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Nutrient absorption in the enterocytes

Nutrient absorption in the enterocytes

Dissecting the multicellular ecosystem of metastatic melanoma by Nutridnt RNA-seq. Importantly, thf optimal nutrition abbsorption enterocytes to appropriately sense enterocjtes absorb Nuutrient nutrients, abdorption underlying molecular Herbal remedies for sinus congestion have remained poorly understood. The digestion Nutrient absorption in the enterocytes protein begins Nutrient absorption in the enterocytes the stomach and is completed in the small intestine. Online ISSN Print ISSN The reduced villi, but not small intestinal length in the Ppara I-KO mice indicate that PPARα is required for optimal adaptive growth of the crypt-villus axis. Consistent with the note that both the small and large intestine are involved in the absorption of essential amino acids, amino acid transporter genes such as SLC3A2, SLC25A39, and SLC25A13 were found in the ileum, colon, and rectum Kobayashi et al.

Nutrient absorption in the enterocytes -

HPRT was used as housekeeper. G Protein expression of SGLT1 and PEPT1 in human organoids cultured in hIC and CCM medium for 5 days, respectively. β-ACTIN serves as loading control. Simple sugars can be taken up by enterocytes via passive or active transport — and exit the enterocyte likewise.

The mechanism of intestinal sugar absorption is still not fully understood, given that a variety of transporters of the sodium glucose co-transporter SGLT family and the family of facilitative glucose transporters GLUT with partly unknown specificities is involved Thorens and Mueckler, Genetic variants of transporters contributing to intestinal sugar transport are associated with human diseases, such as glucose-galactose malabsorption and Fanconi-Bickel syndrome caused by mutations in SGLT1 SLC5A1 and GLUT2 SLC2A2 , respectively Martin et al.

Particularly, fructose uptake gained increasing attention, as fructose consumption is rising over the last decades and is associated with developing cardiovascular diseases and type 2 diabetes Johnson et al.

Furthermore, the molecular basis of fructose malabsorption still remains elusive, but defective absorption is most likely. Hence, intestinal organoids which can be directly derived from patients and allow to picture the complex interaction of transporters might considerably advance science in this field.

Previously, we established a straightforward approach to assess nutrient and drug transport in murine intestinal organoids Zietek et al. By using fluorescently FITC labeled dextrans, we were able to show that molecules of a size of 4 kDa rapidly reach the luminal compartment of murine organoids.

Hence, radiolabeled substrates were simply added to the culture plates, keeping the organoids in their 3-dimensional environment a dome of laminin-rich gel Zietek et al.

As species-specific differences might result in misleading outcomes Youhanna and Lauschke, , we validated experimental procedures for human intestinal organoids, allowing for tackling human-specific research questions.

After confirming translocation of 4 kDa FITC-dextrans also into the lumen of human organoids Supplementary Figure 2A , we applied the experimental procedures established in murine intestinal organoids Supplementary Figure 2B to human organoids.

First investigating uptake of glucose and fructose, we used different inhibitors for functional characterization of monosaccharide transport, the SGLT1 inhibitor phloridzin, the GLUT inhibitor phloretin and rubusoside, inhibiting fructose transport by GLUT5 Figure 2A.

Figure 2. Nutrient and drug transport in human intestinal organoids. A Schematic illustration of the transporters investigated and inhibitors used. B Uptake of radiolabeled glucose in human organoids derived from different small intestinal segments.

C Uptake of radiolabeled fructose in human duodenal organoids. E Left: comparison of detected counts per sample for both approaches using the same amout of radiolabeled substrates and right: reduction of radiolabeled Gly-Sar uptake by the competitive inhibitor Gly-Gly depicted for both approaches.

F Chemical structures and formulas of the peptidomimetics used. G Assessment of transport of peptidomimetics in a competition assay using radiolabeled Gly-Sar in murine small intestinal organoids derived from wild type and Pept1 knockout mice.

H Similar approach to G using human duodenal organoids. I Reduction of radiolabeled Gly-Sar uptake using the antibiotic Cefadroxil as competitive inhibitor. E,I Unpaired t tests. Glucose is a substrate for both, apical and basolateral GLUT transporters, with the electrogenic solute carrier SGLT1 as the main apical glucose transporter in the small intestine.

GLUT5 represents an exception, transporting exclusively fructose at the apical membrane. Opposing, the uniporter GLUT2 mediates glucose and fructose fluxes at the basolateral membrane via facilitated diffusion, providing import as well as export capacities Thorens and Mueckler, ; Roder et al.

Due to the experimental setup, substrates first reach the outside, i. Therefore, it is not possible to target apical or basolateral transporters separately, yet the use of inhibitors enables to illustrate contributions of certain transporters. Thus, using glucose as substrate in combination with either phloridzin or phloretin in human organoids derived from different regions of the small intestine, resulted in the expected pattern of blunted glucose uptake, which was more pronounced with the pan-GLUT inhibitor phloretin as compared to the SGLT1 inhibitor phloridzin Figure 2B.

In line, fructose transport could be diminished by phloretin, and to a lesser extent, by the GLUT5-inhibitor rubusoside Figure 2C as well as glucose Supplementary Figure 2C in human duodenal organoids.

In this case, human duodenal organoids were exposed to the radiolabeled dipeptide glycyl-sarcosin Gly-Sar , a hydrolysis-resistant model substrate of the peptide transporter PEPT1.

Peptide transport over the plasma membrane occurs in cotransport with protons and allows transport of di- and tripeptides against a substrate gradient[24]. Next to PEPT1-mediated substrate fluxes at the apical membrane, a not yet genetically identified system for basolateral peptide uptake with similar features to PEPT1 has been described Berthelsen et al.

Although radiolabeled transport assays are very sensitive, costs of labeled substrates are a major drawback. Hence, reducing the amount of substrates needed for experiments is desirable.

As mentioned before, peptide transporters also play a role in drug uptake, including peptidomimetics. Peptidomimetics are compounds mimicking a peptide or protein, which possess the ability to interact with a biological target to exert agonistic or antagonistic effects Giannis and Kolter, ; Marshall and Ballante, Hence, they have a great potential in drug discovery, exerting drug-like properties Rader et al.

For example, peptidomimetics have been designed for cancer therapy, e. Primary goals in the development of orally available peptides are improving their intestinal transport and enhancing their stability to enzymatic degradation. Common strategies comprise the use of cyclic peptides, as well as D - instead of L -amino acids and N -methylation to increase metabolic stability Rader et al.

