High Dietary Iron Research Articles

Peroxiredoxin 5 deficiency exacerbates iron overload-induced neuronal death via ER-mediated mitochondrial fission in mouse hippocampus

Iron is an essential element for cellular functions, including those of neuronal cells. However, an imbalance of iron homeostasis, such as iron overload, has been observed in several neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Iron overload causes neuronal toxicity through mitochondrial fission, dysregulation of Ca2+, ER-stress, and ROS production. Nevertheless, the precise mechanisms between iron-induced oxidative stress and iron toxicity related to mitochondria and endoplasmic reticulum (ER) in vivo are not fully understood. Here, we demonstrate the role of peroxiredoxin 5 (Prx5) in iron overload-induced neurotoxicity using Prx5-deficient mice. Iron concentrations and ROS levels in mice fed a high iron diet were significantly higher in Prx5−/− mice than wildtype (WT) mice. Prx5 deficiency also exacerbated ER-stress and ER-mediated mitochondrial fission via Ca2+/calcineurin-mediated dephosphorylation of Drp1 at Serine 637. Moreover, immunoreactive levels of cleaved caspase3 in the CA3 region of the hippocampus were higher in iron-loaded Prx5−/− mice than WT mice. Furthermore, treatment with N-acetyl-cysteine, a reactive oxygen species (ROS) scavenger, attenuated iron overload-induced hippocampal damage by inhibiting ROS production, ER-stress, and mitochondrial fission in iron-loaded Prx5−/− mice. Therefore, we suggest that iron overload-induced oxidative stress and ER-mediated mitochondrial fission may be essential for understanding iron-mediated neuronal cell death in the hippocampus and that Prx5 may be useful as a novel therapeutic target in the treatment of iron overload-mediated diseases and neurodegenerative diseases.

Gnpat does not play an essential role in systemic iron homeostasis in murine model.

The GNPAT variant rs11558492 (p.D519G) was identified as a novel genetic factor that modifies the iron‐overload phenotype in homozygous carriers of the HFE p.C282Y variant. However, the reported effects of the GNPAT p.D519G variant vary among study populations. Here, we investigated the role of GNPAT in iron metabolism using Gnpat‐knockout (Gnpat−/−), Gnpat/Hfe double‐knockout (Gnpat−/−Hfe−/− or DKO) mice and hepatocyte‐specific Gnpat‐knockout mice (Gnpatfl/fl;Alb‐Cre). Our analysis revealed no significant difference between wild‐type (Gnpat+/+) and Gnpat−/− mice, between Hfe−/− and DKO mice, or between Gnpatfl/fl and Gnpatfl/fl;Alb‐Cre with respect to serum iron and tissue iron. In addition, the expression of hepcidin was not affected by deleting Gnpat expression in the presence or absence of Hfe. Feeding Gnpat−/− and DKO mice a high‐iron diet had no effect on tissue iron levels compared with wild‐type and Hfe−/− mice, respectively. Gnpat knockdown in primary hepatocytes from wild‐type or Hfe−/− mice did not alter hepcidin expression, but it repressed BMP6‐induced hepcidin expression. Taken together, these results support the hypothesis that deleting Gnpat expression has no effect on either systemic iron metabolism or the iron‐overload phenotype that develops in Hfe−/− mice, suggesting that GNPAT does not directly mediate iron homeostasis under normal or high‐iron dietary conditions.

Iron intake, oxidative stress-related genes and breast cancer risk.

Iron has been suggested to contribute to breast cancer development through oxidative stress generation. Our study investigated associations between iron intake and breast cancer risk, overall and by menopausal and estrogen receptor/progesterone receptor (ER/PR) status, and modification by oxidative stress-related genetic polymorphisms (MnSOD, GSTM1 and GSTT1). A population-based case-control study (3,030 cases and 3,402 controls) was conducted in Ontario, Canada. Iron intake (total, dietary, supplemental, heme, nonheme) was assessed using a validated food frequency questionnaire. Odds ratios (OR) and 95% confidence intervals (CI) were estimated from multivariable logistic regression models. Interactions between iron intake and genotypes were assessed among 1,696 cases and 1,761 controls providing DNA. Overall, no associations were observed between iron intake and breast cancer risk. Among premenopausal women, total, dietary and dietary nonheme iron were positively associated with ER-/PR- breast cancer risk (all ptrend < 0.05). Among postmenopausal women, supplemental iron was associated with reduced breast cancer risk (OR>18 vs. 0 mg/day = 0.68, 95% CI: 0.51-0.91), and dietary heme iron was associated with an increased risk, particularly the ER-/PR- subtype (ORhighest vs. lowest quintile = 1.69, 95% CI: 1.16-2.47; ptrend = 0.02). Furthermore, GSTT1 and combined GSTM1/GSTT1 polymorphisms modified some of the associations. For example, higher dietary iron was most strongly associated with increased breast cancer risk among women with GSTT1 deletion or GSTM1/GSTT1 double deletions (pinteraction < 0.05). Findings suggest that iron intake may have different effects on breast cancer risk according to menopausal and hormone receptor status, as well as genotypes affecting antioxidant capacity.

