Obesogenic diet leads to luminal overproduction of the complex IV inhibitor H2 S and mitochondrial dysfunction in mouse colonocytes.

Obesity is characterized by systemic low-grade inflammation associated with disturbances of intestinal homeostasis and microbiota dysbiosis. Mitochondrial metabolism sustains epithelial homeostasis by providing energy to colonic epithelial cells (CEC) but can be altered by dietary modulations of the luminal environment. Our study aimed at evaluating whether the consumption of an obesogenic diet alters the mitochondrial function of CEC in mice. Mice were fed for 22 weeks with a 58% kcal fat diet (diet-induced obesity [DIO] group) or a 10% kcal fat diet (control diet, CTRL). Colonic crypts were isolated to assess mitochondrial function while colonic content was collected to characterize microbiota and metabolites. DIO mice developed obesity, intestinal hyperpermeability, and increased endotoxemia. Analysis of isolated colonic crypt bioenergetics revealed a mitochondrial dysfunction marked by decreased basal and maximal respirations and lower respiration linked to ATP production in DIO mice. Yet, CEC gene expression of mitochondrial respiration chain complexes and mitochondrial dynamics were not altered in DIO mice. In parallel, DIO mice displayed increased colonic bile acid concentrations, associated with higher abundance of Desulfovibrionaceae. Sulfide concentration was markedly increased in the colon content of DIO mice. Hence, chronic treatment of CTRL mouse colon organoids with sodium sulfide provoked mitochondrial dysfunction similar to that observed in vivo in DIO mice while acute exposure of isolated mitochondria from CEC of CTRL mice to sodium sulfide diminished complex IV activity. Our study provides new insights into colon mitochondrial dysfunction in obesity by revealing that increased sulfide production by DIO-induced dysbiosis impairs complex IV activity in mouse CEC.

Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

Iron overload is one of the secondary osteoporosis etiologies. Cellular and molecular mechanisms involved in iron-related osteoporosis are not fully understood. AIM: The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. MATERIAL AND METHODS: We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. RESULTS: Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 µM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. CONCLUSION: Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.

Hereditary aceruloplasminemia (HA), related to mutations in the ceruloplasmin (Cp) gene, leads to iron accumulation. Ceruloplasmin ferroxidase activity being considered essential for macrophage iron release, macrophage iron overload is expected, but it is not found in hepatic and splenic macrophages in humans. Our objective was to get a better understanding of the mechanisms leading to iron excess in HA. A clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) knockout of the Cp gene was performed on Sprague-Dawley rats. We evaluated the iron status in plasma, the expression of iron metabolism genes, and the status of other metals whose interactions with iron are increasingly recognized. In Cp-/- rats, plasma ceruloplasmin and ferroxidase activity were absent, together with decreased iron concentration and transferrin saturation. Similarly as in humans, the hepatocytes were iron overloaded conversely to hepatic and splenic macrophages. Despite a relative hepcidin deficiency in Cp-/- rats and the loss of ferroxidase activity, potentially expected to limit the interaction of iron with transferrin, no increase of plasma non-transferrin-bound iron level was found. Copper was decreased in the spleen, whereas manganese was increased in the plasma. These data suggest that the reported role of ceruloplasmin cannot fully explain the iron hepatosplenic phenotype in HA, encouraging the search for additional mechanisms.-Kenawi, M., Rouger, E., Island, M.-L., Leroyer, P., Robin, F., Remy, S., Tesson, L., Anegon, I., Nay, K., Derbré, F., Brissot, P., Ropert, M., Cavey, T., Loréal, O. Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.

Spleen iron, molybdenum, and manganese concentrations are coregulated in hepcidin-deficient and secondary iron overload models in mice.

