Effects of carbohydrates on iron metabolism in intestinal and liver cells
Iron deficiency and excess are worldwide public health problems. Studies suggest that carbohydrates such as fructose, and oligo-and polysaccharides (prebiotics), increase iron bioavailability, but results are inconclusive. There is also interest in whether iron and fructose contribute to the pathogenesis of colorectal cancer (CRC), hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). Intake of sugars has increased in the past three decades, as has the prevalence and incidence of HCC and AFLD, while CRC remains one of the top three occurring malignancies in the developed world, making these questions particularly relevant at this time. The aims of this thesis were to study iron-carbohydrate effects on: non-haem iron bioavailability, and on the expression of genes and proteins related to CRC, HCC and NAFLD. The principal carbohydrates investigated were fructose, the related sweetener high fructose corn syrup (HFCS), and fructo- and galacto-oligosaccharide prebiotics (FOS&GOS, respectively). It was hypothesised that fructose, HFCS, and FOS&GOS would increase non-haem iron bioavailability, and that iron and fructose would alter expression of genes and proteins related to the pathogenesis of CRC, HCC and NAFLD. Two human in vitro cell lines were used to investigate these questions: Caco-2 and HepG2 cells, models of the small intestine and liver, respectively. The Caco-2 in vitro digestion model was used to examine fructose, HFCS, and FOS&GOS effects on iron bioavailability with ferritin formation as a surrogate marker for iron uptake. Ferritin formation was also utilised to assess iron uptake in HepG2 cells. Expression of iron homeostasis proteins was analysed by Western Blot and Polymerase Chain Reaction (PCR) to explore biological mechanisms underlying observed effects on iron bioavailability. Lastly, Caco-2 and HepG2 cells were treated with iron and fructose, and the effects on selected genes and proteins involved in CRC, HCC and NAFLD development were evaluated through microarray, PCR and Western Blot analysis. Results indicated that fructose in a water-based matrix with added ferric iron (FeCl3) significantly increased ferritin formation in Caco-2 and HepG2 cells by 40 and 35 %, respectively, in comparison with iron alone treated cells; this effect was negated in the Caco-2 cell line by phytates and polyphenols at 1:5 and 1:1 iron:inhibitor molar ratios, respectively. Fructose treatment alone did not significantly increase ferritin formation in either cell line. Fructose combined with FeCl3 in a pH 7 water-based matrix significantly increased ferrozine-chelatable ferrous iron levels by 320 % in comparison with FeCl3 alone. Two liquid ferrous iron supplements with added fructose had significantly higher ferritin compared with dissolved ferrous sulphate tablets; Spatone Appleâ, the supplement that had the highest fructose to iron molar ratio (62:1), had the highest iron bioavailability with ferritin levels 610 % higher compared with ferrous sulphate alone, although it is important to note that it also had the highest concentration of ascorbic acid. Fructose added to ascorbic acid and FeCl3 in a water-based matrix had an additive effect on ferritin formation in Caco-2 cells, significantly increasing ferritin two-fold compared with FeCl3 and ascorbic acid alone. A high-fructose containing sweet potato (SP) infant complementary food with 240 % more fructose compared with a low fructose SP food had significantly higher ferritin levels by 30 %, but ferritin was still relatively low compared to a commercial weaning food (50 % less), possibly secondary to high levels of phytates and polyphenols in the SP-based foods. Lastly, a mixture of exogenous FOS&GOS prebiotics added to Young Child Formulae (YCF, milk-based products for toddlers) increased ferritin formation by approximately 25%, and eliminated significant differences in ferritin levels in comparison to YCF with manufacturer added FOS&GOS. Gene and protein expression analysis did not support the hypothesis that the observed increase in fructose-induced ferritin was due to changes to iron transporter or homeostasis molecules. Caco-2 and HepG2 cell gene and protein analysis demonstrated significant changes in pathways related to CRC, HCC and NAFLD. Key significant findings of iron and fructose cell treatments were: 1.5 fold or greater changes in HepG2 gene expression of SMADs 2 and 3, STAT3 and NF-κb -- signalling proteins implicated in development of inflammatory liver disease; increased HepG2 mRNA expression of cell cycle related genes, Cyclin D1 and Cyclin D2, and the proto-oncogene Skp2; decreased mRNA expression of HNF4A, a key liver transcription factor, but with increased HNF4A protein expression; increased HepG2 mRNA and protein expression of the “cancer chaperone” Heat Shock Protein 90; and significantly decreased mRNA expression of the tumour suppressor gene APC by 1.3 fold in iron alone treated Caco-2 cells. In conclusion, fructose, HFCS, and a mixture of prebiotics increased iron bioavailability as assessed by ferritin formation in an in vitro intestinal cell model. The observed effects were negated by phyates and polyphenols in a water-based matrix, and also in a complex food matrix (sweet potato-based) containing high levels of these inhibitors, indicating that these carbohydrates would not increase gut iron uptake when eaten as part of a mixed diet containing polyphenols and phytates. Iron/mineral supplements with added fructose and ascorbic acid in a water-based matrix were associated with higher available iron in comparison with ferrous sulphate alone; fructose had an additive effect with ascorbic acid suggesting that iron supplement bioavailability could be improved with the addition of fructose. In a milk-based food matrix added prebiotics FOS&GOS significantly improved available iron suggesting that a mixture of FOS&GOS prebiotics could improve the nutritional benefits of YCF in relation to iron deficiency. Fructose added to iron solutions also increased ferritin in an in vitro hepatocyte cell model. Fructose-induced ferritin increases did not appear to be secondary to altered expression of iron homeostasis molecules, but rather to changed iron oxidation state to the more bioavailable ferrous form. Changes observed in genes and proteins associated with CRC, HCC and NAFLD with iron and fructose treatments related to multiple pathways implicated in these diseases, and require further study. All of the above were obtained in vitro, therefore in vivo confirmation of these findings is required for further evaluation of their impact on human health and disease.
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