For example, Cilengitide, a cyclic pentapeptide with one D -amino acid and one N -methylation is completely stable in humans and is excreted with a half-life of 4 h without any metabolization Becker et al.

Yet, intestinal permeation from the lumen into the bloodstream remains a major challenge. Structural changes affect intestinal and cellular permeability, and a change in one methyl position already can greatly impact permeability properties Ovadia et al.

Oral availability crossing the gastrointestinal wall to reach the circulation can be mediated via paracellular or transcellular mechanisms, including active transporters Rader et al.

Common tools to evaluate permeability properties of peptide drugs include Caco-2 monolayers and the side-by-side diffusion chamber Ussing chamber , however, both systems are poorly correlative Jezyk et al.

Caco-2 cells, even though known to possess a rather small intestinal phenotype Yee, , were originally derived from a colon carcinoma, and phenotypic as well as functional characteristics highly differ from native human enterocytes Harwood et al. For example, Caco-2 cells exhibit tighter junctions compared to the small intestine of human Matsson et al.

In contrast, Ussing chamber approaches, mainly using excised rat tissue better reflect physiology but suffer from potential species differences and large numbers of animals needed for screening. Hence, we tested the applicability of intestinal organoids as a new tool to evaluate the absorption properties of peptidomimetics.

Three different cyclic hexapeptides P1, P2, P3 Figure 2F were tested that were originally developed via a stepwise library approach: First a library of more than 55 different N -methylated alanine peptides of the general structure cyclo D -Ala- L -Ala 5 were synthesized and investigated in a Caco-2 assay Ovadia et al.

Peptides identified as highly permeable including P3 were subsequently functionalized by substitution of neutral Ala residues with the integrin-binding tripeptide sequence RGD.

Among them, one compound P2 , has been identified with similar high activity and selectivity as Cilengitide sub-nanomolar affinity for integrin αvβ3, high selectivity against other integrins Weinmuller et al.

However, P2 lacked permeability due to charges in the cyclic N -methylated alanine-peptides. To overcome this limitation, charged residues were protected with lipophilic protecting moieties two hexyloxycarbonyl Hoc groups and conversion of the carboxylic side chain of Asp into a neutrally charged methyl ester.

The resulting compound P1 showed both, permeability in the Caco-2 assay and biological activity after oral administration in mice Weinmuller et al. To test the involvement of active peptide transporter-mediated uptake in the permeability properties of P1-P3 , we evaluated the ability of the three cyclic hexapeptides to competitively inhibit the uptake of radiolabeled Gly-Sar in murine small intestinal organoids derived from wild type and Pept1-deficient mice.

In this assay, we identified P1 as a potential substrate for active transport mediated by Pept1, P2 to be actively transported independently of Pept1, and in contrast, P3 showed no signs of peptide transporter-mediated uptake in murine organoids Figure 2G.

Subsequently testing P1 and P3 in human duodenal organoids, both peptides were able to significantly reduce radiolabeled Gly-Sar uptake, indicating P1 and P3 to be substrates for peptide transporter-mediated uptake in humans Figure 2H.

These data highlight the suitability of intestinal organoids to screen for transporter-mediated uptake of drug candidates, a process that might have been underappreciated in Caco-2 assays due to lack of physiological transporter expression, but contributes to oral availability.

Additionally, efflux processes that limit drug absorption might be evaluated in detail in organoid systems Schumacher-Klinger et al. Concomitantly, these data point toward potential species-specific transport phenotypes as already described for PEPT1 Kottra et al.

Accordingly, we could also confirm transport of the peptide-like β-lactam antibiotic cefadroxil, that has been previously described as PEPT1 substrate Ganapathy et al. In conclusion, these results underline the superior properties of human intestinal organoids for studying nutrient and drug uptake.

Since organoids retain location-specific properties of their site of origin, absorption could even be determined at an intestinal region-specific resolution. It has been reported that fluorophore-conjugated dipeptides with a high-affinity for PEPT1 were able to block transport of Gly-Sar, however, they failed to be transported Abe et al.

To exclude similar effects, either specific inhibitors can be applied for example Lys-z-NO2-Val, a specific PEPT1-inhibitor or downstream effects of transport processes can be investigated. Thus, we extended our previously established protocol for visualization of intracellular signaling by life-cell imaging of murine intestinal organoids Zietek et al.

As mentioned before, peptide transport over the plasma membrane occurs in cotransport with protons, leading to cytosolic acidification of enterocytes Chen et al. Hence, intracellular changes immediately reflect transport activities and provide direct evidence for substrate fluxes.

A drop in pH can be visualized by live-cell imaging using fluorescent probes Chen et al. Employing the pH-indicator BCECF-AM, intracellular acidification was demonstrated in human duodenal organoids upon exposure to Gly-Sar, Gly-Gly as well as cefadroxil and the carbonyl cyanide m-chlorophenyl hydrazine CCCP, an ionophore used as a positive control Figures 3A—D.

Stimulating organoids with CCCP subsequent to administration of Gly-Sar, Gly-Gly, and cefadroxil caused an additional decline in intracellular pH, indicating the physiological range of observed responses Supplementary Figure 3A.

As expected, neither glucose nor fructose used as negative controls led to an intracellular acidification of enterocytes Supplementary Figure 3B.

In accordance to literature, robust signals were obtained upon ATP-mediated increases in intracellular calcium Figure 3E.

For both dyes, BCECF-AM and FuraAM, excellent dye-loading efficiency was observed Supplementary Figure 3C. Figure 3. Visualization of intestinal peptide transport processes. Intracellular acidification visualized by BCECF-AM induced by transport of peptide-transporter substrates A Gly-Sar and B Gly-Gly, C by the antibiotic Cefadroxil and D the protonophore CCCP.

E Calcium responses to ATP stimulation visualized by Fura Intracellular acidification induced by the antibiotic Cefadroxil. F Schematic illustration of the transporters investigated and inhibitors used.

G Course of intracellular acidification induced by Gly-Sar exposure for an extended time frame left with and middle without the NHE-inhibitor Amilorid; right: overlay of both curves giving relative BCECF ratios.

H Similar approach to G using the NHE3-inhibitor S A—E human duodenal organoids, G,H murine small intestinal organoids. For data analysis, whole organoids were selected and no background correction was applied. Analyses were performed on several organoids derived from independent cultures and representative measurements are shown.