Iron excess increases the hepatic expression of hepcidin, the systemic iron metabolism regulator that favors iron sequestration in the spleen. Genetic iron overload related to hepcidin insufficiency decreases the spleen iron concentration and increases hepatic iron concentration, whereas during secondary iron overload, the hepcidin expression increases together with spleen iron concentration in addition to hepatic iron concentrations increase. Links between iron metabolism and other metals being suggested, our aim was to investigate, during iron overload, the relationships between the hepatic hepcidin expression level and the hepatic and splenic concentrations of iron, manganese, copper, zinc, and molybdenum, determined using inductively coupled plasma mass spectrometry. Hepcidin-deficient mice, secondary iron overload mice models, and their respective controls were studied. Spleen molybdenum and manganese concentrations paralleled the modulation of both spleen iron concentrations, increasing in secondary iron overload and decreasing in hepcidin deficiency related iron overload, as well as hepatic hepcidin mRNA expression. Our data suggest that iron, manganese, and molybdenum metabolisms could share mechanisms controlling their distribution that are associated to hepcidin modulation. In diseases with abnormal hepcidin levels, including chronic inflammation, special attention should be paid to those metals that can participate with the phenotype.-Cavey, T., Latour, C., Island, M.-L., Leroyer, P., Guggenbuhl, P., Coppin, H., Roth, M.-P., Bendavid, C., Brissot, P., Ropert, M., Loréal, O. Spleen iron, molybdenum, and manganese concentrations are coregulated in hepcidin-deficient and secondary iron overload models in mice.

Mid-infrared fibre evanescent wave spectroscopy of serum allows fingerprinting of the hepatic metabolic status in mice.

Non-alcoholic fatty liver disease is associated with obesity, diabetes, and metabolic syndrome. The detection of systemic metabolic changes associated with alterations in the liver status during non-alcoholic fatty liver disease could improve patient follow-up. The aim of the present study was to evaluate the potential of mid-infrared fibre evanescent wave spectroscopy as a minimum-invasive method for evaluating the liver status during non-alcoholic fatty liver disease. Seventy-five mice were subjected to a control, high-fat or high-fat-high carbohydrate diets. We analysed the serum biochemical parameters and mRNA levels of hepatic genes by quantitative RT-PCR. Steatosis was quantified by image analysis. The mid-infrared spectra were acquired from serum, and then analysed to develop a predictive model of the steatosis level. Animals subjected to enriched diets were obese. Hepatic steatosis was found in all animals. The relationship between the spectroscopy-predicted and observed levels of steatosis, expressed as percentages of the liver biopsy area, was not linear. A transition around 10% steatosis was observed, leading us to consider two distinct predictive models (<10% and >10%) based on two different sets of discriminative spectral variables. The model performance was evaluated using random cross-validation (10%). The hypothesis that additional metabolic changes occur beyond this transition was supported by the fact that it was associated with increased serum ALT levels, and Col1α1 chain mRNA levels. Our data suggest that mid-infrared spectroscopy combined with statistical analysis allows identifying serum mid-infrared signatures that reflect the liver status during non-alcoholic fatty liver disease.

Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships.

Iron is reported to interact with other metals. In addition, it has been shown that genetic background may impact iron metabolism. Our objective was to characterize, in mice of three genetic backgrounds, the links between iron and several non-iron metals. Thirty normal mice (C57BL/6, Balb/c and DBA/2; n = 10 for each group), fed with the same diet, were studied. Quantification of iron, zinc, cobalt, copper, manganese, magnesium and rubidium was performed by ICP/MS in plasma, erythrocytes, liver and spleen. Transferrin saturation was determined. Hepatic hepcidin1 mRNA level was evaluated by quantitative RT-PCR. As previously reported, iron parameters were modulated by genetic background with significantly higher values for plasma iron parameters and liver iron concentration in DBA/2 and Balb/c strains. Hepatic hepcidin1 mRNA level was lower in DBA/2 mice. No iron parameter was correlated with hepcidin1 mRNA levels. Principal component analysis of the data obtained for non-iron metals indicated that metals parameters stratified the mice according to their genetic background. Plasma and tissue metals parameters that are dependent or independent of genetic background were identified. Moreover, relationships were found between plasma and tissue content of iron and some other metals parameters. Our data: (i) confirms the impact of the genetic background on iron parameters, (ii) shows that genetic background may also play a role in the metabolism of non-iron metals, (iii) identifies links between iron and other metals parameters which may have implications in the understanding and, potentially, the modulation of iron metabolism.

Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes.

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

Ferroportin diseases: functional studies, a link between genetic and clinical phenotype.

