Induction of arachidonate 12-lipoxygenase (Alox15) in intestine of iron-deficient rats correlates with the production of biologically active lipid mediators

Abstract

To identify novel genes associated with iron metabolism, we performed gene chip studies in two models of iron deficiency: iron-deprived rats and rats deficient in the principal intestinal iron transporter, divalent metal transporter 1 (i.e., Belgrade rats). Affymetrix rat genome gene chips were utilized (RAE230) with cRNA samples derived from duodenum and jejunum of experimental and control animals. Computational analysis and statistical data reduction identified 29 candidate genes, which were induced in both models of iron deficiency. Gene ontology analysis showed enrichment for genes related to lipid homeostasis, and one gene related to this physiological process, a leukocyte type, arachidonate 12-lipoxygenase (Alox15), was selected for further examination. TaqMan real-time PCR studies demonstrated strong induction of Alox15 throughout the small and large intestine, and in the liver of iron-deficient rats. Polyclonal antibodies were developed and utilized to demonstrate that proteins levels are significantly increased in the intestinal epithelium of iron-deprived rats. HPLC analysis revealed altered intestinal lipid metabolism indicative of Alox15 activity, which resulted in the production of biologically active lipid molecules (12-HETE, 13-HODE, and 13-HOTE). The overall effect is a perturbation of intestinal lipid homeostasis, which results in the production of lipids essentially absent in the intestine of control rats. We have thus provided mechanistic insight into the alteration in lipid metabolism that occurs during iron deficiency, in that induction of Alox15 mRNA expression may be the primary event. The resulting lipid mediators may be related to documented alterations in villus structure and cell proliferation rates in iron deficiency, or to structural alterations in membrane lipid composition.

Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes

“…Method I (grinding). Cytosolic and solubilized particulate/membrane fractions were prepared as described (21). All steps were performed at 4°C.…”

“…Thirty micrograms of cytosolic or membrane proteins were resolved by SDS-7.5% PAGE, blotted, and immunoprobed as described earlier (21). The anti-Atp7a antibody (called "54-10") (20,22) and anti-ZnT1 (a kind gift from Robert Cousins, University of Florida, Gainesville, FL) (23) are well validated.…”

D-4F-mediated reduction in metabolites of arachidonic and linoleic acids in the small intestine is associated with decreased inflammation in low-density lipoprotein receptor-null mice

“…The enterocytes may produce free HETEs and HODEs by providing phospholipase activity, which hydrolyzes dietary phospholipids, and/or by enzymatic (e.g., lipoxygenase or cyclooxygenase) or nonenzymatic pathways within the enterocyte that act upon free arachidonic and linoleic acids. Oxidized metabolites of arachidonic and linoleic acids have been reported to be important in the intestine in humans and fi sh with remarkable conservation of some of the enzyme systems that participate (14)(15)(16)(17)(18)(19)(20)(21). It is known that small intestinal ulcers are a complication of treatment with nonsteroidal anti-infl ammatory agents.…”

Role of eicosanoids on intestinal epithelial homeostasis

“…Thus, Kamitani et al [13] suggested that 12-LOX, and/or the lipid products synthesized by this enzyme are involved in intestinal epithelial cell differentiation and apoptosis. Recently, Collins et al [17] reported that 12-LOX was increased by up to > 100-fold throughout the entire length of the intestine of iron-deficient animals, inducing a strong increase in 12-HETE Today, the causal relationship between inflammation, innate immunity and cancer is more widely accepted; nevertheless, many of the molecular and cellular mechanisms mediating this relationship remain unresolved. However, there is now evidence that inflammatory mediators have a powerful effect on tumor development.…”

“…Shoji et al [12] Transformed and non-transformed intestinal epithelial cell lines [13][14][15] as well as the human intestine [16,17] also produce LOX metabolites such as LTs and HETEs, whereas there is no information about HETE/EET production by cytochrome P-450 in intestinal epithelial cells or the intestine. With respect to the presence of LT receptors in the intestine, the CysLT 1 receptor is expressed in intestinal epithelial cells and tumorderived intestinal cells [15] whereas the CysLT 2 receptor is expressed in differentiated intestinal epithelial cells but not in tumor-derived intestinal cell lines [18].…”