For continuous peptide uptake, IECs need to maintain the transmembrane ionic gradients and furthermore, augmented or prolonged acidification of the cell by proton symport of peptide transporters has to be avoided.

In enterocytes, several types of NHEs are expressed, and NHE3 specifically has been shown to be required for proper PEPT1-mediated transport Chen et al. Importantly, NHE-function is targeted by both, clinically relevant drugs as well as bacterial toxins. To illustrate the function of NHEs in general and NHE3 in particular in the context of active peptide transport in organoids, we used two different inhibitors: Amiloride, an FDA-approved inhibitor of NHEs, and S, which predominantly acts on NHE3 Wiemann et al.

As expected, both inhibitors prevented the recovery of intracellular pH to basal levels as observed in non-treated murine organoids following exposure to Gly-Sar Figures 3G,H left.

In accordance to their specific inhibitory spectrum, amiloride led to a continuous influx of protons in the observed time span Figure 3G , while S treatment resulted in a stable intracellular pH level below base line Figure 3H. To decipher biology and functional characteristics of intestinal transporters it is very important not only to quantify transport of substrates, but also to take intracellular downstream effects and signaling into account, as presented above.

These data highlight the high-resolution measurements possible in intestinal organoids. Metabolism in IECs has gained increasing attention, not only due to the expression of key drug metabolizing enzymes, including cytochrome P 3A4 CYP3A4 , in small intestinal epithelial cells, that are prone to diet-drug interactions Lown et al.

IEC and whole body metabolism are tightly interrelated via production of incretine hormones Zietek and Rath, and factors like Fgf15 Kliewer and Mangelsdorf, by enteroendocrine cells and enterocytes, respectively, and vice versa, IECs are targets of remote-tissue metabolic signals such as insulin and leptin signaling Yilmaz et al.

In the gastrointestinal tract, carbohydrates, peptides and lipids are broken down and absorbed by enterocytes. Subsequently, they serve as substrates for cellular energy generation or for interconversions and distribution to the whole organism via transfer into the circulation.

Hence, IEC metabolism also profoundly impacts availability and quality of nutrients, constituting an initial check point between diet and host. In this context, the intestinal microbiota plays an additional key role, as a source of bacterial metabolites such as short chain fatty acids SCFAs including butyrate.

IEC metabolism and exposure to certain nutrients furthermore relates to diseases, for example high-fat diets were shown to enhance tumorigenicity of intestinal progenitors Beyaz et al. Despite the fact that general metabolic functions of enterocytes are understood, many open questions remain, including whether the small intestine can act as a site for gluconeogenesis, which seems to be species-dependent Sinha et al.

Metabolomic approaches are key technologies allowing to tackle such questions by enabling analysis of metabolic events in a large scale and high throughput manner.

First, we determined the effect of insulin on amino acid AA and acylcarnitine levels in small intestinal organoids. All proteinogenic amino acids could be detected in small intestinal organoids at concentration ranges given in Figure 4B.

Insulin is known to promote anabolism, affecting both, processes of protein synthesis and proteolysis. Enterocytes respond to insulin signals and develop insulin resistance under conditions of obesity-related inflammation Monteiro-Sepulveda et al.

In line, concentrations of valine and alanine responded fast to insulin stimulation showing maximal reduction 30 min after addition of insulin Figure 4C , consistently with most other AAs data not shown , indicating a shift in protein turnover toward an enhanced net incorporation of AAs in proteins.

In parallel, tau-methylhistidine, a marker compound for proteolysis and propionylcarnitine C3 , a typical intermediate in the breakdown of valine, isoleucine, methionine and threonine were diminished with lowest levels observed 60 min after insulin stimulation Figure 4D , confirming also the inhibitory effect of insulin on proteolysis in intestinal organoids.

Figure 4. Metabolite analysis in intestinal organoids. A Schematic representation of the experimental setup from which samples were derived for analyses shown in panel B,C. B Range of amino acid AA concentrations detected in organoids. C Concentration of valine and alanine at different time points after insulin stimulation.

D Concentration of tau-methylhistidine, a marker compound for proteolysis, and propionylcarnitine C3 , a typical intermediate in the breakdown of valine, at different time points after insulin stimulation.

E Schematic representation of the experimental setup from which samples were derived for analyses shown in panel F. F Concentration of the acylcarnitine species Acetylcarnitine C2 , Butyrylcarnitine C4 , and Palmitoylcarnitine C16 at different time points after addition of butyrate.

G Proposed mode of action for the effect of butyrate on beta-oxidation. H Schematic representation of the experimental setup from which samples were derived for analyses shown in panel I.

I Appearance of deuterium-labeled acylcarnitines at different time points after addition of deuterium-labeled dpalmitate. J Schematic illustration of carnitine acyltransferases involved in the generation of the acylcarnitine species detected.

B,C,F,I Representative results from three independent organoid cultures. ASCL, long-chain acyl-CoA synthetase; CPT, carnitine palmitoyltransferase; CAT, carnitine acetyltransferase; CACT, carnitine-acylcarnitine translocase.

Next, we depict the effect of butyrate on acylcarnitine profiles in murine large intestinal organoids. In this approach, 1mM butyrate was added and shifts in acylcarnitines were measured 0, 5, 10, 30, and 60 min afterward Figure 4E. Butyrate has been shown to broadly affect colonocyte metabolism, including glucose utilization Donohoe et al.

In accordance to literature, a clear effect of butyrate on saturated acylcarnitines, comprising short-, medium- and long-chain acylcarnitines was observed, with acetylcarnitine, butyrylcarnitine and palmitoylcarnitine increasing to maximal concentrations 60 min after butyrate addition Figure 4F.

A proposed mechanism explaining the effect of butyrate involves the butyrate transporter SLC5A8 and the butyrate receptor GPRA expressed by coloncytes Cresci et al.

Last but not least, we followed the breakdown of dlabeled palmitic acid, in which all 31 hydrogen atoms are replaced by deuterium atoms, in small intestinal organoids. Stable isotope labeling enables following the fate of the labeled fatty acid within the enterocyte, being either subjected to chain-shortening during beta-oxidation and conversion to the respective acylcarnitine species for energy generation, or being reesterified, and incorporated into chylomicrons for systemic supply.

Importantly, sensing dietary fat via fatty acid oxidation in enterocytes has been implicated in the control of eating Langhans et al. Appearance of deuterium-labeled acylcarnitines were determined 0, 10, 30, and 60 min after addition of dpalmitic acid Figure 4H.