Ferroportin (FPN) mediates iron export from cells and this function is modulated by serum hepcidin. Mutations in the FPN gene (SLC40A1) lead to autosomal dominant iron overload diseases related either to loss or to gain of function, and usually characterized by normal or low transferrin saturation versus elevated transferrin saturation, respectively. However, for the same mutation, the phenotypic expression may vary from one patient to another. Using in vitro overexpression of wild-type or mutant FPN proteins, we characterized the functional impact of five recently identified FPN gene mutations regarding FPN localization, cell iron status, and hepcidin sensitivity. Our aim was to integrate functional results and biological findings in probands and relatives. We show that while the p.Arg371Gln (R371Q) mutation had no impact on studied parameters, the p.Trp158Leu (W158L), p.Arg88Gly (R88G), and p.Asn185Asp (N185D) mutations caused an iron export defect and were classified as loss-of-function mutations. The p.Gly204Ser (G204S) mutation induced a gain of FPN function. Functional studies are useful to determine whether or not a FPN gene mutation found in an iron overloaded patient is deleterious and to characterize its biological impact, especially when family studies are not fully informative and/or additional confounding factors may affect bio-clinical expression.

GATA-4 transcription factor regulates hepatic hepcidin expression.

Hepcidin, a hormone mainly synthesized by hepatocytes and secreted in plasma, controls iron bioavailability. Thus, by inducing the internalization of the iron exporter ferroportin, it regulates iron release from macrophages, enterocytes and hepatocytes towards plasma. Abnormal levels of hepcidin expression alter plasma iron parameters and lead to iron metabolism disorders. Understanding the mechanisms controlling hepcidin (HAMP encodes hepcidin) gene expression is therefore an important goal. We identified a potential GATA-binding site within the human hepcidin promoter. Indeed, in hepatic HepG2 cells, luciferase experiments demonstrated that mutation of this GATA-binding site impaired the hepcidin promoter transcriptional activity in basal conditions. Gel-retardation experiments showed that GATA-4 could bind to this site. Co-transfection of a GATA-4 expression vector with a hepcidin promoter reporter construct enhanced hepcidin promoter transcriptional activity. Furthermore, modulation of GATA4 mRNA expression using specific siRNAs (small interfering RNAs) down-regulated endogenous hepcidin gene expression. Finally, we found that mutation of the GATA-binding site impaired the interleukin-6 induction of hepcidin gene expression, but did not prevent the bone morphogenetic protein-6 response. In conclusion, the findings of the present study (i) indicate that GATA-4 may participate in the control of hepcidin expression, and (ii) suggest that alteration of its expression could contribute to the development of iron-related disorders.

Oxidative and glycolytic skeletal muscles deploy protective mechanisms to avoid atrophy under pathophysiological iron overload.

BACKGROUND: Iron excess has been proposed as an essential factor in skeletal muscle wasting. Studies have reported correlations between muscle iron accumulation and atrophy, either through ageing or by using experimental models of secondary iron overload. However, iron treatments performed in most of these studies induced an extra-pathophysiological iron overload, more representative of intoxication or poisoning. The main objective of this study was to determine the impact of iron excess closer to pathophysiological conditions on structural and metabolic adaptations (i) in differentiated myotubes and (ii) in skeletal muscle exhibiting oxidative (i.e. the soleus) or glycolytic (i.e. the gastrocnemius) metabolic phenotypes. METHODS: The impact of iron excess was assessed in both in vitro and in vivo models. Murine differentiated myotubes were exposed to ferric ammonium citrate (FAC) (i.e. 10 and 50 µM) for the in vitro component. The in vivo model was achieved by a single iron dextran subcutaneous injection (1 g/kg) in mice. Four months after the injection, soleus and gastrocnemius muscles were harvested for analysis. RESULTS: In vitro, iron exposure caused dose-dependent increases of iron storage protein ferritin (P < 0.01) and dose-dependent decreases of mRNA TfR1 levels (P < 0.001), which support cellular adaptations to iron excess. Extra-physiological iron treatment (50 µM FAC) promoted myotube atrophy (P = 0.018), whereas myotube size remained unchanged under pathophysiological treatment (10 µM FAC). FAC treatments, whatever the doses tested, did not affect the expression of proteolytic markers (i.e. NF-κB, MurF1, and ubiquitinated proteins). In vivo, basal iron content and mRNA TfR1 levels were significantly higher in the soleus compared with the gastrocnemius (+130% and +127%; P < 0.001, respectively), supporting higher iron needs in oxidative skeletal muscle. Iron supplementation induced muscle iron accumulation in the soleus and gastrocnemius muscles (+79%, P < 0.001 and +34%, P = 0.002, respectively), but ferritin protein expression only increased in the gastrocnemius (+36%, P = 0.06). Despite iron accumulation, muscle weight, fibre diameter, and myosin heavy chain distribution remained unchanged in either skeletal muscle. CONCLUSIONS: Together, these data support that under pathophysiological conditions, skeletal muscle can protect itself from the related deleterious effects of excess iron.

