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.

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.

Antioxidant supplementation accelerates cachexia development by promoting tumor growth in C26 tumor-bearing mice.

More than 50% of patients with advanced stages of colon cancer suffer from progressive loss of skeletal muscle, called cachexia, resulting in reduced quality of life and shortened survival. It is becoming evident that reactive oxygen species (ROS) regulate pathways controlling skeletal muscle atrophy. Herein we tested the hypothesis that antioxidant supplementation could prevent skeletal muscle atrophy in a model of cachectic Colon 26 (C26) tumor-bearing mice. Seven-week-old BALB/c mice were subcutaneously inoculated with colon 26 (C26) cancer cells or PBS. Then C26-mice were daily gavaged during 22 days either with PBS (vehicle) or an antioxidant cocktail whose composition is close to that of commercial dietary antioxidant supplements (rich in catechins, quercetin and vitamin C). We found that antioxidants enhanced weight loss and caused premature death of mice. Antioxidants supplementation failed to prevent (i) the increase in plasma TNF-α levels and systemic oxidative damage, (ii) skeletal muscle atrophy and (iii) activation of the ubiquitin-proteasome system (MuRF-1, MAFbx and polyubiquitinated proteins). Accordingly, immunohistological staining for Ki-67 and the expression of cell cycle inhibitors demonstrated that tumor of supplemented mice developed faster with a concomitant decrease in oxidative damage. Previous studies have shown that the use of catechins and quercetin separately can improve the musculoskeletal function in cachectic animals. However, our results indicate that the combination of these antioxidants reduced survival and enhanced cachexia in C26-mice.