-expressed beta(2)M. C282Y mutant HFE in HC is associated with inadequate hepcidin expression. We found that Huh-7 cells display lower hepcidin messenger RNA levels as compared to HepG2 cells, which carry a wild-type HFE. Interestingly, hepcidin messenger RNA levels increased significantly in Huh-7 cells stably expressing exogenous wild-type HFE at the plasma membrane. Conclusion: Huh-7 cells may represent a novel and valuable tool to investigate the role of altered HFE traffic in iron metabolism and pathogenesis of human HIE HC. (HEPATOLOGY 2010;51:654-659.

Hepatic stellate cells are not subjected to oxidant stress during iron-induced fibrogenesis in rodents

Oxidant stress plays a key role in hepatic fibrogenesis. This study was undertaken to assess whether, during iron overload-associated liver fibrosis in vivo, oxidant stress occurs in hepatic stellate cells (HSC) during active fibrogenesis. Gerbils were treated with iron-dextran, and, after hepatic fibrosis developed, livers were subjected to various combination of in situ hybridization and immunocytochemistry analyses. In iron-treated animals, no specific accumulation of ferritin protein was found in collagen mRNA-expressing cells. Moreover, the activity of the iron regulatory protein, the main sensor of cellular iron status, was unchanged in HSC from iron-treated animals. Although a significant amount of malondialdehyde-protein adducts was detected in gerbil liver during fibrogenesis, accumulation of these lipid peroxidation by-products was restricted to iron-laden cells adjacent to activated HSC. In cultured gerbil HSC, iron, aldehydes, and other pro-oxidants were able to enhance the expression of an oxidant stress-responsive gene, heme oxygenase (HO), with no change in collagen mRNA accumulation. In keeping with these findings, we found that, in vivo, activation of HO gene was present in iron-filled nonparenchymal cell aggregates, but absent in HSC. In conclusion, the data indicate that during iron overload-associated fibrogenesis, HSC are not directly subjected to oxidant stress, but are likely to be activated by paracrine signals arising in neighboring cells

Excess iron into hepatocytes is required for activation of collagen type I gene during experimental siderosis

Background/Aims: Liver fibrosis and cirrhosis represent common pathological findings in humans with iron overload. This study was undertaken to assess whether in vivo targeting of iron to liver parenchymal or nonparenchymal cells would differently affect collagen gene activity. Methods: Rats were treated with an iron diet or intramuscular injections of iron dextran, and in situ hybridization analyses on liver samples were performed. Results: These iron treatments determined parenchymal or reticuloendothelial cell iron overload, respectively. The typical distribution of iron into different liver cells was documented by histochemistry and confirmed by in situ hybridization analysis with a ferritin L complementary RNA probe. In iron-fed rats, in situ hybridization analysis identified a signal for collagen type I messenger RNA into nonparenchymal cells in zones I and ii. In rats with nonparenchymal cell iron overload, no activation of collagen gene expression was detected into or near iron-laden nonparenchymal cells. These findings were also confirmed by quantitative Northern blot analysis. Conclusions: The results of this study indicate that, regardless of the total hepatic iron burden, selective localization of iron into liver cells (i.e., parenchymal cells) is required for the activation of collagen gene during long-term iron overload in rodents