Small and Large Intestine: Composition, Function, and Models

Posted on Feb 5, 2025 in Biotechnology

Composition and Function of the Small Intestine

Digestion within the small intestine produces a mixture of disaccharides, peptides, fatty acids, and monoglycerides. The final digestion and absorption of these substances occur in the villi, which line the inner surface of the small intestine.

• The crypts at the base of the villi contain stem cells that continuously divide by mitosis, producing:
  1. More stem cells
  2. Cells that migrate up the surface of the villus while differentiating
• Columnar epithelial cells (the majority): Responsible for digestion and absorption.
• Goblet cells: Secrete mucus.
• Endocrine cells: Secrete a variety of hormones.
  • Paneth cells: Secrete antimicrobial peptides that sterilize the contents of the intestine.
• The villi increase the surface area of the small intestine. In addition, the apical surface of the epithelial cells of each villus is covered with microvilli (also known as a “brush border”). Thanks largely to these, the total surface area of the intestine is almost 200 square meters.
• Humans with a rare genetic inability to form microvilli die of starvation.

Composition and Function of the Large Intestine

• The large intestine receives the liquid residue after digestion and absorption are complete. This residue consists mostly of water, as well as materials (e.g., cellulose) that were not digested. The colon contains an enormous (~10¹⁴) population of microorganisms. (Our bodies consist of only ~10¹³ cells!)
• Most of the species live there perfectly harmlessly; that is, they are commensals. Some are actually beneficial, e.g.:
  1. By synthesizing vitamins.
  2. By digesting polysaccharides for which we have no enzymes (providing an estimated 10% of the calories we acquire from our food).
• In both obese mice (ob/ob) and humans, the relative proportion of Bacteroidetes (one kind of the MOs) declines and, in mice at least, the efficiency with which residual food is absorbed increases. Putting humans on a diet causes them to regain the normal proportion of Bacteroidetes. Why this relationship exists remains to be discovered.
• Bacteria flourish to such an extent that as much as 50% of the dry weight of the feces may consist of bacterial cells.
• Reabsorption of water is the major function of the large intestine. The large amounts of water secreted into the stomach and small intestine by the various digestive glands must be reclaimed to avoid dehydration. If the large intestine becomes irritated, it may discharge its contents before water reabsorption is complete, causing diarrhea. On the other hand, if the colon retains its contents too long, the fecal matter becomes dried out and compressed into hard masses, causing constipation.

Requirements of an In Vitro Model System of the Intestinal Barrier

• Major problems with freshly excised intestinal segments and everted intestinal sacs:
  1. Maintenance of tissue viability and barrier integrity over time during experiments.
  2. A large quantity of test compounds is required.
  3. Complexity/variability of tissue compounds makes it difficult to interpret the results.
• An easier in vitro model might be helpful.
• Since in vivo absorption studies in animals are complex, time-consuming, ethically challenging, and expensive, there has been a recognized need to develop alternative in vitro methods (EMEA, 1997).
• Cell culture models are used as intermediary complex systems between whole animal studies and isolated enzymes, membrane fractions, or artificial lipid bilayers (Quaroni and Hochman, 1996).
• The simultaneous use of model compounds requires that they do not cause cytotoxicity, do not interact with each other during permeation, and that they are easily detected.
• Therefore, the use of different sets of model compounds has to be validated before the actual experiments with drug candidates can be performed.

Intestinal Cell Systems: Caco-2 Cells

• According to the Biopharmaceutics Classification System (BCS) and FDA approval, Caco-2 cells can be used as a screening method for new drug candidates during drug discovery and development.
• For the suitability and reliability of the method, the permeability of several model compounds with known intestinal absorption in humans has to be demonstrated.
• The FDA recommends the use of compounds with high, low, and zero permeability, passive and active transport, and use of efflux markers for this purpose.
• While active influx transporters have a physiological role in the absorption of essential molecules, efflux proteins in turn act as defense mechanisms, actively protecting the cells by transporting endogenous and xenobiotic toxins or toxic metabolites out of the cells.
• According to the current understanding, the efflux proteins belong to the ABC (ATP [adenosine triphosphate]-binding cassette) transporter superfamily.
• ABC proteins are further divided into subfamilies based on their structural homology. Within these subfamilies, the relevant drug efflux proteins are MDR1 in the MDR/TAP subfamily, several MRPs in the MRP/CFTR subfamily, and BCRP in the White subfamily.