Bacteroides fragilis derived metabolites, identified by molecular networking, decrease Salmonella virulence in mice model.

In the gut microbiota, resident bacteria prevent pathogens infection by producing specific metabolites. Among bacteria belonging to phylum Bacteroidota, we have previously shown that Bacteroides fragilis or its cell-free supernatant inhibited in vitro Salmonella Heidelberg translocation. In the present study, we have analyzed this supernatant to identify bioactive molecules after extraction and subsequent fractionation using a semi-preparative reversed-phase Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS). The results indicated that only two fractions (F3 and F4) strongly inhibited S. Heidelberg translocation in a model mimicking the intestinal epithelium. The efficiency of the bioactive fractions was evaluated in BALB/c mice, and the results showed a decrease of S. Heidelberg in Peyer's patches and spleen, associated with a decrease in inflammatory cytokines and neutrophils infiltration. The reduction of the genus Alistipes in mice receiving the fractions could be related to the anti-inflammatory effects of bioactive fractions. Furthermore, these bioactive fractions did not alter the gut microbiota diversity in mice. To further characterize the compounds present in these bioactive fractions, Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS) data were analyzed through molecular networking, highlighting cholic acid (CA) and deoxycholic acid. In vitro, CA had inhibitory activity against the translocation of S. Heidelberg by significantly decreasing the expression of Salmonella virulence genes such as sipA. The bioactive fractions also significantly downregulated the flagellar gene fliC, suggesting the involvement of other active molecules. This study showed the interest to characterize better the metabolites produced by B. fragilis to make them means of fighting pathogenic bacteria by targeting their virulence factor without modifying the gut microbiota.

Hepcidin induction limits mobilisation of splenic iron in a mouse model of secondary iron overload.

Venesection has been proposed as a treatment for hepatic iron overload in a number of chronic liver disorders that are not primarily linked to mutations in iron metabolism genes. Our aim was to analyse the impact of venesection on iron mobilisation in a mouse model of secondary iron overload. C57Bl/6 mice were given oral iron supplementation with or without phlebotomy between day 0 (D0) and D22, and the results were compared to controls without iron overload. We studied serum and tissue iron parameters, mRNA levels of hepcidin1, ferroportin, and transferrin receptor 1, and protein levels of ferroportin in the liver and spleen. On D0, animals with iron overload displayed elevations in iron parameters and hepatic hepcidin1 mRNA. By D22, in the absence of phlebotomies, splenic iron had increased, but transferrin saturation had decreased. This was associated with high hepatic hepcidin1 mRNA, suggesting that iron bioavailability decreased due to splenic iron sequestration through ferroportin protein downregulation. After 22days with phlebotomy treatments, control mice displayed splenic iron mobilisation that compensated for the iron lost due to phlebotomy. In contrast, phlebotomy treatments in mice with iron overload caused anaemia due to inadequate iron mobilisation. In conclusion, our model of secondary iron overload led to decreased plasma iron associated with an increase in hepcidin expression and subsequent restriction of iron export from the spleen. Our data support the importance of managing hepcidin levels before starting venesection therapy in patients with secondary iron overload that are eligible for phlebotomy.

Hfe Gene Knock-Out in a Mouse Model of Hereditary Hemochromatosis Affects Bodily Iron Isotope Compositions.

Hereditary hemochromatosis is a genetic iron overload disease related to a mutation within the HFE gene that controls the expression of hepcidin, the master regulator of systemic iron metabolism. The natural stable iron isotope composition in whole blood of control subjects is different from that of hemochromatosis patients and is sensitive to the amount of total iron removed by the phlebotomy treatment. The use of stable isotopes to unravel the pathological mechanisms of iron overload diseases is promising but hampered by the lack of data in organs involved in the iron metabolism. Here, we use Hfe -/- mice, a model of hereditary hemochromatosis, to study the impact of the knock-out on iron isotope compositions of erythrocytes, spleen and liver. Iron concentration increases in liver and red blood cells of Hfe -/- mice compared to controls. The iron stable isotope composition also increases in liver and erythrocytes, consistent with a preferential accumulation of iron heavy isotopes in Hfe -/- mice. In contrast, no difference in the iron concentration nor isotope composition is observed in spleen of Hfe -/- and control mice. Our results in mice suggest that the observed increase of whole blood isotope composition in hemochromatosis human patients does not originate from, but is aggravated by, bloodletting. The subsequent rapid increase of whole blood iron isotope composition of treated hemochromatosis patients is rather due to the release of hepatic heavy isotope-enriched iron than augmented iron dietary absorption. Further research is required to uncover the iron light isotope component that needs to balance the accumulation of hepatic iron heavy isotope, and to better understand the iron isotope fractionation associated to metabolism dysregulation during hereditary hemochromatosis.