Indicating beta-oxidation, we could detect chain-shortened, deuterium-labeled acylcarnitine species Figure 4I. The conversion of the long-chain fatty acids to their acylcarnitine species is known to be mediated by carnitine palmitoyltransferase 1 and 2 CPT1 and CPT2 , while short-chain acylcarnitine species are formed by carnitine acetyltransferase CAT Figure 4J.

Carnitine octanoyltransferase COT located in peroxisomes is responsible for the conversion of medium-chain fatty acids Violante et al. Contrarily, CPT1 is located in the outer mitochondrial membrane and thus may convert the added dpalmitic acid directly to dpalmitoylcarnitine Bonnefont et al.

Shorter fatty acid intermediates are formed within the mitochondria and their respective acylcarnitine species are generated by CPT2 and CAT, located in the inner mitochondrial membrane.

Consistent with the sequential removal of 2-carbon units during beta-oxidation, dmyristoylcarnitine dC and to a lesser extent ddodecanoylcarnitine dC could already be seen after 10 min of incubation, whereas ddecanoyl-, doctanoyl- and dhexanoylcarnitine appeared 30 min after addition of dpalmitic acid.

Of note, the larger peaks of dC6, as compared to dC10 and dC8 after 30 and 60 min might be explained by a higher preference of CAT for short-chain fatty acid substrates C2 to C6. In summary, intestinal organoids are an excellent model system close to physiology to explore cellular metabolism and the applied metabolic readouts could be adapted easily to the 3D culture.

Human organoids, constituting the most relevant model, are superior to animal rodent -derived organoids and cancer cell lines, especially in the context of metabolism and diseases, since metabolic properties differ between species and alterations in the cellular metabolism are part of many pathologies.

Thus, human organoids hold great potential to answer remaining questions on intestinal metabolism and to identify drug targets to improve overall metabolic health.

Taken together, our results demonstrate that intestinal organoids cultured in 3D, embedded in a laminin-rich gel dome, the most basic and probably least cost and labor extensive culture protocol, is suitable for a broad range of measurements in the field of intestinal transport and metabolic studies.

Beyond these applications, many other readouts are possible in this setup, for example assessment of proteasome activity Supplementary Figure 2D , which is of interest in the context of proteasome inhibitors, an important class of drugs in the treatment of different types of cancer Fricker, Implementing other culture protocols like organoids with reversed polarity in which the apical side faces outward Co et al.

Paracellular transport of fluorescein, transcellular transport of propranolol, and basolateral efflux of rhodamin, a substrate of p-glycoprotein MDR1 have been measured in a model in which human organoid-derived cells are seeded as a 2D monolayer on a porcine small intestinal scaffold Schweinlin et al.

The field of applications for organoids is still rapidly growing, and there is a trend toward more complex and sophisticated organoid-based model systems. For example, co-cultures with bacterial and viral pathogens and immune cells Yin et al.

These systems provide a microenvironment to study the impact of oxygenation, mechanical stress, and tissue communication via soluble factors and will further advance intestinal research. Yet, to date they remain very expensive tools in highly specialized laboratories not suitable for broad applications Almeqdadi et al.

In contrast, the intestinal organoid culture protocols and methods presented here represent in vitro models that already now allow for partly replacement and reduction of animal numbers needed for research and testing. The pieces were transferred into cold HBSS and vigorously suspended to obtain fractions.

Mesenchymal and immune cells were further removed by discarding supernatant after centrifugation 10 s at rpm. Then, epithelial tissue was enriched through centrifugation 3 min at 1, rpm.

After centrifugation 3 min at 1, rpm , the sediment was incubated in Tryple Invitrogen for 20 min at 37°C to obtain single-cell suspension. The libraries were subjected to high-throughput sequencing on an Illumina Hiseq X Ten PE platform, and bp paired-end reads were generated.

The raw sequencing reads were first demultiplexed using Illumina bcl2fastq software to generate bp paired-end read files in FASTQ format. The reads were then aligned to the GRCh38 human reference genome using the Cellranger toolkit version 2.

The exonic reads uniquely mapped to the transcriptome were then used for unique molecular identifier UMI counting. Selection and filtering of the droplet barcodes for single cells were done using the Cellranger toolkit as described before Haber et al. In brief, the 99th percentile of the total UMI counts divided by 10 was used as cutoff for calling of single cells.

Finally, mesenchyme, immune, and hematopoietic cells were removed based on these marker genes LSP1, MZB1, VIM, CD52, CD78B, and COL3A1. CD45 was not detected in our results.

Library size normalization was performed using Seurat NormalizeData. Next, the six batches of single-cell RNA-seq data were subjected to batch correction, as described previously Mayer et al. In brief, the canonical correlation analysis CCA strategy was used to find linear combinations of features across datasets that are maximally correlated.

The shared correlation structure conserved among the six datasets from the ileum, colon, and rectum were identified. Based on the shared structure, all six batches of data were finally pooled into a single object for downstream analyses Butler et al.

Batch distributions for each dataset were visualized using t-distributed stochastic neighbor embedding t-SNE plots. The R package Seurat was used to combine linear and nonlinear dimensionality reduction algorithms for unsupervised clustering of single cells. Specifically, first, highly variable genes were identified by the FindVariableGenes function, and average expression and dispersion for each gene were calculated.

Subsequently, CCA was performed based on the variable genes in the six intestine samples. The canonical correlation vectors then projected each dataset into the maximally correlated subspaces for downstream analysis.

Graph-based clustering was performed, which allocated cells in a K-nearest neighbor graph structure based on high correlation strength CCA. The cells were then iteratively clustered, and the modularity was optimized with the Louvain algorithm. Finally, we used t-SNE to place cells with similar local neighborhoods in high-dimensional space or low-dimensional space based on scaled expression of variable genes to visualize the clustering results of all the cells.

To identify signature genes of each cell type, the functions FindAllMarkers and FindMarkers in Seurat were used with the following configurations: min. For a given cluster, FindAllMarkers identified positive markers compared with all other cells.

All differentially expressed genes as positive markers of specific cell clusters are listed in Tables S1—S8. Expression heatmaps of the signature genes for each cluster are shown in Fig.

Similarly, the function FindMarkers was used for identification of signature genes by comparing the cell type of interest to another specific group of cells e.