Iron and hepcidin mediate human colorectal cancer cell growth

High dietary iron intake is a risk factor for the development of colorectal cancer. However, how iron subsequently impacts the proliferation of colorectal cancer cells remains unclear. This study determined the expression of six iron regulatory genes in twenty-one human colorectal cancer (CRC) biopsies and matched normal colonic tissue. The results show that only hepcidin and ferritin heavy chain expression were increased in CRC biopsies as compared to matched normal tissues. Four established human CRC cell lines, HT-29, HCT-116, SW-620 and SW-480 were subsequently examined for their growth in response to increasing concentrations of iron, and iron depletion. Real time cell growth assay showed a significant inhibitory effect of acute iron loading in HCT-116 cells (IC50 = 258.25 μM at 72 h), and no significant effects in other cell types. However, ten week treatment with iron significantly reduced HT-29 and SW-620 cell growth, whereas no effect was seen in HCT-116 and SW-480 cells. Intracellular labile iron depletion induced the complete growth arrest and detachment of all of the CRC cell types except for the SW-620 cell line which was not affected in its growth. Treatment of starved CRC cells with hepcidin, the major regulator of iron metabolism, induced a significant stimulation of HT-29 cell growth but did not affect the growth of the other cell types. Collectively these results show that iron is central to CRC cell growth in a manner that is not identical between acute and chronic loading, and that is specific to the CRC cell type.

Iron overload as a risk factor for hepatic ischemia-reperfusion injury in liver transplantation: Potential role of ferroptosis.

Hepatic ischemia-reperfusion (I/R) injury is a major problem in liver transplantation (LT). Although hepatocyte cell death is the initial event in hepatic I/R injury, the underlying mechanism remains unclear. In the present study, we retrospectively analyzed the clinical data of 202 pediatric living donor LT and found that a high serum ferritin level, a marker of iron overload, of the donor is an independent risk factor for liver damage after LT. Since ferroptosis has been recently discovered as an iron-dependent cell death that is triggered by a loss of cellular redox homeostasis, we investigated the role of ferroptosis in a murine model of hepatic I/R injury, and found that liver damage, lipid peroxidation, and upregulation of the ferroptosis marker Ptgs2 were induced by I/R, and all of these manifestations were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) or α-tocopherol. Fer-1 also inhibited hepatic I/R-induced inflammatory responses. Furthermore, hepatic I/R injury was attenuated by iron chelation by deferoxamine and exacerbated by iron overload with a high iron diet. These findings demonstrate that iron overload is a novel risk factor for hepatic I/R injury in LT, and ferroptosis contributes to the pathogenesis of hepatic I/R injury.

High Dietary Iron Disrupts Iron Homeostasis and Induces Amyloid-β and Phospho-τ Expression in the Hippocampus of Adult Wild-Type and APP/PS1 Transgenic Mice

ABSTRACTBackgroundBrain iron deposition is a feature of Alzheimer disease and may contribute to its development. However, the relative contribution of dietary iron remains unclear.ObjectivesWe investigated the impact of high dietary iron on brain pathological changes and cognitive function in adult wild-type (WT) mice and amyloid precursor protein/presenilin 1 (APP/PS1) double transgenic mice.MethodsMale WT mice and APP/PS1 mice aged 10 wk were fed either a control diet (66 mg Fe/kg) (WT-Ctrl and APP/PS1-Ctrl) or a high iron diet (14 g Fe/kg) (WT-High Fe and APP/PS1-High Fe) for 20 wk. Iron concentrations in brain regions were measured by atomic absorption spectrophotometry. Brain iron staining and amyloid-β (Aβ) immunostaining were performed. Protein expressions in the hippocampus were determined by immunoblotting. Superoxide dismutase (SOD) activity and malondialdehyde concentration were examined. Cognitive functions were tested with the Morris water maze system.ResultsIn the hippocampus, APP/PS1-High Fe mice had significantly higher iron concentration (2.5-fold) and ferritin (2.0-fold) than APP/PS1-Ctrl mice (P < 0.001), and WT-High Fe mice had significantly higher ferritin (2.0-fold) than WT-Ctrl mice (P < 0.001). Interestingly, APP/PS1 mice had significantly higher iron concentration (2–3-fold) and ferritin (2–2.5-fold) than WT mice fed either diet (P < 0.001). Histological analysis indicated that iron accumulated in the hippocampal dentate gyrus region in APP/PS1 mice, consistent with the pattern of Aβ deposition. For both mouse strains, iron treatment induced Aβ and phospho-τ expression (1.5–3-fold) in the hippocampus, but had little impact on oxidative stress and cognitive function. Furthermore, APP/PS1 mice had significantly lower SOD activity and higher malondialdehyde concentration than WT mice in the hippocampus (P < 0.0001), paralleled by apparent cognitive dysfunction.ConclusionsDietary iron overload induces iron disorder and Aβ and phospho-τ expression in the hippocampus of adult WT and APP/PS1 transgenic mice.

