As essential cofactor in many proteins and redox enzymes, copper and iron are involved in a wide range of biological processes. and lecithin:cholesterol acyltransferase) in copper deficiency. Furthermore, a severe down-regulation of ApoH was pointed out in iron-deficient animals. value of 0.001 of the underlying statistical test and only genes satisfying the criteria were considered as differentially expressed genes. The fold difference (Sample/Control) was calculated only for these genes. 99755-59-6 supplier Data mining was carried out using the online available software David and OntoExpress (http://david.abcc.ncifcrf.gov, http://vortex.cs.wayne.edu/ontoexpress). The RatRef-12 allows the analysis of 21,910 sequences expressed in rat, including annotated genes and EST sequences. The results were filtered by imposing the fold difference cut-off values as 1.5 (up-regulated genes) or ?1.5 (down-regulated genes). Results Metal deficiencies Dietary mineral deficiencies are frequently associated with significant reductions in food intake. In order to verify that the obtained results were not affected by possible side effects of the treatments on the appetitive behavior of the animals, the daily food intake of each animal was registered and the group means of rats fed on metal-deficient diets were compared with that of metals adequate control group. As reported in Tables?1 and ?and2,2, the dietary administration of low contents of copper and iron did not affect the mean food intakes, and mean body weights were substantially unaffected. Moreover, the animals were fed ad libitum and did not show any evident physiological or behavioral alteration during the period of treatment and before killing. Table?1 Daily mean food intake of rats during dietary treatments Table?2 Body weight, liver and intestine metals content of control (C), copper-deficient (CuD) and iron-deficient (FeD) rats Atomic absorption spectrometry carried out on mineralized diets and on liver and small intestine samples showed that the lower content of copper and iron in the diets mirrored a significantly lower amount of each metal in CuD and FeD 99755-59-6 supplier rat tissues (Table?2). Furthermore, to confirm the homeostatic response to low dietary iron, we analyzed the expression of the divalent metal transporter 1 (DMT1) . This protein is able to transport dietary non-heme iron, and other divalent metal cations, and is localized on the apical surface of absorptive enterocytes. Its expression is positively regulated in the proximal duodenum of mice fed on iron-deficient diet. Real-time PCR analysis showed a positive regulation of DMT1 in the intestines of both FeD and CuD rats. Finally, microarray data confirmed the up-regulation of DCytb (FeD?=?+6.25; CuD?=?+1.70) and ferroportin (FeD?=?+1.29; CuD?=?n.d.) genes. On the other hand, current copper biomarkers are not reliable to detect minor but biologically significant variations of copper status . Microarray analysis As reported in Table?3, taking into account the generally accepted cut-off value of 1 1.5 for the fold induction, both metal deficiencies cause the down-regulation of the most part of the affected genes. Furthermore, by comparing the effects of dietary treatments, the data show that the intestinal transcriptome is considerably more sensitive to the iron deprivation. Table?3 Summary of the regulated genes As it can be inferred by the summary of the regulated genes ordered by biological process (Fig.?1a, b), both dietary deficiencies affect the energetic metabolism of the cell and modulate the FA and CL metabolism. Moreover, other processes are affected by both metals (e.g. intracellular transport of protein, G-protein coupled receptor protein signaling pathway, phospholipids transport, etc.), thus confirming their biological importance and entwined metabolic relationship. Fig.?1 a Biological processes affected by copper deficiency. b Biological processes affected by iron deficiency Tables?4 and ?and55 show that genes involved in the lipid metabolism 99755-59-6 supplier generally present smaller fold values (threshold set to 1 1.2), 99755-59-6 supplier although they are still statistically significant. Table?4 Copper deficiency: down-regulated (A) and DNAPK up-regulated (B) sequences in fatty acids and cholesterol metabolic pathways Table?5 Iron deficiency: down-regulated (A) and up-regulated (B) sequences in fatty acids and cholesterol metabolic pathways Discussion The overall results of the microarray analysis show that copper deficiency down-regulates the mitochondrial and peroxisomal beta-oxidation of FA. In fact, Acyl-CoA synthetase (concurs with the above-mentioned enzymes in slowing down the process, with a reduced production of acetyl-CoA and energy, and a consequent cytoplasmic accumulation of FA. 99755-59-6 supplier The down-regulation of acyl-CoA synthetase, delta-2-enoyl-CoA isomerase ((twofold).