Significance of non-esterified fatty acids in iron uptake by intestinal brush-border membrane vesicles

Iron uptake from Fe/ascorbate by mouse brush-border membrane vesicles is not greatly inhibited by prior treatment with a variety of protein-modification reagents or heat. Non-esterified fatty acid levels in mouse proximal small intestine brush-border membrane vesicles show a close positive correlation with initial Fe uptake rates. Loading of rabbit duodenal brush-border membrane vesicles with oleic acid increases Fe uptake. Depletion of mouse brush-border membrane vesicle fatty acids by incubation with bovine serum albumin reduces Fe uptake. Iron uptake by vesicles from Fe/ascorbate is enhanced in an O2-free atmosphere. Iron uptake from Fe/ascorbate and Fe3+-nitrilotriacetate (Fe3+-NTA) were closely correlated. Incorporation of oleic acid into phosphatidylcholine/cholesterol (4:1) liposomes leads to greatly increased permeability to Yb3+, Tb3+, Fe2+/Fe3+ and Co2+. Ca2+ and Mg2+ are also transported by oleic acid-containing liposomes, but at much lower rates than transition and lanthanide metal ions. Fe3+ transport by various non-esterified fatty acids was highest with unsaturated acids. The maximal transport rate by saturated fatty acids was noted with chain length C14-16. It is suggested that Fe transport can be mediated by formation of Fe3+ (fatty acid)3 complexes.     

In vitro measurement and adaptive response of Fe3+ uptake by mouse intestine

In order to define the importance of the mucosal uptake step in the intestinal regulation of iron absorption, unidirectional uptake rates of Fe3+ from a nitrilotriacetic acid chelate were measured in duodenal fragments from mice using an in vitro technique. [57Co]-Cyanocobalamin was used as a marker of adherent incubation medium. Uptake showed saturation kinetics over the concentration range 18-450 microM. Uptake was increased in fragments from hypoxic, dietary iron-deficient and pregnant mice. The enhanced uptake was due to an increase in Vmaxapp. However, the modest increase in uptake rates in pregnancy and the gross changes observed in iron-deficiency make the hypoxic model the most convenient. The increase in uptake in hypoxic animals was located to the duodenal region and was not associated with changes in either total mucosal iron content or epithelial cell turnover. The rate of uptake of iron via the serosa did not change with hypoxia. This study implies that flux of Fe3+ across the brush border is subject to adaptive regulation. The hypoxic model is suitable for investigation into the regulation of iron homeostasis.     

Subcellular localization of recently-absorbed iron in mouse duodenal enterocytes: identification of a basolateral membrane iron-binding site

The subcellular distribution of newly absorbed iron in isolated mouse duodenal enterocytes was investigated by analytical subcellular fractionation using sucrose density gradient centifugation. Two major peaks of mucosal 59Fe activity were observed: one soluble and one particulate (density 1.18-1.20 g ml-1). The latter was increased following prior exposure of animals to chronic hypoxia. The particulate 59Fe was localized to the basolateral membranes using the marker enzyme Na+, K+ activated, Mg2+ dependent, ATPase and by washing intact enterocytes with the selective plasma membrane perturbant digitonin. The basolateral membrane can be selectively labelled by in vitro incubation of intact enterocytes at 0 degrees C with 59Fe(III)-nitrilotriacetate complex, confirming the presence of a 59Fe binding site on this membrane. No significant difference in in vitro iron binding to this site was observed between normal and chronically hypoxic animals. Iron binding to the basolateral membrane was significantly higher in disrupted, compared to intact enterocytes, indicating that this site is present on both sides of the basolateral membrane. It is therefore suggested that the increased labelling of this site in hypoxia, in vivo, is a consequence of an increase in a mucosal Fe pool which is available for binding to a membrane receptor.     

Surface iron polymers and hydroxy acids. A model of iron supply in sideramine-free fungi

