Mn bioavailability by polarized Caco-2 cells: comparison between Mn gluconate and Mn oxyprolinate

Abstract

Background: Micronutrient inadequate intake is responsible of pathological deficiencies and there is a need of assessing the effectiveness of metal supplementation, frequently proposed to rebalance poor diets. Manganese (Mn) is present in many enzymatic intracellular systems crucial for the regulation of cell metabolism, and is contained in commercially available metal supplements.

Methods: We compared the effects of two different commercial Mn forms, gluconate (MnGluc) and oxyprolinate (MnOxP). For this purpose we used the polarized Caco-2 cells cultured on transwell filters, an established in vitro model of intestinal epithelium. Since micronutrient deficiency may accelerate mitochondrial efficiency, the mitochondrial response of these cells, in the presence of MnGluc and MnOxP, by microscopy methods and by ATP luminescence assay was used.

Results: In the presence of both MnOxP and MnGluc a sustained mitochondrial activity was shown by mitoTraker labeling (indicative of mitochondrial respiration), but ATP intracellular content remained comparable to untreated cells only in the presence of MnOxP. In addition MnOxP transiently up-regulated the antioxidant enzyme Mn superoxide dismutase more efficiently than MnGluc. Both metal treatments preserved NADH and βNADPH diaphorase oxidative activity, avoided mitochondrial dysfunction, as assessed by the absence of a sustained phosphoERK activation, and were able to maintain cell viability.

Conclusions: Collectively, our data indicate that MnOxP and MnGluc, and primarily the former, produce a moderate and safe modification of Caco-2 cell metabolism, by activating positive enzymatic mechanisms, thus could contribute to long-term maintenance of cell homeostasis.

Figure 1

Microscopy characterization of the in vitro intestinal epithelium model. Cross-sectional images from Caco-2 grown on Transwell filters to form confluent monolayers of differentiated cell are in panels A and B. A junction -rich, organized field is in panel A (JAM white, DAPI blue). Another field with well-developed apical brush border (DPPIV red) is in panel B. Inset in the bottom right corner of panel B displays the 2D free projection max of the same. In panel C Hematoxylin and Eosin stain shows a transversal cryosection from a filter with cells treated with MnOxP for 30 min. The morphology, i.e. columnar cells forming a tight and regular monolayer, appears preserved.

Figure 2

Confocal microscopy of MT on polarized Caco-2 monolayers. Root Mean Square fluorescence intensity quantification from dose-response experiment using cells incubated with MnGluc or MnOxP is shown. Bars indicate standard deviation, lowest at the intermediate concentration.

Figure 3

Confocal microscopy of polarized Caco-2 cells treated with MnGluc or MnOxP- analysis of MT fluorescence. Quantification and distribution of MT in response to 12.5 μM MnGluc or MnOxP are shown. Root Mean Square fluorescence intensity quantification from a representative long experiment is in panel A. Symbols (o, #, *, §) indicate significant differences (p < 0.05) between Caco-2 cells responses. Panel B shows confocal MT images from the same samples at 30 min and 24 h, in agreement with data in panel A and displaying that the regular mitochondria distribution is preserved in treated cells. Transversal 2D free projection max (panel C) from a brief experiment displays both AO and MT signals in the presence of MnOxP. MT shows a transient redistribution of mitochondria close to the apex of cells at 15 min. AO helps in localize the nuclei respect to MT and demonstrates the cells viability.

Figure 4

ATP measurement in Caco-2 cell treated with MnGluc or MnOxP. Panel A shows the quantification of ATP in cell homogenates. No difference between MnOxP -treated compared to untreated cells was measured. Treatment with MnGluc diminished the intracellular content of ATP at 24 h, respect to both MnOxP-treated and untreated Caco-2 cells. ATP determination in apical supernatants of Caco-2 cells in transwell system cell displays that ATP release during the early times (5 min) of MnGluc supplementation is significantly higher in comparison with both MnOxP-supplemented and untreated cells (panel B). In the basal supernatants the release of ATP is in nanomolar range, which renders the statistical difference between MnGluc-treated and untreated cells showed in the histogram not physiologically relevant (panel C). Symbols: * indicates MnGluc differences (p < 0.05) respect to MnOxP, # respect to untreated cells.

Figure 5

Confocal microscopyof pERK, MnSOD and diaphorase histochemistryon Caco-2 treated with MnGluc or MnOxP. Single plane cross-sectional images of a z-series stack from Caco-2 cells doubly labeled for MnSOD (green) and pERK (red) are in panel A left. Cross sections show x-y (squared), x-z and y-z (both rectangular) projections of the same field. Bright granular fluorescence for MnSOD with both metal supplements is shown, but only faint granules of pERK are visible, indicative of mitochondria metabolically active and of healthy cells, respectively. In panel A right the 2D free projection max obtained from all z-planes of the stack are shown, to evidence the contribution of whole cells to pERK signal and to underline the scarce total expression of pERK. βNADPH- and NADH diaphorase assays (representative experiment in panel B) demonstrates by the presence of stable blue formazan precipitate a prevalent activation of NADH- respect to βNADPH-related enzymes upon incubation with MnGluc or MnOxP, a slightly different time-course with the two compounds and a moderate but non-homogenous activation of NADH diaphorase in treated compared to untreated cells.