Hepcidin secretion was not directly proportional to intracellular iron-loading in recombinant-TfR1 HepG2 cells: short communication

Hepcidin is the master regulator of systemic iron homeostasis and its dysregulation is observed in several chronic liver diseases. Unlike the extracellular iron-sensing mechanisms, the intracellular iron-sensing mechanisms in the hepatocytes that lead to hepcidin induction and secretion are incompletely understood. Here, we aimed to understand the direct role of intracellular iron-loading on hepcidin mRNA and peptide secretion using our previously characterised recombinant HepG2 cells that over-express the cell-surface iron-importer protein transferrin receptor-1. Gene expression of hepcidin (HAMP) was determined by real-time PCR. Intracellular iron levels and secreted hepcidin peptide levels were measured by ferrozine assay and immunoassay, respectively. These measurements were compared in the recombinant and wild-type HepG2 cells under basal conditions at 30 min, 2 h, 4 h and 24 h. Data showed that in the recombinant cells, intracellular iron content was higher than wild-type cells at 30 min (3.1-fold, p<0.01), 2 h (4.6-fold, p<0.01), 4 h (4.6-fold, p<0.01) and 24 h (1.9-fold, p<0.01). Hepcidin (HAMP) mRNA expression was higher than wild-type cells at 30 min (5.9-fold; p=0.05) and 24 h (6.1-fold; p<0.03), but at 4 h, the expression was lower than that in wild-type cells (p<0.05). However, hepcidin secretion levels in the recombinant cells were similar to those in wild-type cells at all time-points, except at 4 h, when the level was lower than wild-type cells (p<0.01). High intracellular iron in recombinant HepG2 cells did not proportionally increase hepcidin peptide secretion. This suggests a limited role of elevated intracellular iron in hepcidin secretio

A Novel Human Neuronal Cell Model to Study Iron Accumulation in Parkinson’s Disease

Objectives: With an estimated seven to ten million sufferers worldwide, Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder. Progress in elucidating its causes has been slow, partly due to the lack of human-relevant models. Similarly, while the contribution of iron is increasingly advocated, identifying its role in disease progression remains challenging mainly due to the lack of valid model. In this study, we created Parkinson-like conditions in a human neuron model and conducted preliminary studies on iron-related parameters to assess whether these cells replicated iron accumulation observed in Parkinsonism. Methods: ReNcell VM (human neural progenitor) were differentiated into dopaminergic neurons (dDCNs) and treated with neurotoxin 6-hydroxy dopamine (100 μM) to mimic Parkinsonism. Total intracellular, mitochondrial and cytoplasmic iron was measured by ferrozine assay. Expression of iron-related genes TFRC, SLC40A1, HAMP and SLC25A37 were assessed through real-time PCR. Results: Data showed that the treated dDCNs accumulated iron over time and exceeded levels measured in untreated dDCNs by 2.5-fold at 48 h (p<0.02). Following the treatment, the treated cells showed lower expression of TFRC (p<0.05), but substantially higher mRNA expressions of SLC40A1 (9-fold; p<0.02) and HAMP (5.7-fold; p<0.05), along with higher intracellular iron (p<0.05). Higher iron accumulation in the mitochondria than cytosol (p<0.05), was also observed with increased expression of the mitochondrial iron-importer SLC25A37 (p=0.08). Conclusion: Our Parkinsonian model demonstrates iron accumulation and elevated HAMP expression as previously described in PD phenotype. The observed mitochondrial iron shuttling, which is proposed to be one of the primary contributors of oxidative stress in PD, calls for further investigation. The differences observed in distribution of iron in our human model and with the expression of major iron-related proteins, indicate that our model reproduces the disease state successfully, and suggests that further study could help in advancing our understanding of PD

HFE mRNA expression is responsive to intracellular and extracellular iron loading:short communication

In liver hepatocytes, the HFE gene regulates cellular and systemic iron homeostasis by modulating cellular iron-uptake and producing the iron-hormone hepcidin in response to systemic iron elevation. However, the mechanism of iron-sensing in hepatocytes remain enigmatic. Therefore, to study the effect of iron on HFE and hepcidin (HAMP) expressions under distinct extracellular and intracellular iron-loading, we examined the effect of holotransferrin treatment (1, 2, 5 and 8 g/L for 6 h) on intracellular iron levels, and mRNA expressions of HFE and HAMP in wild-type HepG2 and previously characterized iron-loaded recombinant-TfR1 HepG2 cells. Gene expression was analyzed by real-time PCR and intracellular iron was measured by ferrozine assay. Data showed that in the wild-type cells, where intracellular iron content remained unchanged, HFE expression remained unaltered at low holotransferrin treatments but was upregulated upon 5 g/L (p < 0.04) and 8 g/L (p = 0.05) treatments. HAMP expression showed alternating elevations and increased upon 1 g/L (p < 0.05) and 5 g/L (p < 0.05). However, in the recombinant cells that showed higher intracellular iron levels than wild-type cells, HFE and HAMP expressions were elevated only at low 1 g/L treatment (p < 0.03) and were repressed at 2 g/L treatment (p < 0.03). Under holotransferrin-untreated conditions, the iron-loaded recombinant cells showed higher expressions of HFE (p < 0.03) and HAMP (p = 0.05) than wild-type cells. HFE mRNA was independently elevated by extracellular and intracellular iron-excess. Thus, it may be involved in sensing both, extracellular and intracellular iron. Repression of HAMP expression under simultaneous intracellular and extracellular iron-loading resembles non-hereditary iron-excess pathologies