Biochemical and electron microscopic evidence is presented that sideramine-free fungi form iron hydroxide polymer layers on the cell surface when grown in an iron containing medium.Iron hydroxide polymer formation on the cell surface is completely prevented in sideramine producing strains of Neurospora crassa. After feeding a sideramine-free mutant of Neurospora crassa with ornithine in order to restore the sideramine synthesis the iron hydroxide coat is gradually dissolved.The addition of excess citrate and malate to the incubation medium also prevents iron polymer adsorption, suggesting that hydroxy acids may be involved in iron supply, when sideramine-free organisms are grown in iron containing media.In order to study the interaction between iron hydroxide polymer deposition upon the cell surface and iron chelating acids in Neurospora crassa, the amount and the proportion of excreted acids was studied under various experimental conditions. Gas chromatographic analysis of the acids produced under iron deficient conditions revealed that succinate, malate and citrate were present within the cells in the early growth phase. The acids were sequentially excreted into the medium in the order succinate, malate and citrate. The amount of succinate decreased after 2 days of cultivation, whereas the amount of malate and citrate continually increased. Although citrate was present within the cells from the 1st day, excretion occurred very late, generally after the 3rd day.It is suggested that sideramine-free fungi first adsorb iron as a hydroxide polymer on the cell surface, and that it is gradually solubilized by excreted hydroxy acids such as citrate or malate. Thus high local concentrations of iron chelated by hydroxy acids provide sideramine-free fungi with a continuous iron supply.Abbreviations BSTFA N,O-Bis(trimethylsilyl)-trifluoracetamide - GC Gaschromatography - EGTA Ethylenglykol-bis(2-aminoethylether) N,N-tetraacetic acid - TMS Trimethylsilyl     

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.     

Haemochromatosis protein is expressed on the terminal web of enterocytes in proximal small intestine of the rat

The haemochromatosis protein (HFE) is an important regulator of body iron stores. In the liver, HFE is required for appropriate expression of hepcidin, a humoral mediator of iron absorption. HFE is also present in enterocytes, though its function in the intestine is unknown; it is not intrinsically required for iron absorption, but can augment iron absorption when over-expressed—independent of hepcidin regulation by the liver. In this study, an antibody was raised against rat HFE and validated by enzyme-linked immunosorbent assay, Western blot and quenching of antibody function by the immunising peptide. The sub-cellular location of HFE in enterocytes of iron-deficient and control rats was determined by double-labelling experiments with markers for the microvillus membrane, terminal web, early endosomes, lysosomes and the transferrin receptor. Parallel studies were performed for the primary iron absorption protein, divalent metal transporter 1 (DMT1). HFE co-localised exclusively with the terminal web of intestinal enterocytes. HFE expression was increased in iron deficiency, consistent with a second regulatory role for HFE in iron absorption, independent of hepcidin from the liver. DMT1 was localised primarily on the microvillus membrane, but did partially co-localise with HFE raising the possibility that the two proteins may interact to regulate iron absorption.     

Iron binding proteins and influx of irom across the duodenal brush border: Evidence for specific lactotransferrin receptors in the human intestine

The ability of a range of homologous transferrin-like proteins to donate iron to pieces of human duodenal mucosa, was examined with an in vitro incubation technque. In contrast to serum transferrin and ovotransferrin, only lactotransferrin was able to yield its iron to intestinal tissue, but in an autologous system this protein was unable to donate iron to human reticulocyte preparations. Studies with ¹²⁵I-labelled lactotransferrin and lactotransferrin dual-labelled with ⁵⁹Fe and ¹²⁵I, indicated that the intact protein is excluded from entry into the enterocytes. The experiments suggest that iron may be transported across the brush border after delivery to specific protein binding sites at the cell surface.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Abstract

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Brush border membrane proteins in experimental Fanconi's syndrome induced by 4-pentenoate and maleate.

Fanconi's syndrome was investigated using brush border membrane (BBM) vesicles isolated from dog kidney. Sodium-dependent uptake of glucose, phosphate, and amino acids and protein phosphorylation were studied in BBM isolated from normal and from 4-pentenoate- and maleate-treated animals. The time course of D-glucose and phosphate uptake, in BBM vesicles, remained unchanged, indicating that both treatments had no effect on carrier properties, and that permeabilities to these substrates and to sodium were not modified. Furthermore, sodium-dependent transport of alanine, phenylalanine, proline, glycine, and glutamate into vesicles remained unaltered by either treatment. 4-Pentenoate treatment caused modifications of the phosphorylation pattern of BBM proteins: the phosphorylation of two proteins (61 and 74 kDa) was increased and that of two others (48 and 53 kDa) was decreased. Maleate treatment caused an increase in the phosphorylation for the same 61-kDa protein, which was also affected by 4-pentenoate treatment, suggesting that phosphorylation of this protein could be related to a mechanism involved in both 4-pentenoate- and maleate-induced Fanconi's syndrome. These changes were also observed in the presence of sodium fluoride and L-bromotetramisole, indicating that the modification of phosphorylation was not due to a difference in phosphatase activities. These results suggest that Fanconi's syndrome induced by 4-pentenoate or maleate is not caused by an inhibition of BBM Na(+)-dependent transport systems. Our results also suggest that protein phosphorylation may play an important role in the molecular defect involved in Fanconi's syndrome.     

