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Objective: Oxidative stress is one of the major mechanisms of cyclophosphamide (CPX)-induced toxicities. However, it is unknown how CPX induces oxidative stress. Based on the available information, we speculated that CPX could increase iron content in the tissues and then induce oxidative stress. Method: We tested this hypothesis by investigating the effects of CPX on iron and ferritin contents, expression of transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1), iron regulatory proteins (IRPs), hepcidin, and nuclear factor erythroid 2-related factor-2 (Nrf2) in the liver and spleen, and also on reticulocyte count, immature reticulocyte fraction, and hemoglobin (Hb) in the blood in c57/B6 mouse. Results: We demonstrated that CPX could induce a significant increase in iron contents and ferritin expression in the liver and spleen, notably inhibit erythropoiesis and Hb synthesis and lead to a reduction in iron usage. The reduced expression in TfR1 and Fpn1 is a secondary effect of CPX-induced iron accumulation in the liver and spleen and also partly associated with the suppressed IRP/iron-responsive element system, upregulation of hepcidin, and downregulation of Nrf2. Conclusions: The reduced iron usage is one of the causes for iron overload in the liver and spleen and the increased tissue iron might be one of the mechanisms for CPX to induce oxidative stress and toxicities.

Background/Aims: The studies in the patients with iron deficiency anemia (IDA) implied the existence of the association of ghrelin with iron or hepcidin levels in the plasma under the pathophysiological conditions. We hypothesized that fasting may be able to affect iron metabolism via ghrelin under the physiological conditions. Methods: We investigated the effects of fasting on serum ghrelin and iron contents in healthy volunteers (23–31 years) and C57BL/6 male mice (8-week-olds) under the physiological conditions. Results: Fasting induced a significant elevation in both total ghrelin and acylated ghrelin and a reduction in iron levels in the serum of both human and mice. Correlation analysis demonstrated that total ghrelin or acylated ghrelin is negatively correlated with iron in the serum in human and mice. Conclusion: Ghrelin has a role to reduce serum iron under the conditions of fasting.

Prolonged, isocaloric, time-restricted feeding (TRF) protocols can promote weight loss, improve metabolic dysregulation, and mitigate non-alcoholic fatty liver disease (NAFLD). In addition, 3-day, severe caloric restriction can improve liver metabolism and glucose homeostasis prior to significant weight loss. Thus, we hypothesized that short-term, isocaloric TRF would improve NAFLD and characteristics of metabolic syndrome in diet-induced obese male mice. After 26 weeks of ad libitum access to western diet, mice either continued feeding ad libitum or were provided with access to the same quantity of western diet for 8 h daily, over the course of two weeks. Remarkably, this short-term TRF protocol modestly decreased liver tissue inflammation in the absence of changes in body weight or epidydimal fat mass. There were no changes in hepatic lipid accumulation or other characteristics of NAFLD. We observed no changes in liver lipid metabolism-related gene expression, despite increased plasma free fatty acids and decreased plasma triglycerides in the TRF group. However, liver Grp78 and Txnip expression were decreased with TRF suggesting hepatic endoplasmic reticulum (ER) stress and activation of inflammatory pathways may have been diminished. We conclude that two-week, isocaloric TRF can potentially decrease liver inflammation, without significant weight loss or reductions in hepatic steatosis, in obese mice with NAFLD.

Inhibition of eukaryotic elongation factor 1A1 (EEF1A1) with the marine compound didemnin B decreases lipotoxic HepG2 cell death in vitro and improves early stage non-alcoholic fatty liver disease (NAFLD) in young genetically obese mice. However, the effects of didemnin B on NAFLD in a model of long-term diet-induced obesity are not known. We investigated the effects of didemnin B on NAFLD severity and metabolic parameters in western diet-induced obese mice, and on the cell types that contribute to liver inflammation and fibrosis in vitro. Male 129S6 mice were fed either standard chow or western diet for 26 weeks, followed by intervention with didemnin B (50 μg/kg) or vehicle by intraperitoneal (i.p.) injection once every 3 days for 14 days. Didemnin B decreased liver and plasma triglycerides, improved oral glucose tolerance, and decreased NAFLD severity. Moreover, didemnin B moderately increased hepatic expression of genes involved in ER stress response (Perk, Chop), and fatty acid oxidation (Fgf21, Cpt1a). In vitro, didemnin B decreased THP-1 monocyte proliferation, disrupted THP-1 monocyte-macrophage differentiation, decreased THP-1 macrophage IL-1β secretion, and decreased hepatic stellate cell (HSteC) proliferation and collagen secretion under both basal and lipotoxic (high fatty acid) conditions. Thus, didemnin B improves hepatic steatosis, glucose tolerance, and blood lipids in obesity, in association with moderate, possibly hormetic, upregulation of pathways involved in cell stress response and energy balance in the liver. Furthermore, it decreases the activity of the cell types implicated in liver inflammation and fibrosis in vitro. These findings highlight the therapeutic potential of partial protein synthesis inhibition in the treatment of NAFLD.

Background: We recently demonstrated that the reduced iron usage due to suppressed erythropoiesis is a major cause of the systemic iron overload in CBS knockout (CBS-/-) mice. However, the molecular mechanisms by which CBS deficiency suppresses erythropoiesis are unknown. Methods: Here, we therefore examined the changes in the ratio of granulocyte/erythroid cells, iron content, the expression of erythropoietin (EPO), iron-metabolism proteins including transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1) and iron regulatory proteins (IRPs), and other molecules that affect iron-metabolism, including hypoxia inducible factor-2 subunit α (HIF-2α), nuclear factor erythroid 2-related factor-2 (Nrf2), and Kelch-like ECH-associated protein 1 (Keap1) in the blood, bone marrow, bone marrow-derived macrophages, liver or spleen of CBS-/- (homozygous), CBS+/- (heterozygous) and CBS+/+ (Wild Type) mice. Findings & Interpretation: Our findings demonstrated that CBS deficiency suppressed erythropoiesis by disrupting iron supply mainly via EPO/IRP/TfR1, Hepcidin/Fpn1, HIF-2α/EPO/Fpn1 and Keap1/Nrf2/Fpn1 pathways in macrophage or erythroid progenitors of rats. Funding: Supported by the National Natural Science Foundation of China (NSFC31571195), and the Competitive Earmarked Grants of the Hong Kong Research Grants Council (GRF14111815, GRF 14167817, GRF14107616). Declaration of Interest: The authors have no financial or competing interests to disclose. Ethical Approval: All animal care and experimental protocols were performed according to the Animal Management Rules of the Ministry of Health of China, and approved by the Animal Ethics Committees of Fudan University (NDFC31271132) and The Chinese University of Hong Kong (GRF14111815).