Deferiprone and efonidipine mitigated iron-overload induced neurotoxicity in wild-type and thalassemic mice

AimsWe previously demonstrated that iron-overload in non-thalassemic rats induced neurotoxicity and cognitive decline. However, the effect of iron-overload on the brain of thalassemic condition has never been investigated. An iron chelator (deferiprone) provides neuroprotective effects against metal toxicity. Furthermore, a T-type calcium channels blocker (efonidipine) effectively attenuates cardiac dysfunction in thalassemic mice with iron-overload. However, the effects of both drugs on brain of iron-overload thalassemia has not been determined. We hypothesize that iron-overload induces neurotoxicity in Thalassemic and wild-type mice, and not only deferiprone, but also efonidipine, provides neuroprotection against iron-overload condition. Main methodsMice from both wild-type (WT) and β-thalassemic type (HT) groups were assigned to be fed with a standard-diet or high-iron diet containing 0.2% ferrocene/kg of diet (HFe) for 4 months consecutively. After three months of HFe, 75-mg/kg/d deferiprone or 4-mg/kg/d efonidipine were administered to the HFe-fed WT and HT mice for 1 month. Key findingsHFe consumption caused an equal impact on circulating iron-overload, oxidative stress, and inflammation in WT and HT mice. Brain iron-overload and iron-mediated neurotoxicity, such as oxidative stress, inflammation, glial activation, mitochondrial dysfunction, and Alzheimer's like pathologies, were observed to an equal degree in HFe fed WT and HT mice. These pathological conditions were mitigated by both deferiprone and efonidipine. SignificanceThese findings indicate that iron-overload itself caused neurotoxicity, and T-type calcium channels may play a role in this condition.

Combined iron chelator with N-acetylcysteine exerts the greatest effect on improving cardiac calcium homeostasis in iron-overloaded thalassemic mice

The morbidity and mortality in thalassemia patients are predominantly caused by iron overload cardiomyopathy (IOC). Iron-induced cardiac intracellular Ca2+ ([Ca2+]i) dysregulation is among the core pathophysiological processes in IOC-related heart failure. Although cardioprotective roles of deferiprone (DFP) and N-acetylcysteine (NAC) have been reported, their effect on cardiac [Ca2+]i transients and Ca2+-regulatory protein expression in thalassemic mice is unknown. In the present study, iron overload condition was induced in wild-type (WT) and heterozygous β-thalassemic (HT) mice by a high-iron diet. The iron-overloaded mice subsequently received a vehicle, DFP, NAC, or DFP plus NAC co-therapy. In both WT and HT iron-overloaded mice, DFP and NAC had similar efficacy in decreasing plasma non-transferrin-bound iron, decreasing cardiac iron concentration (CIC) and relieving systolic dysfunction. DFP plus NAC co-therapy, however, was better than the monotherapy in reducing CIC and restoring cardiac [Ca2+]i transient amplitude and rising rate. All regimens produced no change in cardiac Ca2+-regulatory protein expression. We provided the first evidence regarding the synergistic effect of combined iron chelator-antioxidant therapy on cardiac [Ca2+]i homeostasis in iron-overloaded thalassemic mice, with consistent improvement of cardiac contractility.