Single-cell transcriptome data of mouse ileum was obtained from Gene Expression Omnibus GEO accession no. GSE Haber et al. Altogether, we compared gene expression matrices of 6, human ileum cells in this study to the 3, mouse ileum cells, which were subjected to the same process of quality control and filtering.

We only considered the homologous genes between human and mouse, which eventually generated a scaled expression matric for 11, genes of 10, cells. For each pair of cells from human and mouse, the Pearson correlation was calculated with the scaled expression data of the genes in the two cells.

We obtained single-cell transcriptome data of mouse ileum from GEO accession no. Altogether, we analyzed gene expression matrices of 6, human ileal cells in this study and 3, mouse ileal cells after removing the low-quality cells using the same filtering strategy with the human data.

We considered the expression data of all 11, homologous genes with identical gene names between the human and mouse datasets and then performed CCA as implemented in the Seurat Butler et al. t-SNE, cell cycle, and differential expression analyses were performed using the same methods described above.

Immunofluorescence and immunohistochemistry were performed as previously described Qi et al. Then, the sections were incubated overnight with the primary antibody at 4°C. The fluorescein-labeled secondary antibodies ; Life Technologies for immunofluorescence or secondary horseradish peroxidase—conjugated anti-rabbit antibody ; Invitrogen for immunohistochemistry were added for 2 h at room temperature.

Lgr5-EGFP mice were obtained from The Jackson Laboratory. Animals were then euthanized, and tissue was processed immediately. All animal experiments were conducted in accordance with the relevant animal regulations with approval of the Institutional Animal Care and Use Committee of Tsinghua University.

Rabbit anti-LYZ , ab; Abcam , mouse anti-MUC2 , ab; Abcam , rabbit anti-NUSAP1 , —1-AP; Proteintech , mouse-anti Ki67 , s; CST , mouse anti-E-Cad ,, ; BD Biosciences , rabbit anti-ITLN1 , —1-AP; Proteintech , rabbit anti-TFF1 , —1-AP; Proteintech , rabbit anti-APOB , —1-AP; Proteintech , rabbit anti-APOA4 , —1-AP; Proteintech , rabbit anti-SLC26A2 , —1-AP; Proteintech , rabbit anti-SCNN1B , —1-AP; Proteintech , rabbit anti-SLC35A1 , —1-AP; Proteintech , rabbit anti-FABP6 , —1-AP; Proteintech , rabbit anti-SLC38A1 , —1-AP; Proteintech , and rabbit anti-SLC44A1 , —1-AP; Proteintech.

The sections were prepared with vibrating blade microtome HM; Microm and endogenous peroxidase blocking was performed by RNAscope Hydrogen Peroxide ; ACD for 10 min at room temperature.

Then, RNAscope Protease Plus ; ACD was used for 10 min at 40°C before probe hybridization. ITLN1 probe ; ACD and LYZ probe ; ACD were hybridized for 2 h at 40°C, AMP 1—6, and signal detection was performed as described in the user manual ; ACD. RNeasy Mini Kit Qiagen was used to extract total RNA, and cDNA was obtained by Revertra Ace Toyobo.

Then, real-time PCR reactions were performed in triplicate on a LightCycler Roche. Primers of selected genes are listed in Table S Human intestinal tissue was washed in cold HBSS and removed muscle tissue. The pieces were transferred into cold HBSS and vigorously suspend to get fraction, and epithelial tissue was enriched through centrifugation 3 min at 1, rpm.

Crypts were then embedded in Matrigel BD Biosciences and seeded on a well plate. To find out amino acid uptake per cell, organoids were incubated in Tryple Invitrogen for 20 min at 37°C to obtain a single-cell suspension.

The amino acid uptake per cell was calculated by combining amino acid changes and cell number. For choline, succinic and citric acid uptake, 20 mM choline C; MACLIN , succinic acid S; MACLIN , and citric acid C; MACLIN were added to the crypt culture medium 8 h later after the first passage.

Organic solute uptake per cell was calculated by combining amino acid changes and cell number. For sugar uptake, GlutaMAX-I was removed from the medium 8 h later after the first passage.

Then, 20 mM fructose S; Selleck , galactose S; Selleck , and mannose S; Selleck were added to the medium, and 50 μl medium was selected 24 h later to detect uptake changes, and the cell number was counted by FACS for PI-negative cells as described above.

Sugar uptake per cell was calculated by combining amino acid changes and cell number. The fold change from ileum, colon, and rectum organoids was calculated by comparing with ileum organoids.

All experiments with quantitation were performed independently at least three times with three replicates within each experiment, and data are represented as mean ± SD. All statistical analysis was performed with GraphPad Prism 7 win. All data have been deposited in the GEO under accession no.

R markdown scripts enabling the main steps of the analysis to be performed are available from the corresponding authors on reasonable request. S1 shows general information of clinical samples and annotations of cell types of single-cell RNA-seq data. S2 shows expression patterns of cell markers.

S3 shows characterization of stem cells, TA cells, progenitor cells, and transcription factor analysis. S4 shows expression patterns of transporter genes and validation by immunofluorescence or immunohistochemistry.

Table S1 shows all cell type—specific genes. Table S2 shows ileum, colon, and rectum cell type—specific genes. Table S3 shows stem cell and TA subset genes. Table S4 shows progenitor subset-specific genes. Table S5 shows enterocyte cell subset-specific genes.

Table S6 shows enteroendocrine cell subset-specific genes. Table S7 shows PLC subset signature genes. Table S8 shows goblet cell subset signature genes.

Table S9 shows signature genes involved in the cell cycle. Table S10 shows quantitative PCR primers. We thank Drs. Ligong Chen and Xin Zhou for critical reading of the manuscript, Yuxin Sun for information consolidation, and the Metabolomics Facility at Tsinghua University for LC-MS analyses.

This work was supported by the National Key Research and Development Program of China grant YFA to Y. Chen and grant YFC to X. Yang and the National Natural Science Foundation of China grant to Y. Chen and grants and to X. Author contributions: Y.

Wang and Y. Chen designed the study and analyzed the data; Y. Wang performed the experiments; W. Song and X. Yang performed the bioinformatics analysis and analyzed the data; J. Wang and W. Fu provided samples and selected clinical information and analyzed the data; T.

Wang and X. Xiong helped with functional experiments; Z. Qi helped with single-cell isolation; and Y. Wang, W. Song, X. Yang, and Y. Chen wrote the manuscript. Cell landscapes of human intestines based on single-cell transcriptome profiles.

