Protective effects of Acetobacter ghanensis against gliadin toxicity in intestinal epithelial cells with immunoregulatory and gluten-digestive properties.

PURPOSE: The aim of this study was to establish whether Acetobacter ghanensis, the probiotic characteristics of which were evaluated previously, attenuates gliadin-induced toxicity in intestinal epithelial cells with gluten-digestive and immunoregulatory properties. METHODS: A co-culture model of human intestinal epithelial cell (Caco-2) monolayers on top of peripheral blood mononuclear cells (PBMCs) obtained from patients with celiac disease (CD) was established. The gluten-digestive properties of A. ghanensis were determined by checking bacterial growth in a medium containing gluten as the main nitrogen source. The mRNA levels of genes encoding TJ-associated proteins were measured by quantitative real-time PCR (qRT-PCR). The concentrations of IL-6 and TNFα were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that PT-gliadin disrupted intestinal barrier integrity by modulating the expression of TJ-associated genes encoding zonulin (increased by ~ 60%), zonula occludens-1 (ZO-1) (decreased by ~ 22%), and occludin (decreased by ~ 28%) in Caco-2 cells. Furthermore, PT-gliadin treatment in Caco-2 cells was associated with increased concentrations of IL-6 (~ 1.6-fold) and TNFα (~ twofold) from PBMCs. These modulatory effects of PT-gliadin, however, were suppressed when Caco-2 cells were subjected to A. ghanensis in the presence of PT-gliadin. As a factor underlying these protective effects, we showed that A. ghanensis could digest gluten peptides. CONCLUSIONS: To our knowledge, the current study is the first to demonstrate that A. ghanensis improves intestinal barrier functions by attenuating the modulatory effects of PT-gliadin with immunoregulatory and gluten-digestive properties.

Intersection of Iron and Copper Metabolism in the Mammalian Intestine and Liver.

Iron and copper have similar physiochemical properties; thus, physiologically relevant interactions seem likely. Indeed, points of intersection between these two essential trace minerals have been recognized for many decades, but mechanistic details have been lacking. Investigations in recent years have revealed that copper may positively influence iron homeostasis, and also that iron may antagonize copper metabolism. For example, when body iron stores are low, copper is apparently redistributed to tissues important for regulating iron balance, including enterocytes of upper small bowel, the liver, and blood. Copper in enterocytes may positively influence iron transport, and hepatic copper may enhance biosynthesis of a circulating ferroxidase, ceruloplasmin, which potentiates iron release from stores. Moreover, many intestinal genes related to iron absorption are transactivated by a hypoxia-inducible transcription factor, hypoxia-inducible factor-2α (HIF2α), during iron deficiency. Interestingly, copper influences the DNA-binding activity of the HIF factors, thus further exemplifying how copper may modulate intestinal iron homeostasis. Copper may also alter the activity of the iron-regulatory hormone hepcidin. Furthermore, copper depletion has been noted in iron-loading disorders, such as hereditary hemochromatosis. Copper depletion may also be caused by high-dose iron supplementation, raising concerns particularly in pregnancy when iron supplementation is widely recommended. This review will cover the basic physiology of intestinal iron and copper absorption as well as the metabolism of these minerals in the liver. Also considered in detail will be current experimental work in this field, with a focus on molecular aspects of intestinal and hepatic iron-copper interplay and how this relates to various disease states. © 2018 American Physiological Society. Compr Physiol 8:1433-1461, 2018.

Intestinal DMT1 Is Essential for Optimal Assimilation of Dietary Copper in Male and Female Mice with Iron-Deficiency Anemia.