Early-Life Iron Deficiency Alters Glucose Transporter-1 Expression in the Adult Rodent Hippocampus

Early-life iron deficiency (ID) impairs hippocampal energy production. Whether there are changes in glucose transporter (GLUT) expression is not known. The aim of this study was to investigate whether early-life ID and the treatment iron dose alter brain regional GLUT expression in adult rats and mice. In Study 1, ID was induced in male and female Sprague Dawley rat pups by feeding dams a 3-mg/kg iron diet during gestation and the first postnatal week, followed by treatment using low-iron [3-10 mg/kg; formerly iron-deficient (FID)-10 group], standard-iron (40-mg/kg; FID-40 group), or high-iron (400-mg/kg; FID-400 group) diets until weaning. The control group received the 40 mg/kg iron diet. GLUT1, GLUT3, hypoxia-inducible factor (HIF)-1α, and prolyl-hydroxylase-2 (PHD2) mRNA and protein expression in the cerebral cortex, hippocampus, striatum, cerebellum, and hypothalamus were determined at adulthood. In Study 2, the role of hippocampal ID in GLUT expression was examined by comparing the Glut1, Glut3, Hif1α, and Phd2 mRNA expression in adult male and female wild-type (WT) and nonanemic hippocampal iron-deficient and iron-replete dominant negative transferrin receptor 1 (DNTfR1-/-) transgenic mice. In Study 1, Glut1, Glut3, and Hif1α mRNA, and GLUT1 55-kDa protein expression was upregulated 20-33% in the hippocampus of the FID-10 group but not the FID-40 group, relative to the control group. Hippocampal Glut1 mRNA (-39%) and GLUT1 protein (-30%) expression was suppressed in the FID-400 group, relative to the control group. Glut1 and Glut3 mRNA expression was not altered in the other brain regions in the 3 FID groups. In Study 2, hippocampal Glut1 (+14%) and Hif1α (+147%) expression was upregulated in the iron-deficient DNTfR1-/- mice, but not in the iron-replete DNTfR1-/- mice, relative to the WT mice (P<0.05, all). Early-life ID is associated with altered hippocampal GLUT1 expression in adult rodents. The mouse study suggests that tissue ID is potentially responsible.

Abstract 96: High iron or high fat diet show similar effects in inducing hepatocellular carcinoma in a mouse model

Abstract Introduction: Hepatocellular carcinoma (HCC) has multiple risk factors such as Hepatitis virus infection and obesity. Iron overload may be another risk factor as patients with hemochromatosis have high levels of liver iron accumulation and higher incidence of HCC than the general population. This study aims to assess the risk of high iron diet in comparison to high fat diet in the development of HCC. Materials &amp; Methods: We used C3HeB/FeJ male mice, which have been shown to spontaneously develop HCC. Two-month old mice were fed for 15 months with a control diet (10 kcal% from fat; 48 ppm Iron), high iron diet (HID) (10 kcal% from fat; 200 ppm Iron), high fat diet (HFD) (61.1 kcal% from fat; 48 ppm Iron), or high iron and fat diet (HIFD) (61.1 kcal% from fat; 200 ppm Iron). The presence of liver tumor was confirmed by ultrasound imaging and histology analysis. At termination, normal and tumor liver tissues were harvested for tumor size measurement, metal ion quantification, immunohistochemistry, and gene expression with RNA-seq. Results: HID like HFD induced significant body weight gain. All three diets induced HCC (4 out of 7 by HID, 7 out of 9 by HFD, and 8 out of 10 by HIFD). Yet, HCC was not found in the control diet group. The three diets showed no significantly different effects on tumor burden and grade. The concentrations of iron and copper were increased in the normal liver tissues of HID and HIFD groups. Interestingly, they were significantly lower in the tumor tissues than in the paired adjacent non-tumor tissues. Analyses of RNA-seq data revealed extensive overlap of tumor-specific differentially expressed genes (DEGs) among the three diet groups. The extensive overlap was also observed in the DEG-associated gene otology (GO) terms among the three diet groups. All three diets, but not the control diet, were shown to activate mTORC1 pathway in the tumors according to our RNA-seq data. The activation of mTORC1 pathway in the three diet-induced tumors was also confirmed with Real-time RT-PCR assays revealing upregulation of mTORC1 target genes and with immunohistochemistry showing increased phosphorylation of an mTORC1 substrate. Conclusions: The spontaneous hepatocarcinogenesis in male C3HeB/FeJ mice requires HID or HFD. The lack of additive effect of HID and HFD on HCC incidence and the extensive overlap of DEGs and GO terms among tumors induced by HID and HFD suggest they may regulate common mechanisms such as mTORC1 in inducing HCC. Citation Format: Hakim Bouamar, Kalyan Kakarla, Matyas Cserhati, Fatima Ezzahra El Mroussi, Francis E Sharkey, Francisco Cigarroa, Lu-Zhe Sun. High iron or high fat diet show similar effects in inducing hepatocellular carcinoma in a mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 96.