A, C, E, and G t-SNE plots of single-cell clusters. The Seurat algorithm was used to visualize the clustering of all 14, intestine epithelial cells from six donors A , 6, ileum cells from two donors C , 4, colon cells from two donors E , and 3, rectum cells from two donors G.

B, D, F, and H Expression heatmaps of cell type—specific genes were obtained by analyzing all cells pooled together B or based on cells from one of the three intestine segments: ileum D , colon F , and rectum H.

EC, enterocytes; EEC, enteroendocrine cells; G, goblet cells; PRO, progenitor cells; SC, stem cells. Differential nutrient absorption preferences in the human small and large intestine. A Expression heatmap of signature genes in enterocytes and gene functional enrichments.

B Violin plots showing distributions of mean expression of transporter genes in different segments il, ileum; co, colon; re, rectum. C Expression patterns of specific genes involved in nutrient absorption and transport in different intestine segments.

Each dot represents a gene, of which the color saturation indicates the average expression level scaled by Z-score in an intestine segment, and the size indicates the percentage of cells expressing the gene. D Relative mRNA expression of the gene marked in red in C was confirmed by qPCR in the organoids derived from human ileum, colon, and rectum.

E Nutrient uptake was measured by LC-MS in the organoids derived from human ileum, colon, and rectum. Data are represented as mean ± SD of at least three independent experiments.

Differential expression of signaling molecules, hormones, and immunity-related genes. A Violin plots showing expression distributions of signaling genes in enterocytes from the ileum il , colon co , and rectum re.

B Expression heatmap of signature genes in enteroendocrine cells. C Expression patterns of specific hormone genes in enteroendocrine cells from different intestine segments.

D Expression patterns of immune-related genes in cells from different intestine segments and gene functional enrichments.

Each dot represents a gene, of which the color saturation indicates the average expression level scaled by Z-score in an intestine segment and the size indicates the percentage of cells expressing the gene.

PLCs identified in the human colon and rectum. A Expression heatmap of signature genes in PLCs and gene functional enrichments. B Lysozyme LYZ expression indicated by color saturation in single cells from the ileum, colon, and rectum. C Immunofluorescence was performed to confirm LYZ expression in the ileum, colon and rectum.

Scale bars, µm. D smFISH results of LYZ in human ileum, colon, and rectum. Scale bar, µm. E mRNA expression levels of growth factors in PCs and PLCs in the ileum and PLCs in the colon and rectum. F Specific expression of GNPTAB and SOD3 in PCs and PLCs of the human large intestine.

G Violin plots showing expression distributions of transcription factors in PCs or PLCs. H Kit expression in all 14, cells. Potential new markers of human TA cells and goblet cells. A NUSAP1 expression indicated by color saturation in single cells from the ileum, colon, and rectum.

B Immunofluorescence was performed to confirm the NUSAP1 expression in the ileum, colon, and rectum. C Statistics of the NUSAP1 immunofluorescence data shown in B. D Expression heatmap of signature genes in goblet cells and gene functional enrichments.

E ITLN1 expression indicated by color saturation in single cells from the ileum, colon, and rectum. F Immunofluorescence was performed to confirm ITLN1 expression.

G TFF1 expression indicated by color saturation in single cells from the ileum, colon, and rectum. H Immunofluorescence was performed to confirm the TFF1 expression. Comparison of gene expression profiles between the epithelial cells of the human and mouse ilea.

C Cell cycle analysis. Cell Metab. Tough, I. Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y1 and Y2 receptors. Moodaley, R.

Agonism of free fatty acid receptors 1 and 4 generates peptide YY-mediated inhibitory responses in mouse colon. Bilchik, A. Peptide YY augments postprandial small intestinal absorption in the conscious dog.

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Download references. We thank Dr. Gerard Gradwohl and Dr. Mary Estes, Dr. Sarah Blutt and Ms. Xi-Lei Zeng for training in generating HIO-derived enteroid monolayer culture systems; Ms. Catherine Martini for technical assistance. We acknowledge support provided by the Confocal Imaging Center, the Pluripotent Stem Cell Facility, and Research Flow Cytometry Core at CCHMC.

We would like to thank the members of the Wells, Zorn, and Helmrath laboratories for reagents and feedback. This work was supported by the grants from the NIH, U19 AI J. and the Allen Foundation J. We also received support from the Digestive Disease Research Center P30 DK Heather A.

McCauley, Jacob R. Enriquez, Jonah T. Nichol, J. Guillermo Sanchez, William J. Stone, Michael A. Department of Pharmacology and Systems Physiology, University of Cincinnati College of Medicine, Albert Sabin Way, Cincinnati, OH, , USA. Andrea L. Matthis, Marshall H. You can also search for this author in PubMed Google Scholar.

and J. conceived and initiated the project, designed experiments, and wrote the paper, with conceptual input from M. and E. performed all experiments in collaboration with: J. and W. on mouse transplantation; N. and M. in generating HIO-derived enteroids; A.

on electrophysiological studies. interpreted data. supervised the project. All authors have edited and approved the paper. Correspondence to James M. Peer review information Nature Communications thanks the anonymous reviewer s for their contribution to the peer review of this work.

Open Access This article is licensed under a Creative Commons Attribution 4. Reprints and permissions. Enteroendocrine cells couple nutrient sensing to nutrient absorption by regulating ion transport.

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nature nature communications articles article. Download PDF. Subjects Gastrointestinal hormones Gastrointestinal models Mechanisms of disease Physiology. Abstract The ability to absorb ingested nutrients is an essential function of all metazoans and utilizes a wide array of nutrient transporters found on the absorptive enterocytes of the small intestine.

Introduction Enteroendocrine cells EECs are a rare population of cells found in the gastrointestinal epithelium that sense nutrients that are passing through the gut and in response secrete more than 20 distinct biologically active peptides.

Results The PYY-VIP axis regulates ion transport in small intestine If EECs were required for regulating the normal electrophysiology of the small intestine, we would expect to see deranged ion transport in intestinal tissues lacking EECs.

Full size image. Discussion In this study, we found that loss of all EECs resulted in profound imbalance of ion transport in the small intestine, with subsequent impairment of nutrient absorption.