Hibernation enhancesd-glucose uptake by intestinal brush border membrane vesicles in ground squirrels

The ability to actively transport nutrients is maintained in intestinal tissues of hibernating ground squirrels compared with their active counterparts, and shows apparent upregulation in hibernators when transport rates are normalized to tissue mass. To identify the mechanisms responsible for the preservation of transport function during the extended fast of hibernation, we studiedd-glucose uptake into jejunal brush border membrane vesicles prepared from active and hibernating 13-lined ground squirrels. Hibernators were without food and showing regular bouts of torpor for at least 6 weeks before sacrifice. Electron micrographs indicated similar microvillus heights of jejunal enterocytes in the two activity states, whereas microvillus density was slightly greater in the hibernators. Glucose uptake into brush border membrane vesicles was inversely related to medium osmolarity, indicating negligible binding of substrate to brush border membrane vesicles surfaces, and intravesicular spaces were similar in hibernating and active squirrels. Glucose uptake showed strong Na⁺ dependency in both groups, with equivalent overshoot values in the presence of Na⁺. Kinetic analysis revealed a significant increase in the maximal velocity of transport (J _max) in hibernators (55.9±5.6 nmol·min^-1·mg^-1) compared with active squirrels (36.7±5.1 nmol·min^-1·mg^-1,P<0.05), with no change inK _m. Thus, the structure and absorptive capacity of the intestinal brush border persists in fasted hibernators, and the increase inJ _max for glucose uptake during hibernation likely contributes to the enhanced Na⁺-dependent glucose absorption previously observed at the tissue level.Abbreviations BBM brush border membrane(s) - BBMV brush border membranes vesicles - SGLT1 Na⁺-glucose transporter - 3-OMG 3-orthomethylglucose - J _max maximal velocity of transport - K _m transporter affinity for substrate - T _b body temperature     

Interactions in vivo between oxidation of non-esterified fatty acids and gluconeogenesis in the newborn rat.

Metabolic interactions between fatty acid oxidation and gluconeogenesis were investigated in vivo in 16h-old newborn rats under various nutritional states. As the newborn rat has no white adipose tissue, starvation from birth induces a low rate of hepatic fatty acid oxidation. Hepatic gluconeogenesis in inhibited in the starved newborn rat when compared with the suckling rat, which receives fatty acids through the milk, at the steps catalysed by pyruvate carboxylase and glyceraldehyde 3-phosphate dehydrogenase. These inhibitions are rapidly reversed by triacylglycerol feeding. Inhibition of fatty acid oxidation by pent-4-enoate in the suckling animal mimics the effect of starvation on the pattern of hepatic gluconeogenic metabolites. It is concluded that, in the newborn rat in vivo, hepatic fatty acids oxidation can increase the gluconeogenic flux by providing the acetyl-CoA necessary for the reaction catalysed by pyruvate carboxylase and the reducing equivalents (NADH) to displace the reversible reaction catalysed by glyceraldehyde 3-phosphate dehydrogenase in the direction of gluconeogenesis.     

Crusade for iron: iron uptake in unicellular eukaryotes and its significance for virulence

The effective acquisition of iron is a pre-requisite for survival of all organisms, especially parasites that have a high iron requirement. In mammals, iron homeostasis is meticulously regulated; extracellular free iron is essentially unavailable and host iron availability has a crucial role in the host-pathogen relationship. Therefore, pathogens use specialized and effective mechanisms to acquire iron. In this review, we summarize the iron-uptake systems in eukaryotic unicellular organisms with particular focus on the pathogenic species: Candida albicans, Tritrichomonas foetus, Trypanosoma brucei and Leishmania spp. We describe the diversity of their iron-uptake mechanisms and highlight the importance of the process for virulence.     

Effect of polyunsaturated fatty acids on endocannabinoid and N-acyl-ethanolamine levels in mouse adipocytes