Abstract 623: Iron intake, oxidative stress-related genes, and breast cancer risk

Abstract Background: Iron has been shown to enhance breast carcinogenesis in animal models through generation of oxidative stress, although epidemiological evidence of the association between iron intake and breast cancer risk remains inconclusive. This study investigated associations between different types of iron intake and breast cancer risk, and whether these associations were modified by genetic polymorphisms in antioxidant enzymes, including manganese superoxide dismutase (MnSOD) and glutathione S-transferases M1 (GSTM1) and T1 (GSTT1). Methods: A population-based case-control study in Ontario, Canada recruited 3,030 breast cancer cases identified from the Ontario Cancer Registry and 3,402 controls from random digit dialing. Iron intake from foods and supplements was assessed using a 178-item food frequency questionnaire. Multivariable logistic regression analyses were used to evaluate associations between breast cancer risk and intakes of dietary, supplemental, and total (dietary plus supplemental) iron, among all women and stratified by menopausal status. Associations were also examined by hormone receptor [estrogen receptor (ER)/progesterone receptor (PR)] tumor subtype. Among women providing saliva (DNA) samples (1,696 cases and 1,761 controls), interactions between iron intake and genetic polymorphisms were assessed using the likelihood ratio test. Results: Among all women, intakes of dietary, supplemental, and total iron were not associated with breast cancer risk, overall or by tumor subtype. Among premenopausal women, there was an increase in breast cancer risk for the ER–PR– subtype associated with higher intakes (highest vs. lowest quintile) of dietary [odds ratio (OR) = 1.4; 95% confidence interval (CI): 0.9–2.4] and total (OR = 1.8; 95% CI: 1.1–2.9) iron (P for trend &amp;lt; 0.05). Among postmenopausal women, supplemental iron intake (&amp;gt;18 vs. 0 mg/day) was associated with reduced breast cancer risk (OR = 0.7; 95% CI: 0.5–0.9), with similar associations across tumor subtypes. Associations of dietary and total iron intake with overall breast cancer risk were modified by GSTT1 and/or GSTM1/T1 combined genotypes (P for interaction &amp;lt; 0.05). For example, among women with deletions in both GSTM1 and GSTT1 loci, higher dietary iron intake was associated with increased breast cancer risk (OR = 2.1; 95% CI: 1.1–4.2), whereas null or inverse associations were found among women with other GSTM1/T1 genotypes. Conclusions: Results from this study suggest that higher dietary and total iron intake may be associated with increased risk of ER–PR– breast cancer among premenopausal women, whereas higher supplemental iron intake may be associated with reduced postmenopausal breast cancer risk. In addition, associations between iron intake and breast cancer risk may be modified by polymorphisms in oxidative stress-related genes. Our ongoing work will investigate heme and non-heme iron intake in relation to breast cancer risk. Citation Format: Vicky C. Chang, Michelle Cotterchio, Susan J. Bondy, Joanne Kotsopoulos. Iron intake, oxidative stress-related genes, and breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 623.

1755-P: Transferrin Role in Systemic Iron Influence on Insulin Sensitivity and Adipocyte Function

Systemic iron overload is associated with decreased insulin sensitivity (Si) and diabetes. We previously characterized the genetic regulation of iron and its effect on Si in humans, which demonstrated that expression of the iron transport protein transferrin (Tf) in adipose tissue plays a role in regulating insulin sensitivity. To further characterize this effect at the tissue and cellular levels, we generated heterozygous Tf knock out (KO) mice and human adipose cells (ASC52Telo) with knock down (KD) of Tf. Heterozygous Tf KO and wild type (WT) mice were fed a 40% fat “fast-food” diets, supplemented with glucose in the water, containing either 35mg or 2000mg iron per kg chow. Fasting glucose levels were not affected in the Tf KO mice compared to WT (p=0.3889) on the low iron diet, but increased in the KO mice on 2gm/kg iron (13% increase, p=0.0511) suggesting the attenuation of the protective effect of transferrin in blood sugar regulation at high iron diet. We previously reported that adipose cells transfected with TF shRNA compared to a control vector had decreased intracellular iron and exhibited insulin resistance. We also performed mitochondrial function studies (oxygen consumption analyzed by Seahorse) in these cells. Tf KD cells exhibited decreased maximal oxygen consumption compared to controls (p:0.0004). Iron treatment of the cells augmented respiration in the control cells but worsened maximal oxygen consumption in the Tf KD cells (p=0.0011). We conclude that transferrin-mediated iron transport in adipocytes plays an important role in determining insulin sensitivity, allowing beneficial responses to high iron, namely protection from hyperglycemia and augmented mitochondrial function. Disclosure F. Lorenzo: None. S. Jain: None. D. McClain: None. S.K. Das: None. Funding U.S. Department of Veterans Affairs