Background: Divalent metal-ion transporter 1 (DMT1) may transport copper, but studies to date on this topic have been equivocal. Previously, an ex vivo experiment showed that intestinal copper transport was impaired in Dmt1-mutant Belgrade rats. Objective: In this study, we tested the hypothesis that intestinal DMT1 transports copper in vivo. Methods: Intestine-specific Dmt1 knockout (Dmt1int/int) mice and normal (control) littermates (Dmt1fl/fl) were used. In study 1, intestinal copper absorption was assessed in 7-wk-old mice of both sexes and genotypes by oral-intragastric gavage of 64Cu under normal and iron-deficiency anemia (IDA) conditions. In study 2, both sexes and genotypes of 8-wk-old mice were fed diets with adequate iron concentrations [72 parts per million (ppm)] plus adequate (9 ppm) or excessive (183 ppm) copper concentrations for 4 wk. Iron- and copper-related physiologic variables were subsequently assessed. Results: Study 1 showed that intestinal copper transport was enhanced in normal (∼11% increase in males, 35% in females) and anemic (∼42% increase in males, 35% in females) Dmt1int/int mice. Study 2 showed that, with adequate copper intakes, serum ceruloplasmin (Cp) activity was decreased (by ∼29% in males and 20% in females) and spleens were enlarged (by 3-fold in both sexes) in Dmt1int/int mice. Higher dietary copper increased hepatic copper concentrations (by ∼3.3-fold in males and 1.5-fold in females), restored serum Cp activity, and mitigated the noted splenomegaly in Dmt1int/int mice. Conclusions: Copper homeostasis was disrupted in Dmt1int/int mice, particularly during IDA, despite the noted increases in intestinal copper transport. This was exemplified by the fact that extra dietary copper was required to restore serum Cp activity (a biomarker of copper status) and reduce the severity of the noted splenomegaly (which could reflect changes in erythropoietic demand) in Dmt1int/int mice. Collectively, these observations show that intestinal DMT1 is essential for the assimilation of sufficient quantities of dietary copper to maintain systemic copper homeostasis during IDA.

Copper supplementation reverses dietary iron overload-induced pathologies in mice.

Dietary iron overload in rodents impairs growth and causes cardiac hypertrophy, serum and tissue copper depletion, depression of serum ceruloplasmin (Cp) activity and anemia. Notably, increasing dietary copper content to ~25-fold above requirements prevents the development of these physiological perturbations. Whether copper supplementation can reverse these high-iron-related abnormalities has, however, not been established. The current investigation was thus undertaken to test the hypothesis that supplemental copper will mitigate negative outcomes associated with dietary iron loading. Weanling mice were thus fed AIN-93G-based diets with high (>100-fold in excess) or adequate (~80 ppm) iron content. To establish the optimal experimental conditions, we first defined the time course of iron loading, and assessed the impact of supplemental copper (provided in drinking water) on the development of high-iron-related pathologies. Copper supplementation (20 mg/L) for the last 3 weeks of a 7-week high-iron feeding period reversed the anemia, normalized serum copper levels and Cp activity, and restored tissue copper concentrations. Growth rates, cardiac copper concentrations and heart size, however, were only partially normalized by copper supplementation. Furthermore, high dietary iron intake reduced intestinal 64Cu absorption (~60%) from a transport solution provided to mice by oral, intragastric gavage. Copper supplementation of iron-loaded mice enhanced intestinal 64Cu transport, thus allowing sufficient assimilation of dietary copper to correct many of the noted high-iron-related physiological perturbations. We therefore conclude that high- iron intake increases the requirement for dietary copper (to overcome the inhibition of intestinal copper absorption).

Progressive Increases in Dietary Iron Are Associated with the Emergence of Pathologic Disturbances of Copper Homeostasis in Growing Rats.