Catalina Cosovanu Nutrlent, Philipp ReschStefan JordanNutrient absorption in the enterocytes LehmannMarkus RalserNutrient absorption in the enterocytes AbxorptionJoachim Stress management for blood pressureMichael Mülleder absrption, Sebastian BrachsChristian Neumann; Intestinal epithelial c-Maf expression determines enterocyte differentiation and nutrient uptake in mice. J Exp Med 5 December ; 12 : e The primary function of the small intestine SI is to absorb nutrients to maintain whole-body energy homeostasis. Enterocytes are the major epithelial cell type facilitating nutrient sensing and uptake. However, the molecular regulators governing enterocytes have remained undefined.

Nutrient absorption in the enterocytes -

In total, 5—10 villi per section from 2—3 sections per jejunum were measured, and the average length reported. Microvilli were imaged in the samples from the proximal jejuna by the transmission electron microscope Morgagni FEI Company, Eindhoven, Netherlands at EM Core Facility of University of Geneva.

Antibodies : LysC sc, Santa Cruz , anti-BrdU ab, Abcam , chromogranin A sc, Santa Cruz , Alcian blue was used as previously described 3. For histological experiments, researchers were blinded to group allocation during processing and quantification of the sample through labelling of samples by the numeric codes.

Intestinal crypts were isolated and the cultured as described before For WENR medium, murine WNT3A Peprotech was added to ENR in the concentration indicated. Crypts were harvested for RNA or protein isolation on days 7—10 by dissolving matrigel in ice-cold PBS. Crypts from Lgr5-GFP mice were isolated as above, and suspension of villi in PBS was obtained by gently scraping longitudinally opened section of proximal-to-mid jejunum by microscope cover glass.

The cells were sorted on BD FACS ARIA Fusion, with software BD FACS Diva Software version 8. FACS plots have been produced by FlowJoTM version Cells were sorted in RTL solution Qiagen.

MatTek obtained tissue samples from accredited institutions after informed consent of the donor or next of kin for use of cells or tissues for research purposes. Biopsy cultures were maintained in proprietary medium supplanted on day 2 on apical side with the inhibitors in the concentrations described in the previous section, and renewed every day.

Free fatty acids in sampled aliquots were measured by NEFA-HR kit Wako. Membranes with the tissues were excised from the insets, cut in half and saved for RNA isolation and histology. Statistical tests are specified in the figure legends. To calculate significances, we used: for normally distributed continuous data e.

body weight, plasma triglycerides, qPCR etc. For the multiple comparisons, we used non-paired one-way ANOVA, with Dunnet post-hoc correction, the alpha 0. For the gene expression levels by RNA sequencing, significances are calculated by general linear model with negative binomial distribution; P values without correction are shown in the figures.

Graphs and statistical analysis were done in GraphPad Prism 8. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Sequencing data associated with this study have been deposited to the Gene Expression Omnibus with accession codes GSE , GSE , and GSE Source data are provided with this paper.

Metabolomic source data are provided in the Supplementary Data 1 of the paper. All other data used in this study are available from the corresponding author upon reasonable request.

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Using reporter animals with mosaic loss of EECs we found that regions of jejunal epithelium that escaped recombination had normal pH as measured by pHrodo MFI. We have demonstrated that inhibiting PYY signaling in isolated wild-type small intestinal tissues was sufficient to perturb normal electrophysiology in both human and mouse.

This suggests that in vivo the mechanism of action of PYY could be paracrine rather than endocrine. PYY-expressing EECs are abundant in mouse and human small intestine 24 Supplementary Fig. Moreover, PYY-expressing EECs extend long basal processes which underlie several neighboring epithelial cells 25 , 26 , raising the possibility that they may exert paracrine effects on whole populations of nearby enterocytes.

We therefore investigated whether the effects of PYY on ion transport in the small intestine occurred via paracrine mechanisms. To do this, we exploited the mosaicism of VillinCre mice to determine if regions of EEC-deficient epithelium had different transporter activities as compared to regions of epithelium that still had EECs.

Our data suggested that treatment with PYY might restore normal carbohydrate and protein absorption in the intestines of EEC-deficient animals. PYY can be converted to PYY 3—36 by the protease DPP4 27 , and this form of PYY has potent anorexic effects in the brain We therefore co-injected PYY 1—36 and a DPP4 inhibitor to prevent PYY cleavage and to better target the epithelial NPY1R receptor that preferentially binds the 1—36 form 10 , 12 , We simultaneously treated another group of mutant mice with vehicle, DPP4 inhibitor diluted in water.

Patients with EEC-deficiency die without total parenteral nutrition, and similarly very few EEC-deficient mice survive without treatment within the first few weeks. Treatment of mutant mice with vehicle or with PYY significantly improved survival, consistent with therapeutic administration of supportive fluids in diarrheal disease Fig.

However, only PYY injections helped animals gain body weight Fig. PYY also resulted in reduced diarrhea and improved fecal output to be nearly indistinguishable from wild-type, which was independent of intestinal motility Fig. Statistics calculated by one-way ANOVA. g PYY improved dipeptide transport in EEC-deficient mouse and human intestine.

All wild-type and untreated mutant mouse data points are the same as shown in Figs. PYY has a well-established anti-secretory role in the colon, which was likely an important factor contributing to the improvement in diarrheal symptoms observed in EEC-deficient mice.

However, our data suggested that PYY may have additional roles in nutrient and ion transport in the small intestine. Therefore, we investigated if the animals that received PYY injections had restored electrophysiology and improved nutrient absorption in the small intestine.

We found that PYY injections restored the basal I sc of jejunum to normal Fig. In addition, the response to VIP Fig.

The rescue of EEC-deficient intestinal tissue also extended to the human model, where EEC-deficient HIOs were grown and matured in vivo and then host animals were injected with exogenous PYY for 10 days prior to harvest.

These EEC-deficient HIOs exposed to PYY demonstrated electrogenic response to glucose that was indistinguishable from wild-type Fig. By administering PYY to the mosaic EEC-deficient reporter mice, we found PYY injections restored intracellular pH in EEC-deficient intestinal cells to normal levels which would support PEPT1-mediated dipeptide absorption Fig.

Consistent with this, PYY-injected mouse and human small intestine displayed a significantly improved electrogenic response to dipeptides Fig.

These data demonstrated functional efficacy of PYY on improved ion and nutrient transport in EEC-deficient intestine.

In this study, we found that loss of all EECs resulted in profound imbalance of ion transport in the small intestine, with subsequent impairment of nutrient absorption. We demonstrated that the peptide hormone PYY functions in the small intestine to regulate normal electrophysiology and absorption.