A Piglet Model Studying the Role of Dietary Iron on Host Resilience to Enterotoxigenic E. Coli Infection in Early Childhood (P24-057-19)

ObjectivesAlthough iron fortification/supplementation is recommended for infants (6 to 12 months) and toddlers by pediatrics, the optimal dose is still largely debated. One of controversies centers on whether iron fortification at recommended dose increases the risk of infection. Using a weanling piglet model, we aim to assess the dose of iron fortification on host susceptibility to enterotoxigenic E. coli (ETEC) infection. MethodsThirty-two weanling piglets were randomly assigned to four treatments on PD21, including normal iron diet (250 mg/kg) without ETEC challenge (CON), and Low (125 mg/kg; LOI), normal (COI), or high iron (750 mg/kg; HII) diets with ETEC challenge. On study day 10 (d10), piglets were orally inoculated with 1010 cfu/dose of F18 ETEC once daily for 3 consecutive days. The pathogen colonize in small intestine and cause watery diarrhea in weanling pigs. Piglets were euthanized on d16 for tissue sampling. A 5-scale fecal score were recorded daily. Feces collected on d10, 13 and 16 were plated on blood and MacConkey agars to verify absence or presence of the pathogen. Blood sampled on d0, 10, 13 and 16 were analyzed for hemoglobin, hematocrit, iron and cytokines. Intestinal tissue sections will be stained for iron using Prussian blue and spatial localization of E. coli through fluorescence in situ hybridization. ResultsThe pathogen was absent in feces of all piglets on d10 before inoculation, but was detected at different abundances only in ETEC-inoculated piglets on d13. By d16, only 2 fecal samples from each ETEC-inoculated group were positive for the pathogen, which was accompanied by improved fecal scores. Piglets in LOI suffered more days of diarrhea (fecal score >2) as compared to CON and COI (P < 0.05). Iron content did not affect BW by d10. Nonetheless, piglets in LOI had lower BW (P < 0.05) than those in HII and CON on d16. Hemoglobin was consistently higher in HII than that in COI and LOI. Enteric infection reduced hemoglobin regardless of dietary iron content. ConclusionsOur preliminary results showed both iron inadequacy and excess exacerbated ETEC-induced diarrheal illness. However, high iron improved growth during transient ETEC infection in weanling pigs. Funding SourcesUC Davis; NIFA.

Iron down-regulates leptin by suppressing protein O-GlcNAc modification in adipocytes, resulting in decreased levels of O-glycosylated CREB

We previously reported that iron down-regulates transcription of the leptin gene by increasing occupancy of phosphorylated cAMP response element-binding protein (pCREB) at two sites in the leptin gene promoter. Several nutrient-sensing pathways including O-GlcNAcylation also regulate leptin. We therefore investigated whether O-glycosylation plays a role in iron- and CREB-mediated regulation of leptin. We found that high iron decreases protein O-GlcNAcylation both in cultured 3T3-L1 adipocytes and in mice fed high-iron diets and down-regulates leptin mRNA and protein levels. Glucosamine treatment, which bypasses the rate-limiting step in the synthesis of substrate for glycosylation, increased both O-GlcNAc and leptin, whereas inhibition of O-glycosyltransferase (OGT) decreased O-GlcNAc and leptin. The increased leptin levels induced by glucosamine were susceptible to the inhibition by iron, but in the case of OGT inhibition, iron did not further decrease leptin. Mice with deletion of the O-GlcNAcase gene, either via whole-body heterozygous deletion or through adipocyte-targeted homozygous deletion, exhibited increased O-GlcNAc levels in adipose tissue and increased leptin levels that were inhibited by iron. Of note, iron increased the occupancy of pCREB and decreased the occupancy of O-GlcNAcylated CREB on the leptin promoter. These patterns observed in our experimental models suggest that iron exerts its effects on leptin by decreasing O-glycosylation and not by increasing protein deglycosylation and that neither O-GlcNAcase nor OGT mRNA and protein levels are affected by iron. We conclude that iron down-regulates leptin by decreasing CREB glycosylation, resulting in increased CREB phosphorylation and leptin promoter occupancy by pCREB.