MRI quantification of splenic iron concentration in mouse.

PURPOSE: To quantify hepatic and splenic iron load, which is a critical issue for iron overload disease diagnosis. MRI is useful to noninvasively determine liver iron concentration, but not proven to be adequate for robust evaluation of splenic iron load. We evaluated the usefulness of MRI-derived parameters to determine splenic iron concentration in mice. MATERIALS AND METHODS: A mouse model of experimental iron load was used. Multi-echo spin-echo images of liver and spleen were acquired at 4.7 Tesla. The parameters were tested at all echoes with and without an external reference. Splenic and hepatic iron concentrations were determined using biochemical assay as the gold standard. RESULTS: Our results show that (i) use of an internal or external reference is essential; (ii) optimal echo times were TE = 19.5 ms and TE = 32.5 ms for the liver and spleen, respectively; (iii) in the liver, the relationship between biochemical and MRI iron concentration determinations is logarithmic; (iv) in the spleen, the best relationship is an inverse function. CONCLUSION: A single spin-echo sequence allows robust estimation of hepatic and splenic iron content. Parameters classically used for hepatic iron concentration cannot be applied to splenic iron determination, which requires both the specific sequence and the adapted fitting function.

Oral dysbiosis induced by Porphyromonas gingivalis is strain-dependent in mice.

Background: Porphyromonas gingivalis strain W83, one of the most widely investigated, is considered virulent in the context of periodontitis. The recently isolated P. gingivalis TDC60 has been reported to be highly pathogenic, although it has not yet been investigated in a mouse periodontitis model by oral gavage. Aim: Our aim was to compare the virulence of both strains by evaluating their impact on alveolar bone loss and the composition of oral microbiota. Methods: We inoculated by oral gavage C57BL/6 mice with either one of the two P. gingivalis strains and compared to a sham-treated group, without antibiotics pre-treatment. The mandibular alveolar bone of treated mice and controls were assessed, one month after the final inoculation, by microCT measurements. Moreover, at this time, we characterized their oral microbiota by 16S rRNA gene sequencing. Results: While P. gingivalis W83 successfully initiated periodontitis, TDC60-treated mice only experienced moderate lesions. Furthermore, only W83-treated mice exhibited a specific distinct microbiota, with significantly lower richness and evenness than other samples, and decreased proportions of taxa usually found in healthy individuals. Conclusion: This association between alveolar bone loss and a major persistent shift of the oral microbiota gives insights into virulence discrepancies among these bacterial strains.

Transcripts of ceruloplasmin but not hepcidin, both major iron metabolism genes, exhibit a decreasing pattern along the portocentral axis of mouse liver.

BACKGROUND/AIMS: During iron overload of dietary origin, iron accumulates predominantly in periportal hepatocytes. A gradient in the basal and normal transcriptional control of genes involved in iron metabolism along the portocentral axis of liver lobules could explain this feature. Therefore, we aimed at characterizing, by quantitative RT-PCR, the expression of iron metabolism genes in adult C57BL/6 mouse hepatocytes regarding lobular localisation, with special emphasis to cell ploidy, considering its possible relationship with lobular zonation. METHODS: We used two methods to analyse separately periportal and perivenous liver cells: 1) a selective liver zonal destruction by digitonin prior to a classical collagenase dissociation, and 2) laser capture microdissection. We also developed a method to separate viable 4N and 8N polyploid hepatocytes by flow cytometer. RESULTS: Transcripts of ceruloplasmin, involved in iron efflux, were overexpressed in periportal areas and the result was confirmed by in situ hybridization study. By contrast, hepcidin 1, hemojuvelin, ferroportin, transferrin receptor 2, hfe and L-ferritin mRNAs were not differentially expressed according to either lobular zonation or polyploidisation level. CONCLUSIONS: At variance with glutamine or urea metabolism, iron metabolism is not featured by a metabolic zonation lying only on a basal transcriptional control. The preferential periportal expression of ceruloplasmin raises the issue of its special role in iron overload disorders involving a defect in cellular iron export.