Chemical inhibition of the epithelial NPY1R receptor in wild-type small intestine isolated from HIOs and mouse demonstrated the requirement of this pathway in the modulation of VIP-induced ion secretion.

Administration of PYY to EEC-deficient animals resulted in improvements in survival, diarrheal symptoms, glucose absorption, and protein absorption in the absence of all other EEC peptides. Historically, mouse models have been exceedingly tolerant of loss of individual EEC populations, largely due to functional overlap between EEC-derived peptides This has rendered it difficult to assign roles of individual EEC peptides to physiologic functions.

Here, we were able to exploit a model which lacks all EECs to functionally evaluate the role of one EEC peptide, PYY. However, other peptides like somatostatin have similar activities to PYY and likely play a similar regulatory role in vivo.

Somatostatin has many systemic targets 30 and the use of the somatostatin-analogue octreotide in the treatment of chylous effusion and hyperinsulinemia causes an increased risk of necrotizing enterocolitis in infants We therefore chose to use PYY in our preclinical model of ion-coupled nutrient absorption and diarrhea.

PYY has been classically defined as a satiety hormone that acts in an endocrine manner wherein the DPP4-cleaved PYY 3—36 signals to the brain to reduce food intake However PYY 1—36 has been shown to act in a paracrine manner in the colon using combination of genetic and pharmacological approaches 10 , 12 , These findings lend some clarity on how EECs integrate their nutrient sensing function with nutrient absorption, providing us with a new way to approach management of absorptive diseases and those in which EECs are commonly dysregulated.

Human embryonic stem cell ESC line WA01 H1 was purchased from WiCell. CDH1-mRuby2 and non-reporter hESCs were used interchangeably. hESCs were maintained in feeder-free culture. Cells were passaged routinely every 4 days using Dispase STEMCELL Technologies.

NSG mice were maintained on Bactrim chow for a minimum of 2 weeks prior to transplantation and thereafter for the duration of the experiment 8—14 weeks.

After ~10 weeks of in vivo growth, crypts were isolated from transplanted HIOs and plated in 3D Images were acquired using a Nikon A1 GaAsP LUNV inverted confocal microscope and NIS Elements software Nikon. qPCR primers were designed using NCBI PrimerBlast.

Primer sequences are listed in the table below. qPCR was performed using Quantitect SYBR ® Green PCR kit QIAGEN and a QuantStudio 3 Flex Real-Time PCR System Applied Biosystems. Relative expression was determined using the ΔΔCt method and normalizing to PPIA cyclophilin A.

Samples from at least three independent passages were used for quantification. The area of ten representative enteroids per well was quantified using NIS Elements software at both time points. The outline of individual enteroids was traced manually and the area calculated by NIS Elements.

Data include a minimum of three independent experiments per condition on three wild-type and three EEC-deficient HIO-derived enteroid lines.

NHE3 activity was determined by confocal live imaging of enteroids with a ratiometric pH-sensitive dye Intracellular pH was calibrated using the Intracellular pH Calibration Buffer kit Invitrogen at pH 7. A minimum of three enteroids in three wells over two independent passages were quantified.

Electrophysiological experiments were conducted using a modified Ussing chamber 22 , Mouse jejunum and transplanted HIOs were dissected and immediately placed in ice-cold Krebs-Ringer solution. Tissues were opened to create a flat epithelial surface. Because seromuscular stripping is associated with release of cyclooxygenases and prostaglandins 22 , and prostaglandins can stimulate L-cells to release GLP1, GLP2 and PYY 37 , we performed the Ussing chamber experiments in intestinal tissue with an intact muscular layer.

Tissues were mounted into sliders 0. D-glucose and Gly-Sar were added to the luminal side of the chamber once the VIP-induced I sc had stabilized at a maximum value. HIOEs were differentiated for 5—7 days, then were removed from Matrigel and enzymatically dissociated into single-cell suspension using 0.

Monolayers were then excised from the plastic Transwell insert and mounted on a glass slide for live confocal Z-stack imaging using a Nikon A1 GaAsP LUNV inverted confocal microscope and NIS Elements software Nikon.

On the final day, enteroids were removed from Matrigel and enzymatically dissociated into single-cell suspension using 0. In all experiments, samples were labeled with either CDH1-mRuby2 or Anti-EpCam-APC BD Biosciences to distinguish epithelial cells and incubated with SYTOX Blue dead cell stain Life Technologies or 7-AAD BD Pharmingen.

Forward scatter and side scatter were used to discriminate doublets and cellular debris. A minimum of 50, events per sample was recorded using an LSR Fortessa flow cytometer BD Biosciences and data were analyzed using FACSDiva software BD Biosciences.

Mice were housed in a specific pathogen-free barrier facility in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. We established a diarrhea score, with 3 representing wet, yellow feces that smeared the perianal fur, and 0 representing normal, dry, brown, well-defined pellets.

Mutant mice which suffered from diarrhea score 3 were included in the rescue experiment. Mice were given access to solid chow on the floor of the cage beginning at postnatal day 10 and weaned at postnatal day Mice were treated daily for a minimum of 10 days after HIOs had been maturing for 8 weeks, then dissected and analyzed.

Data represents measurements taken from individual mice and biological replicates of HIOs from two human pluripotent stem cell lines. Enteroid experiments were conducted on three independent lines. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

All data generated or analyzed during this study are included in the published article and Supplementary Information files. Source data are available in the Source Data file, and available upon reasonable request from the corresponding author.

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E nterocytes are Nutrient absorption in the enterocytes Detoxify your liver that form most of the epithelium of the gut intestine Nytrient 1 enterocyte 2. Absorptiom are Electrolyte Powder Nutrient absorption in the enterocytes in the small intestine absorpfion in the large intestine and appendix. The main function rnterocytes enterocytes is to absorb molecules Weight management for diabetes the gut enyerocytes and Nutrienr transport toward the surrounding absortion tissue and blood vessels. It is of notice that the gut epithelium is the larger surface of the body in contact with the external environment the lumen of the gut is the external environmentlarger than the skin. E nterocytes have microvilli in their apical free surface Figure 3many mitochondria in the basal part, and well-developed Golgi apparatus and endoplasmic reticulum. The mechanical integrity of the intestine epithelium, that is, the cohesion between enterocytes and the lack of intercellular passages, depends on the cell junctions between adjacent enterocytes Figure 4. Tight junctions and adherent junctions are found close to the apical domain of the enterocyte. Nutrient absorption in the enterocytes

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