Iron-load exacerbates the severity of atherosclerosis via inducing inflammation and enhancing the glycolysis in macrophages.

Atherosclerosis is still the major cause of morbidity and mortality all over the world. Recently, it has been reported increased levels of tissue iron increase the risk of atherosclerosis. However, the detailed mechanism of iron-induced atherosclerosis progression is barely known. Here, we used apoE-deficient mice models to investigate the effects of low iron diet (<0 mg iron carbonyl/kg), high iron diet (25,000 mg iron carbonyl/kg) on atherosclerosis in vivo. As exhibited, we observed that CD68 was significant enriched by high iron diet in apoE-deficient mice. In addition, transforming growth factor β, tumor necrosis factor α, interleukin 6 (IL-6), IL-23, IL-10, and IL-1β levels were also greatly induced by high iron diet. Then, we found that the iron load promoted the inflammation response in macrophages. Moreover, macrophage polarization is a process by which macrophage can express various functional programs in activating macrophages. Here, we observed that iron-load macrophages were polarized toward a proinflammatory macrophage phenotype. The polarization of M1 macrophage was promoted by ferric ammonium citrate (FAC) in bone marrow derived macrophages (BMDMs). Furthermore, ECAR and cellular OCR in BMDM with or without FAC was examined. As shown, BMDM indicated with 50 μM FAC showed a significant increase in basic state and maximal ECAR in contrast to the control group. However, there was no significant difference in OCR. This indicated that the glycolysis was involved in the polarization of M1 macrophage triggered by iron-load. In conclusion, we indicated that the iron load exacerbates the progression of atherosclerosis via inducing inflammation and enhancing glycolysis in macrophages.

The Role of Iron and Nerve Inflammation in Diabetes Mellitus Type 2-Induced Peripheral Neuropathy

Peripheral diabetic neuropathy (PDN) is one of the most common complications of diabetes mellitus. Previous studies showed an association between dietary iron load and inflammation in the development of PDN in a rat model of type 1 diabetes (T1D). Here we investigated the role of iron and neural inflammation in development of PDN in a animal model of obesity and type 2 diabetes (T2D). 3-month-old db/db mice were fed with a high, standard or low iron diet for 4 months. High iron chow lead to a significant increase in motor nerve conduction velocities compared to mice on standard and low iron chow. Direct beneficiary effects on lowering blood glucose and HbA1c concentrations were shown in the high iron treated diabetic mice. Numbers of pro-inflammatory M1 macrophages were reduced in nerve sections, and anti-inflammatory M2 macrophages were increased in db/db mice on high iron diet compared to other groups. These results confirm and extend our previous findings in STZ-diabetic rats by showing that dietary non-hem iron supplementation may partly prevent the development of PDN in opposition to iron restriction. The identification of these dietary iron effects on the metabolic and inflammatory mechanisms of PDN supports a role of dietary iron and leads us to suggest testing for iron levels in human diabetic patients.

Iron Exposure and the Cellular Mechanisms Linked to Neuron Degeneration in Adult Mice.

Although the causal relationship between Alzheimer’s disease (AD) and iron overload remains unclear, iron dyshomeostasis or improper transport mechanisms are speculated to lead to the accumulation of this neurotoxic metal in the hippocampal formation and other cerebral areas related to neurodegenerative diseases, resulting in the formation of reactive oxygen species (ROS) and, ultimately, cell death. In this study, exposure to high dietary iron (HDI) revealed no significant difference in the number of iron-positive cells and iron content in the cortex and hippocampal region between wild-type (WT) and APP/PS1 mice; however, compared with the control mice, the HDI-treated mice exhibited upregulated divalent metal transporter 1 (DMT1) and ferroportin (Fpn) expression, and downregulated transferrin receptor (TFR) expression. Importantly, we confirmed that there were significantly fewer NeuN-positive neurons in both APP/PS1 and WT mice given HDI, than in the respective controls. Moreover, this iron-induced neuron loss may involve increased ROS and oxidative mitochondria dysfunction, decreased DNA repair, and exacerbated apoptosis and autophagy. Although HDI administration might trigger protective antioxidant, anti-apoptosis, and autophagy signaling, especially in pathological conditions, these data clearly indicate that chronic iron exposure results in neuronal loss due to apoptosis, autophagy, and ferroptosis, hence increasing the risk for developing AD.