The tissue concentrations of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoyl-ethanolamine (anandamide), are altered in the adipose tissue of mice fed a high fat diet. We have investigated here the effect on endocannabinoid levels of incubation of mouse 3T3-F442A adipocytes with several free polyunstaurated fatty acids (PUFAs), including linolenic acid (LA), alpha-linolenic acid (ALA), arachidonic acid (AA) and docosahexaenoic acid (DHA), as well as oleic acid (OA) and palmitic acid (PA). By using mass spectrometric methods, we quantified the levels of endocannabinoids, of two anandamide congeners, N-palmitoyl-ethanolamine (PEA) and N-oleoyl-ethanolamine (OEA), and of fatty acids esterified in triacylglycerols or phospholipids, which act as 2-AG and/or N-acyl-ethanolamine precursors. Incubation with AA strongly elevated 2-AG levels and the amounts of AA esterified in triacylglycerols and on glycerol carbon atom 2 (sn-2), but not 1 (sn-1), in phospholipids. Incubation with DHA decreased 2-AG and anandamide levels and the amounts of AA esterified on both the sn-2 and sn-1 position of phospholipids, but not on triacylglycerols. PEA levels augmented following incubation of adipocytes with OA and PA, with no corresponding changes in phospholipids and triacylglycerols. We suggest that dietary PUFAs might modulate the levels of adipocyte phospholipids that act as endocannabinoid precursors.     

Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells

It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km 7.73 × 10⁻⁶ M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10 kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of <1500 Da. Interestingly, only these fractions containing ferric reductase activity also stimulated the uptake of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.     

Changes in mRNA levels of intracellular fatty acid metabolism regulators in human hepatoma HepG2 cells following their treatment with non-esterified fatty acids and dehydroepiandrosterone

The effects of non-esterified fatty acids (NEFA) and hormone dehydroepiandrosterone (DHEA) on the levels of mRNAs of protein kinase C (PKC) -delta and -epsilon isoforms and those of liver fatty acid binding protein (L-FABP) were investigated in the human hepatoma HepG2 cell line. The cells were kept in low-serum, low-albumin medium during experiments. Low FA levels (100 microM) and time intervals of 4 h and 20 h were used. In these conditions, the saturated (palmitic, stearic) and monounsaturated (oleic) acids rather selectively stimulated PKC-epsilon mRNA levels. Unexpectedly, we found that these acids also suppressed liver fatty-acid binding protein (L-FABP) mRNA levels. DHEA in pharmacological doses (100 microM) produced a significant increase in PKC-delta and -epsilon mRNA levels. Although molecular mechanisms underlying the identified changes have not been investigated in this paper, our findings emphasize that NEFA-induced modulation of mRNA levels of key signalling components represent an additional mechanism for how the ambient NEFA can influence metabolic homeostasis in cells.     

The relationship between iron status and lead absorption in rats

The absorption of lead from loops of small intestinein situ was investigated in rats in which iron absorption was increased by stimuli varying in type, intensity, or duration. Lead absorption was increased by a short period of severe iron restriction before any change in hematological indices became apparent. A period of hypoxia, which markedly increased iron absorption, did not influence absorption of lead. An extended period of moderate iron restriction resulted in a marked reduction in liver iron stores and increased iron absorption throughout the 17-wk experiment. Under these conditions lead absorption was initially also increased, but after 12 wk, when iron intake had become adequate to meet essential requirements, lead absorption was similar to that in iron-supplemented rats. These results are discussed in the light of evidence for a receptor-mediated absorption process for iron.     

Organ culture of adult mouse intestine. III. Behavior of the proteins, DNA content and brush border membrane enzymatic activities.

The proteins, the DNA content and the brush border membrane enzyme activities of adult mouse intestinal explants have been measured during a 24-hr organ culture. These activities were not modified in comparison with the controls at the beginning of the culture. The activity of the enzymes secreted in the medium during the 24-hr culture was equal to the activity present in the explants at the start of the culture. These results show that several metabolic functions of the intestine are fully preserved in organ culture.     

Iron-limited growth and kinetics of iron uptake in Magnetospirillum gryphiswaldense

Growth and magnetite formation in Magnetospirillum gryphiswaldense MSR-1 were found close to the maximum at an extracellular iron concentration of 15–20 μM. Ferrous iron was incorporated by a slow, diffusion-like process. Several iron chelators including various microbial siderophores were unable to promote transport of iron into the cells. In contrast, spent culture fluids stimulated the uptake of ferric iron in iron-depleted cells at a high rate, whereas fresh medium and transport buffer were unable to promote iron uptake. However, no siderophore-like compound could be detected in spent culture fluids by the Chrome Azurol S assay. Ferric iron uptake followed Michaelis-Menten kinetics with a K _m of 3 μM and a V _max of 0.86 nmol min^–1 (mg dry weight)^–1, suggesting a comparatively low-affinity, but high-velocity transport system. Iron incorporation was sensitive to 2,4-dinitrophenol and carbonylcyanide-m-chlorophenylhydrazone, indicating an energy-dependent transport process. Received: 21 May 1996 / Accepted: 7 August 1996