Background: Consumption of a high-iron diet causes copper deficiency in weanling rodents; however, the minimum amount of dietary iron that disrupts copper homeostasis has not been established. Objective: We tested the hypothesis that dietary iron at only several-fold above physiologic requirements would cause copper depletion. Methods: Weanling male Sprague-Dawley rats (n = 6/group) were fed AIN-93G-based diets with adequate (88 µg Fe/g = 1×), or excessive (4×, 9.5×, 18.5×, 38×, or 110×) iron content for 7 wk (110× group, due to notable morbidity) or 8 wk (all other groups). Copper-related physiologic parameters were then assessed. Results: A hierarchy of copper-related, pathologic symptoms was noted as dietary iron concentrations increased. All statistical comparisons reported here refer to differences from the 1× (i.e., control) group. The highest iron concentration (110×) impaired growth (final body weights decreased ∼40%; P < 0.0001), and caused anemia (blood hemoglobin and hematocrit decreased ∼65%; P < 0.0001) and hepatic copper depletion (>85% reduction; P < 0.01). Cardiac hypertrophy occurred in the 110× (∼130% increase in mass; P < 0.0001) and 38× (∼25% increase; P < 0.05) groups, whereas cardiac copper content was lower in the 110× (P < 0.01), 38× (P < 0.01), and 18.5× (P < 0.05) groups (∼70% reductions). Splenic copper was also depleted in the 110× (>90% reduction; P < 0.0001), and in the 38× (P < 0.001) and 18.5× (P < 0.01) groups (∼70% reductions). Moreover, serum ceruloplasmin activity was decreased in the 110× and 38× (>90% reductions; P < 0.0001), and 18.5× (P < 0.001) and 9.5× (P < 0.05) (∼50% reductions) groups, typifying moderate to severe copper deficiency. Conclusions: Increasing dietary iron intakes to ∼9.5-fold above dietary recommendations caused copper deficiency. Importantly, human iron supplementation is common, and recommended intakes for at-risk individuals may be ≤10-fold above the RDA. Whether these iron intakes perturb copper metabolism is worth considering, especially since copper defi-ciency can impair iron utilization (e.g., by decreasing the ferroxidase activity of ceruloplasmin).

Consumption of a High-Iron Diet Disrupts Homeostatic Regulation of Intestinal Copper Absorption in Adolescent Mice.

High-iron feeding of rodents has been commonly used to model human iron-overload disorders. We recently noted that high-iron consumption impaired growth and caused severe systemic copper deficiency in growing rats, but the mechanism by which this occurred could not be determined due to technical limitations. In the current investigation, we thus utilized mice; first to determine if the same phenomenon occurred in another mammalian species, and secondly since we could assess in vivo copper absorption in mice. We hypothesized that excessive dietary iron impaired intestinal copper absorption. Weanling, male mice were thus fed AIN-93G-based diets containing high (HFe) (~8800 ppm) or adequate (AdFe) (~80 ppm) iron in combination with low (~0.9 ppm), adequate (~9 ppm) or high (~180 ppm) copper for several weeks. Iron and copper homeostasis was subsequently assessed. Mice consuming the HFe diets grew slower, were anemic, and had lower hepatic copper levels and serum ceruloplasmin activity. These physiologic perturbations were all prevented by higher dietary copper, demonstrating that copper depletion was the underlying cause. Furthermore, homeostatic regulation of copper absorption was noted in the mice consuming the AdFe diets, with absorption increasing as dietary copper decreased. HFe-fed mice did not have impaired copper absorption (disproving our hypothesis), but homeostatic control of absorption was disrupted. There were also noted perturbations in the tissue distribution of copper in the HFe-fed mice, suggesting that altered storage and thus bioavailability contributed to the noted copper deficiency. Dietary iron loading thus antagonizes copper homeostasis leading to pathological symptoms of severe copper depletion.

Intestinal hephaestin potentiates iron absorption in weanling, adult, and pregnant mice under physiological conditions.

Regulation of intestinal iron absorption is crucial to maintain body iron levels because humans have no regulated iron-excretory system. Elucidating molecular events that mediate intestinal iron transport is thus important for the development of therapeutic approaches to modify iron absorption in pathological states. The process of iron uptake into duodenal enterocytes is relatively well understood, but less is known about the functional coupling between the iron exporter ferroportin 1 and the basolateral membrane iron oxidase hephaestin (Heph). Initial characterization of intestine-specific Heph knockout (Hephint) mice demonstrated that adult male mice were mildly iron deficient; however, the specific role of intestinal Heph has not been determined in weanling mice, in female mice, or during physiological states which stimulate iron absorption. Furthermore, because ferroportin 1-mediated iron export from some tissues (eg, liver) is impaired in the absence of the Heph homolog, ceruloplasmin, we hypothesized that Heph is rate limiting for intestinal iron absorption, especially when iron demands increase. Our experimental approach was to assess various physiological parameters and iron (59Fe) absorption and tissue distribution in weanling, adult, and pregnant Hephint mice (and controls) under physiological conditions and in adult Hephint mice after dietary iron deprivation or acute hemolysis. Results demonstrate that intestinal Heph is essential for optimal iron transport in weanlings and adults of both sexes and during pregnancy, but not in adult mice with iron-deficiency or hemolytic anemia. Moreover, activation of unidentified, intestinal ferroxidases was noted, which may explain why intestinal Heph is not always required for optimal iron absorption.

Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

Intestinal iron absorption is highly regulated since no mechanism for iron excretion exists. We previously demonstrated that expression of an intestinal copper transporter (Atp7a) increases in parallel with genes encoding iron transporters in the rat duodenal epithelium during iron deprivation (Am. J. Physiol.: Gastrointest. Liver Physiol., 2005, 288, G964-G971). This led us to postulate that Atp7a may influence intestinal iron flux. Therefore, to test the hypothesis that Atp7a is required for optimal iron transport, we silenced Atp7a in rat IEC-6 and human Caco-2 cells. Iron transport was subsequently quantified in fully-differentiated cells plated on collagen-coated, transwell inserts. Interestingly, 59Fe uptake and efflux were impaired in both cell lines by Atp7a silencing. Concurrent changes in the expression of key iron transport-related genes were also noted in IEC-6 cells. Expression of Dmt1 (the iron importer), Dcytb (an apical membrane ferrireductase) and Fpn1 (the iron exporter) was decreased in Atp7a knockdown (KD) cells. Paradoxically, cell-surface ferrireductase activity increased (>5-fold) in Atp7a KD cells despite decreased Dcytb mRNA expression. Moreover, increased expression (>10-fold) of hephaestin (an iron oxidase involved in iron efflux) was associated with increased ferroxidase activity in KD cells. Increases in ferrireductase and ferroxidase activity may be compensatory responses to increase iron flux. In summary, in these reductionist models of the mammalian intestinal epithelium, Atp7a KD altered expression of iron transporters and impaired iron flux. Since Atp7a is a copper transporter, it is a logical supposition that perturbations in intracellular copper homeostasis underlie the noted biologic changes in these cell lines.

High-Iron Consumption Impairs Growth and Causes Copper-Deficiency Anemia in Weanling Sprague-Dawley Rats.

Iron-copper interactions were described decades ago; however, molecular mechanisms linking the two essential minerals remain largely undefined. Investigations in humans and other mammals noted that copper levels increase in the intestinal mucosa, liver and blood during iron deficiency, tissues all important for iron homeostasis. The current study was undertaken to test the hypothesis that dietary copper influences iron homeostasis during iron deficiency and iron overload. We thus fed weanling, male Sprague-Dawley rats (n = 6-11/group) AIN-93G-based diets containing high (~8800 ppm), adequate (~80) or low (~11) iron in combination with high (~183), adequate (~8) or low (~0.9) copper for 5 weeks. Subsequently, the iron- and copper-related phenotype of the rats was assessed. Rats fed the low-iron diets grew slower than controls, with changes in dietary copper not further influencing growth. Unexpectedly, however, high-iron (HFe) feeding also impaired growth. Furthermore, consumption of the HFe diet caused cardiac hypertrophy, anemia, low serum and tissue copper levels and decreased circulating ceruloplasmin activity. Intriguingly, these physiologic perturbations were prevented by adding extra copper to the HFe diet. Furthermore, higher copper levels in the HFe diet increased serum nonheme iron concentration and transferrin saturation, exacerbated hepatic nonheme iron loading and attenuated splenic nonheme iron accumulation. Moreover, serum erythropoietin levels, and splenic erythroferrone and hepatic hepcidin mRNA levels were altered by the dietary treatments in unanticipated ways, providing insight into how iron and copper influence expression of these hormones. We conclude that high-iron feeding of weanling rats causes systemic copper deficiency, and further, that copper influences the iron-overload phenotype.