Major Efflux Proteins and Their Function

• The general structure of ABC transporters consists of 12 transmembrane regions and two nucleotide-binding domains, but there are exceptions: for example, MRPs 1-3 and 6-7 have five additional transmembrane regions and BCRP is a “half-transporter.”
• MDR1 (ABCB1, commonly referred to as P-glycoprotein, P-gp), the classic example, is probably the most studied efflux transporter and, although initially discovered in tumor tissues, it is also present in normal human intestinal epithelium, blood-brain barrier, and hepatic canalicular membranes.
• Predicted secondary structures of drug efflux transporters of the ATP-binding cassette family. MRP8 is similar to MRP4 and MRP5, while MRP6 and MRP7 are structurally similar to MRP1-3.
• In the human genome, nine genes encoding MRP transporters (as well as one non-functional pseudo-gene, ABCC13) have been identified (Dean et al., 2001; Kruh and Belinsky, 2003; Toyoda et al., 2008). MRP1 (ABCC1) was discovered first, in 1992, and it is expressed in the basolateral membrane of several normal human tissues, transporting a variety of hydrophilic compounds.
• MRP2 (ABCC2, cMOAT) also transports a wide range of organic anions (Nies and Keppler, 2007). It was originally found in the canalicular membranes of hepatocytes, excreting especially conjugated compounds into the bile (Jansen et al., 1993). It is also expressed in the apical membranes of the polarized cells in, e.g., kidney, intestine, gallbladder, bronchi, and placenta.
• MRP2 is probably clinically the most important MRP with respect to intestinal absorption.
• The physiological role of MRP3 (ABCC3) appears, indeed, to be the function of an alternative detoxification route if MRP1 and MRP2 are not working properly.
• On the other hand, the role of MRP4-6 (ABCC4-6) and MRP7-9 (ABCC10-12) in the intestine is still not clear.
• However, according to the current knowledge on the expression levels and tissue localization, the clinical significance of the other members of the MRP family than MRP1-3 is minor for the intestinal absorption of xenobiotics.
• Like MDR1 and MRP2, BCRP (ABCG2) is localized in the apical surface of intestinal epithelial cells, suggesting a role in reducing the absorption of xenobiotics.
• The most relevant known active transport, efflux, and metabolism processes in the enterocytes. ISBT = Ileal bile salt transporter, OATP-B = Organic anion transporting polypeptide B, HPT1 = Human oligopeptide transporter, OCTN2 = Organic cation/carnitine transporter, PEPT1 = Oligopeptide transporter, MCT = Monocarboxylic acid transporter, OCT1 = Organic cation transporter, MDR1 = Multidrug resistance protein 1, MRP = Multidrug resistance-related protein, BCRP = Breast cancer-related protein, CYP = Cytochrome P450 enzyme, UGT = UDP-glucuronosyltransferase, SULT = Sulfotransferase, GST = Glutathione-S-transferase.

Clinical Relevance of Efflux Proteins

• Clinically, both ethnic and interindividual differences have been reported between the intestinal efflux proteins.
• Why is that important? Because those proteins decide the efficiency of absorption of the drug.

Molecular Mechanisms of Transport Activity

• Active transport of drugs and nutrients is mediated by several membrane transporter proteins located in the cell membranes.
• The membrane transporter carriers can be classified based on their energy requirements into facilitated diffusion, primary active, and secondary active transport.
  1. Facilitated diffusion does not require energy for its function.
  2. Primary active transport is an energy-demanding process.
  3. Secondary active transport uses energy from ion gradients (mostly Ca²⁺, Na²⁺, and H⁺ -gradients) across the cell membranes generated by primary active ion pumps (for example, Na⁺/K⁺-ATPase) and are also called symporters or antiporters.

Schematic figure of intestinal epithelium as a selective barrier against the entry of compounds to circulation. A: passive trans- and paracellular diffusion; B: carrier-mediated absorption at apical and basolateral membranes; C: active efflux transporter on apical membrane, acting during absorption; D: active efflux transporter on apical membrane, offering an additional route for drug clearance from the circulation; E: intracellular metabolizing enzymes localized inside the enterocytes, possibly combined with an active efflux transporter on apical and basolateral membranes.