Effects of n-3 fatty acid and iron deficiency, alone and in combination, on hepcidin regulation and lipid mediators in rats

MSc (Nutrition), North-West University, Potchefstroom Campus Background: Globally, iron deficiency is the most common nutrient deficiency and can lead to various adverse health consequences. Iron absorption, circulation and storage are regulated by the hormone hepcidin, which is expressed by the HAMP gene in the liver. Iron cannot be actively excreted by the human body, thus hepcidin is released to decrease iron absorption and recycling to prevent iron overload. Hepcidin expression is activated by high iron stores and by inflammation via the inflammatory cytokine interleukin-6 (IL-6). This occurs via two main pathways known as the bone morphogenetic protein–small mothers against decapentaplegic (BMP/SMAD) pathway (induced by iron levels) and the JAK/STAT3 pathway (induced by IL-6). Long chain omega-3 (n-3) fatty acids can resolve inflammation by acting as a precursor for the synthesis of anti-inflammatory and pro-resolving lipid mediators. Low n-3 fatty acid status can lead to a decreased ability by the host to resolve inflammation and sets off pro-inflammatory signalling and increased IL-6 levels. Multiple nutrient deficiencies, such as a combined iron and n-3 fatty acid deficiency, often co-exist in populations and individuals. The presence of a combined iron deficiency with a low n-3 fatty acid status may lead to an inability to effectively resolve inflammation and increased circulating IL-6 levels, therefore potentially leading to the upregulation of hepcidin in an already iron deficient state, further hindering mechanisms to improve iron status. Aim and Objectives: Therefore, the aim of this study was to determine the effects of n-3 fatty acid and iron deficiency, alone and in combination, on hepcidin regulation and lipid mediator (docosahexaenoic acid (DHA)-, eicosapentaenoic acid (EPA)- and arachidonic acid (ARA)- derived) signalling in rats. The objectives of this study were to investigate the effects of n-3 fatty acid and iron deficiency, alone and in combination, on (A) Expression of the hepcidin regulatory pathway genes HAMP, BMP2 and TFR2 in rat liver. (B) ARA-derived 5-, 8-, 11-, 12- and 15- hydroxyeicosatetraenoic acid (HETE); EPA-derived 12-hydroxyeicosapentaenoic acid (HEPE) and DHA-derived 17-hydroxydocosahexaenoic acid (HDHA) lipid mediators in rat plasma. (C) To determine the correlations of HAMP, BMP2 and TFR2 expression with plasma lipid mediator concentrations in rats. Design: Female Wistar rats were fed a standard or n-3 fatty acid deficient (FAD) diet. The female rats were mated, and 39 male rats were included in the study with a 2x2 factorial. At weaning (postnatal day 21), the male rats were fed either a 1) control, 2) iron deficient (ID), 3) n-3 FAD or 4) ID + n-3 FAD diet for five weeks. Expression of HAMP and the HAMP regulatory genes BMP2 and TFR2 was analysed in hepatic tissue using quantified real time polymerase chain reaction (RT-qPCR). Peripheral lipid mediators such as ARA-derived pro-inflammatory lipid mediators, 5-, 8-, 11-, 12- and 15-hydroxyeicosatetraenoic acid (HETE), EPA-derived anti-inflammatory 12-hydroxyeicosapentaenoic acid (HEPE), and DHA-derived pro-resolving lipid mediator 17-hydroxydocosahexaenoic acid (HDHA) were measured in plasma using liquid chromatography-tandem mass spectrometry (LCMSMS). Results: HAMP expression was significantly upregulated by ID, whilst BMP2 and TFR2 expression were both downregulated. There was a significant effect of ID for higher ARA-derived 15-HETE, but lower 5-HETE. Furthermore, there was a significant effect of ID for higher DHA-derived 17-HDHA, whereas 17-HDHA was lowered by n-3 FAD. When compared to the control group, 17-HDHA was significantly higher in the ID group but lower in the n-3 FAD group. 17-HDHA concentrations in the ID+n-3 FAD group did not differ from the control group (p = 0.99). HAMP expression correlated positively with 15-HETE and 17-HDHA and negatively with 5-HETE. Conclusion: As expected, both iron level-dependent genes BMP2 and TFR2 were downregulated by ID. However, the upregulation of HAMP expression by ID was unexpected and suggestive of inflammation. The presence of inflammation in an ID state was confirmed by the effect of ID for increased ARA-derived pro-inflammatory lipid mediators and DHA-derived inflammation-resolving 17-HDHA levels. N-3 FAD alone did not induce HAMP expression, but may compromise the inflammation-resolving capacity of rats with a concomitant ID. Masters