Decreased Bone Formation Explains Osteoporosis in a Genetic Mouse Model of Hemochromatosiss.

Osteoporosis may complicate iron overload diseases such as genetic hemochromatosis. However, molecular mechanisms involved in the iron-related osteoporosis remains poorly understood. Recent in vitro studies support a role of osteoblast impairment in iron-related osteoporosis. Our aim was to analyse the impact of excess iron in Hfe-/- mice on osteoblast activity and on bone microarchitecture. We studied the bone formation rate, a dynamic parameter reflecting osteoblast activity, and the bone phenotype of Hfe-/- male mice, a mouse model of human hemochromatosis, by using histomorphometry. Hfe-/- animals were sacrificed at 6 months and compared to controls. We found that bone contains excess iron associated with increased hepatic iron concentration in Hfe-/- mice. We have shown that animals with iron overload have decreased bone formation rate, suggesting a direct impact of iron excess on active osteoblasts number. For bone mass parameters, we showed that iron deposition was associated with bone loss by producing microarchitectural impairment with a decreased tendency in bone trabecular volume and trabecular number. A disorganization of trabecular network was found with marrow spaces increased, which was confirmed by enhanced trabecular separation and star volume of marrow spaces. These microarchitectural changes led to a loss of connectivity and complexity in the trabecular network, which was confirmed by decreased interconnectivity index and increased Minkowski's fractal dimension. Our results suggest for the first time in a genetic hemochromatosis mouse model, that iron overload decreases bone formation and leads to alterations in bone mass and microarchitecture. These observations support a negative effect of iron on osteoblast recruitment and/or function, which may contribute to iron-related osteoporosis.

Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload.

In contrast to the human genome, the mouse genome contains two HEPC genes encoding hepcidin, a key regulator of iron homeostasis. Here we report a comparative analysis of sequence, genomic structure, expression and iron regulation of mouse HEPC genes. The predicted processed 25 amino acid hepcidin 2 peptide share 68% identity with hepcidin 1 with perfect conservation of eight cysteine residues. Both HEPC1 and HEPC2 genes have similar genomic organization and have probably arisen from a recent duplication of chromosome 7 region, including the HEPC ancestral gene and a part of the adjacent USF2 gene. Insertion of a retroviral intracisternal A-particle element was found upstream of the HEPC1 gene. Both genes are highly expressed in the liver and to a much lesser extent in the heart. In contrast to HEPC1, a high amount of HEPC2 transcripts was detected in the pancreas. Expression of both genes was increased in the liver during carbonyl-iron and iron-dextran overload. Overall our data suggest that both HEPC1 and HEPC2 genes are involved in iron metabolism regulation but could exhibit different activities and/or play distinct roles.

Natural and synthetic STAT3 inhibitors reduce hepcidin expression in differentiated mouse hepatocytes expressing the active phosphorylated STAT3 form.

During the inflammatory process, hepcidin overexpression favours the development of anaemia of chronic diseases which represents the second most common form of anaemia worldwide. The identification of therapeutic agents decreasing hepcidin expression is therefore an important goal. The aim of this study was to target the STAT3 signalling involved in the development of increased hepcidin expression related to chronic inflammation. In a co-culture model associating mouse hepatocytes and rat liver epithelial cells, the mRNA levels of hepcidin1, albumin, aldolase B, Cyp3a4, Stat3, Smad4 and iron regulatory genes were measured by real-time PCR. STAT3 and phosphorylated SMAD1/5/8 proteins were analysed by Western blot. At variance of hepatocyte pure culture, co-culture provided high levels of hepcidin1 mRNA, reaching 400% of the freshly isolated hepatocyte values after 6 days of culture. Hepcidin expression was associated with the maintenance of hepatocyte phenotype, STAT3 phosphorylation and functional BMP/SMAD pathway. Stat3 siRNAs inhibited the hepcidin1 mRNA expression. STAT3 inhibitors, including curcumin, AG490 and a peptide (PpYLKTK), reduced hepcidin1 mRNA expression even when cells were additionally exposed to IL-6. Hepcidin1 mRNA was expressed at high levels by hepatocytes in the co-culture model, and STAT3 pathway activation was controlled through STAT3 inhibitors. Such inhibitors could be useful to prevent anaemia related to hepcidin overexpression during chronic inflammation.