DEVELOPMENT AND PATHOPHYSIOLOGICAL CHARACTERIZATION OF AN IN VIVO MODEL OF IRON OVERLOAD ASSOCIATED TO INSULIN RESISTANCE AND REPRODUCTIVE IMPAIRMENT
Creators: name: MACCHI, CHIARA
Publisher: name: Universit� degli Studi di Milano
Year: 2018

description: Introduction. Iron is an essential micronutrient required for fundamental biochemical activities, such as oxygen and energy metabolism, mitochondrial function and brain development. However, it may catalyze the formation of highly reactive hydroxyl radicals, leading to oxidative stress, lipid peroxidation, and DNA damage with, finally, cell and tissue damages. Given its potential high toxicity, a condition of iron overload can promote multiple organ damages, associated to acute and chronic diseases. Among the several complications associated with iron overload syndromes, hypogonadism is the second most common endocrinopathy although the role of iron in its pathophysiology is still debated. Aim. To explore in a dysmetabolic murine model, the molecular determinants of hypogonadism induced by iron overload, with a specific focus on hypothalamic derangement. Material and methods. Male C57BL/6J mice fed standard iron concentration diet or iron-enriched diet (IED, 3% carbonyl-iron) and HFE-/- mice, these last resembling a murine model of human genetic hemochromatosis; cell-based models of gonadotropin-releasing hormone (GnRH) neurons (GN-11 and GT1-7 cell lines); radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA); histological analysis and immunostaining; image processing and quantitation; atomic absorption spectrometry; ATPliteTM 1step assay; Trypan Blue exclusion test; qRT-PCR; Boyden?s chamber assay; Western blot analysis. Results. In vivo models. IED led to a hypogonadal phenotype as shown by micro- and macroscopic alterations at the testicular level. Iron accumulation in testes and pituitary significantly reduced serum levels of testosterone (-83%) and luteinizing hormone (-86%). Although, hypothalamic iron concentration did not differ in mice fed IED compared to controls, a significant increment in GnRH gene expression (+34%) and in intensity of GnRH-neuron innervation of the median eminence (+1.5-fold) were found; similar changes were obtained in HFE-/- mice. Hypothalamic gene expression of tumor necrosis factor ? was increased in IED mice. Moreover, a series of metabolic impairments, such as (i) increment in glycemia and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index and (ii) reduction in body weight and fat as well as in plasma leptin was found upon IED. In vitro models. Treatment of GN-11 and GT1-7 cells with ferric ammonium citrate, as a source of iron, significantly increased its intracellular concentration; as such, the genes involved in iron homeostasis were changed: transferrin receptor, -75%; ferritin H, +92%. Furthermore, GN-11 cell chemomigration was inhibited by iron overload with an apparent involvement of the extracellular signal-regulated kinase (ERK) 1/2 cell signaling pathway. Finally, iron overload induced oxidative stress in GN-11 cells. Conclusions. In adult male mice, iron overload leads to a severe impairment of the hypothalamic-pituitary-gonadal axis possibly resulting in a hypogonadal condition, a feature possibly deriving from iron deposition in pituitary and/or gonads via extrahypothalamic mechanisms. This finding represents a further step in understanding how iron overload leads to this endocrinopathy. In this context, the use of in vitro GnRH neurons, which functions were impaired by iron accumulation, leaves open questions relative to the role of brain blood barrier in the protection of the central region (hypothalamus).