Comparison study of iron preparations using a human intestinal model

Iron deficiency and related iron deficiency anaemia (IDA) are the most prevalent nutritional disorders worldwide. The standard treatment involves supple-mentation with solid or liquid iron supplement preparations, usually based on a ferrous salt such as ferrous sulphate, ferrous fumarate, or ferrous gluconate. In the present study, we compared iron uptake and absorption from various solid and liquid iron supplement preparations currently available in the United Kingdom using the well-characterised human epithelial adenocarcinoma cell line Caco-2. Intracellular ferritin protein formation by the Caco-2 cell was considered an indicator of cellular iron uptake and absorption. We investigated the effects of formulation ingredients at a defined pH on iron uptake and absorption, and designed a novel two-stage dissolution-absorption protocol that mimicked physiological conditions. Our experiments revealed wide variations in the rate of dissolution between the various solid iron preparations. Conventional-release ferrous iron tablets dissolved rapidly (48 ± 4 mins to 64 ± 4 mins), whereas modified-released tablets and capsules took significantly longer to undergo complete dissolution (274 ± 8 to 256 ± 8 mins). Among the solid iron preparations, ferrous sulphate conventional-release tablets demon-strated the highest iron absorption, whereas modified-release ferrous prepa-rations demonstrated uniformly low iron absorption, as compared to the control (P < 0.05). Taken together, our results demonstrate that there are wide-ranging variations in dissolution times and iron uptake from oral iron preparations, with the physical characteristics of the preparation as well as the form of iron playing a key role

Identification of an iron–hepcidin complex

Following its identification as a liver-expressed antimicrobial peptide, the hepcidin peptide was later shown to be a key player in iron homoeostasis. It is now proposed to be the 'iron hormone' which, by interacting with the iron transporter ferroportin, prevents further iron import into the circulatory system. This conclusion was reached using the corresponding synthetic peptide, emphasizing the functional importance of the mature 25-mer peptide, but omitting the possible functionality of its maturation. From urine-purified native hepcidin, we recently demonstrated that a proportion of the purified hepcidin had formed iron-hepcidin complexes. This interaction was investigated further by computer modelling and, based on the sequence similarity of hepcidin with metallothionein, a three-dimensional model of hepcidin, containing one atom of iron, was constructed. To characterize these complexes further, the interaction with iron was analysed using different spectroscopic methods. Monoferric hepcidin was identified by MS, as were possibly other complexes containing two and three atoms of iron respectively, although these were present only in minor amounts. UV/visible absorbance and CD studies identified the iron-binding events which were facilitated at a physiological pH. EPR spectroscopy identified the ferric state of the bound metal, and indicated that the iron-hepcidin complex shares some similarities with the rubredoxin iron-sulfur complex, suggesting the presence of Fe(3+) in a tetrahedral sulfur co-ordination. The potential roles of iron binding for hepcidin are discussed, and we propose either a regulatory function in the maturation of pro-hepcidin into active hepcidin or as the necessary link in the interaction between hepcidin and ferroportin

Influence of vitamin D supplementation on immune function of healthy aging people:A pilot randomized controlled trial

Objectives: This study aims to investigate the influence of vitamin D supplementation on immune function of healthy older adults. Materials and methods: Designed as a randomized controlled trial, 21 participants (55–85 years) completed the study during May–November 2018 in Coventry, England. The participants were randomized into vitamin D or the control group, stratified by age, gender and body mass index. The vitamin D group (n = 12) took vitamin D3 tablets of 1,000 IU/day for 12 weeks plus vitamin D education leaflet, while the control group (n = 9) were only provided with the leaflet. At baseline, 6 and 12 weeks, plasma 25(OH)D levels and immunological and metabolic parameters including phagocytic activity of granulocytes and monocytes, tumor necrosis factor, interleukin 6, lymphocyte subsets and fasting blood glucose and lipid were measured. Dietary vitamin D intake was analyzed at baseline and week 12. Data were presented as mean ± SD. Two-way repeated measures ANOVA and independent t-test were used to analyze the data. Results: At baseline, 42.9% of the participants were vitamin D deficiency (25(OH)D 50 nmol/L. Overweight/obese participants (n = 9) had significantly lower mean plasma 25(OH)D concentration (22.3 ± 8.7 nmol/L) than normal weight participants (48.1 ± 34.3 nmol/L) (P = 0.043). There was a significant increase in plasma 25(OH)D concentration in vitamin D group compared with that in control group (P = 0.002) during the intervention period. The plasma 25(OH)D concentration in vitamin D group was increased at 6 weeks (from 38.4 ± 37.0 nmol/L at baseline to 51.0 ± 38.2 nmol/L) with little change observed between 6 and 12 weeks (51.8 ± 36.4 nmol/L). The plasma creatinine concentration in vitamin D group was significantly decreased compared with the control group (P = 0.036) (79.8 ± 7.0 μmol/L at baseline vs 75.1 ± 5.4 μmol/L at week 12). No significant effect of vitamin D supplementation was determined on immunological parameters. Conclusion: Vitamin D deficiency is common among the aging population in the UK even during the summertime. Vitamin D supplementation at 1,000 IU/day for 12 weeks significantly increased plasma 25(OH)D concentration but showed no effect on metabolic and immunological parameters except decreased plasma creatinine