Dietary iron interacts with genetic background to influence glucose homeostasis

BackgroundIron is a critical component of metabolic homeostasis, but consumption of dietary iron has increased dramatically in the last 30 years, corresponding with the rise of metabolic disease. While the link between iron metabolism and metabolic health is well established, the extent to which dietary iron contributes to metabolic disease risk is unexplored. Further, it is unknown how dietary iron interacts with genetic background to modify metabolic disease risk.MethodsLG/J and SM/J inbred mouse strains were used to investigate the relationship between genetic background and metabolic function during an 8-week high iron diet. Glucose tolerance and adiposity were assessed, colorimetric assays determined levels of circulating metabolic markers, and hepatic iron content was measured. RNA sequencing was performed on white adipose tissue to identify genes differentially expressed across strain, diet, and strain X diet cohorts. Hepatic Hamp expression and circulating hepcidin was measured, and small nucleotide variants were identified in the Hamp genic region.ResultsLG/J mice experienced elevated fasting glucose and glucose intolerance during the high iron diet, corresponding with increased hepatic iron load, increased circulating ferritin, and signs of liver injury. Adipose function was also altered in high iron-fed LG/J mice, including decreased adiposity and leptin production and differential expression of genes involved in iron and glucose homeostasis. LG/J mice failed to upregulate hepatic Hamp expression during the high iron diet, resulting in low circulating hepcidin levels compared to SM/J mice.ConclusionsThis study highlights the importance of accounting for genetic variation when assessing the effects of diet on metabolic health, and suggests dietary iron’s impact on liver and adipose tissue is an underappreciated component of metabolic disease risk.

The role of dietary non-heme iron load and peripheral nerve inflammation in the development of peripheral neuropathy (PN) in obese non-diabetic leptin-deficient ob/ob mice

ABSTRACTIntroduction: Here, we investigated inflammatory signs of peripheral nerves in leptin-deficient obese ob/ob mice and the modulating effects of the exogenous iron load.Methods: Ob/ob and ob/+ control mice were fed with high, standard, or low iron diet for four months.Results: We found intraepidermal nerve fiber degeneration in foot skin and low-grade neuropathic abnormalities including mildly slowed motor and compound sensory nerve conduction velocities and low-grade macrophage and T-cell infiltration without overt neuropathology in sciatic nerves of all ob/ob mice. Low dietary iron load caused more pronounced abnormalities than high iron load in ob/ob mice.Discussion: Our data suggest that dietary non-heme iron deficiency may be a modulating factor in the pathogenesis of peripheral neuropathy in obese ob/ob mice with metabolic syndrome. Once the mechanisms can be further elucidated, how low dietary iron augments peripheral nerve degeneration and dysfunction via pro-inflammatory pathways and new therapeutic strategies could be developed.Abbreviations: CMAP: compound muscle action potential; cSNCV: compound sensory nerve conduction velocity; IENFD: intraepidermal nerve fiber density; LDL: low-density lipoprotein; MetS: metabolic syndrome; MNCV: motor conduction velocity; NCV: nerve conduction velocity; PN: peripheral neuropathy; PNS: peripheral nervous system; STZ: streptozotocin; T2D: type 2 diabetes mellitus; TNF alpha: tumor necrosis factor alpha; WHO: World Health Organization

The role of iron overload in the progression of nonalcoholic steatohepatitis (NASH)

Non-alcoholic steatohepatitis (NASH), one of the most common chronic liver diseases (CLD), is getting the most important cause of cirrhosis and hepatocellular carcinoma. Iron is an essential micronutrient for organisms. Once excess iron is accumulated in vital organs, dysfunctions of these organs can occur via the generation of reactive oxygen species. Hepatic iron overload is often seen in CLD patients. In NASH patients, iron accumulation in the liver is positively correlated with histological severity. Thus iron overload can contribute to progression of nonalcoholic fatty liver disease (NAFLD) to NASH. In a rat model of NASH, feeding of high-fat and high-iron diet increases hepatic inflammation with increased hepatic cytokine expression compared with feeding of high-fat diet only. In this model, iron is intensely accumulated in Kupffer cells/macrophages within the lesion, raising the possibility that iron-laden Kupffer cells/macrophages can play a key role in the enhancement of hepatic inflammation in NASH condition. On the other hand, in a rat model of liver cirrhosis, dietary iron overload clearly abrogates the development and progression of liver cirrhosis induced by repeated administration of thioacetamide (TAA). These findings suggest that iron overload can promote or suppress chronic liver diseases depending on the tissue microenvironment. Here we review and introduce the recent findings on the pathological roles of iron overload in the development and progression of NAFLD/NASH.