Gastrointestinal Functions, edited by Edgard E. Delvin and Michael J. Lentze. Nestle Nutrition Workshop Series. Pediatric Program. Vol. 46. Nestec Ltd.. Vevey/Lippincott Williams & Wilkins, Philadelphia © 2001. Human Intestinal Nutrient Transporters Ernest M. Wright Department of Physiology, UCLA School of Medicine, Los Angeles, California, USA Over the past decade, advances in molecular biology have revolutionized studies on intestinal nutrient absorption in humans. Before the advent of molecular biology, the study of nutrient absorption was largely limited to in vivo and in vitro animal model systems. This did result in the classification of the different transport systems involved, and in the development of models for nutrient transport across enterocytes (1). Nutrients are either absorbed passively or actively. Passive transport across the epithelium occurs down the nutrient's concentration gradient by simple or facilitated diffusion. The efficiency of simple diffusion depends on the lipid solubility of the nutrient in the plasma membranes—the higher the molecule's partition coefficient, the higher the rate of diffusion. Facilitated diffusion depends on the presence of simple carriers (uniporters) in the plasma membranes, and the kinetic properties of these uniporters. The rate of facilitated diffusion depends on the density, turnover number, and affinity of the uniporters in the brush border and basolateral membranes. The ' 'active'' transport of nutrients simply means that energy is provided to transport molecules across the gut against their concentration gradient. It is now well recog- nized that active nutrient transport is brought about by Na+ or H+ cotransporters (symporters) that harness the energy stored in ion gradients to drive the uphill trans- port of a solute. The rate of active transport of a nutrient depends on the density and kinetics of these cotransporters. The models for passive and active nutrient absorption across the intestinal mucosa have evolved from those derived for sugar absorption (Fig. 1). Fructose is passively absorbed across the intestine by two different uniporters in the brush border and basolateral membranes of enterocytes (2), GLUTS in the brush border and GLUT2 in the basolateral membrane (Table 1) (5-26). In humans, the amount of fructose absorption is limited by the number of brush border GLUT5 transporters, and this accounts for the fact that 80% of the population shows malabsorption symptoms after ingesting 50 g of the sugar (27). Other nutrients (e.g., organic ions) are also passively absorbed across the gut by having two uniporters expressed in series in the brush border and basolateral membranes. Glucose and galactose are actively absorbed by a cotransporter and a uniporter expressed in series. These sugars are accumulated within the cell across the brush 77 78 HUMAN INTESTINAL NUTRIENT TRANSPORTERS Lumen Cell Blood Galactose/ducose Fructose GLUTS tight J lateral intercellular space FIG. 1. A model for nutrient absorption across enterocytes of the small intestine. The passive transport of fructose across the brush border and basolateral membranes of the epithelium is accomplished by two uniporters, GLUT5 and GLUT2. The active transport of glucose and galac- tose across the epithelium occurs in two stages: the first is the accumulation of the sugar within the epithelium by the brush border Na+/glucose cotransporter, SGLT1; the second stage is the transport of sugar out of the cell across the basolateral membrane by the uniporter, GLUT2. Similar models account for the passive and active absorption of other nutrients across the intes- tine. border membrane by an Na+/glucose cotransporter (SGLTJ), and then leave the cell across the basolateral membrane into the blood by facilitated diffusion through GLUT2 (28). Similar models have been proposed to explain the active absorption of vitamins, bile salts, phosphate, dicarboxylates, amino acids, peptides, and trace ions. In some cases, it is the pH gradient across the brush border membrane rather than the Na+ gradient that drives uptake, for example the PEPTI and NRAMP cotransporters (Table 1). The actual identification of the transport proteins proved to be elusive, owing to the inherent problems in working with rare integral membrane proteins. For example, the brush border Na+/glucose cotransporter only accounts for 0.1% of the total protein in the brush border, and it was very difficult to purify and assay by conven- tional biochemical techniques. In fact, it took almost 25 years to identify the trans- porter protein (29). The rapid advances of the past decade have come through the power of molecular biology and the development of heterologous expression systems for transport pro- teins. Nevertheless, it took the development of a novel cloning strategy by Coady, Hediger, and Ikeda in my laboratory—expression cloning (30,31)—to remove the major bottleneck to cloning membrane transporters. Rather than screening comple- mentary DNA (cDNA) libraries with oligonucleotide probes or antibodies, which required the purification of rare membrane proteins, a functional screening approach HUMAN INTESTINAL NUTRIENT TRANSPORTERS 79 TABLE 1. Human intestinal nutrient transporters Gene Substrate Location Reference 1. Na+ cotransporters IA. Na+/glucose family 1. SGLT1 (SLC5A1) Glucose, galactose BBM 3 2. SMVT1 (SLC5A6) Biotin, pantothenate, lipoate BBM 4 3. /V/S(SLC5A5) Iodide BBM 5 4. SMIT (SLC5A3) Myoinositol BBM 6 IB. Na+/ascorbate 1. Sl/C72(SLC23A1) Ascorbate BBM 7 IC. Na+/bile salt 1. ASBT (SLC10A2) Cholate, taurocholate BBM 8 ID. Na+/phosphate 1. NaPi-IIB (SLC34A3) Phosphate BBM 9 IE. Na"7dicarboxylate 1. NaDC 1 (SLC13A2) Citrate, succinate BBM 10 2. ratNaDC2 (SLC13A3) Citrate, succinate BLM 11 IF. Na+/nucleoside 1. SPNT1ICNT2 (SLC28A2) Inosine, uridine BBM 12 2. CNT2 (SLC28AZ) Inosine, uridine BBM 13 IG. Na+/camitine 1. OCTN2 (SLC22A5) Carnitine BBM 14, 15 IH. Na+/H+/K+/glutamate 1. EAAT3/EAAC1 (SLC1A1) Glutamate, aspartate BBM 16 II. H+/peptide 1. PEPT1 (SLC15A1) Di- and tripeptides BBM 17 MB. HVdivalent cations 1. DCT1/NRAMP2 (SLC11A2) Zn, Fe, Cd, Mn, Co, Ni BBM 18 III. Uniporters IMA. Multiple facilitator family (MFS) 1. GLUT2 (SLC2A2) Glucose, galactose, fructose BBM 19 2. GLUT5 (SLC2A5) Fructose BBM 20 11 IB. Organic cation 1. OCT1 (SLC22A2) TEA, choline BLM 21 2. OCT2 (SLC22A) TEA, choline BLM 21 IMC. Folate 1. RFC1/F0LT1 (SLC19A1) Reduced folate BBM? 22 HID. Nucleoside 1. ENT1 (SLC29A1) Purine and pyrimidine 23 nucleosides 2. ENT2 (SLC29A2) Purine and pyrimidine 24, 25 nucleosides HIE. Neutral amino acids 1. M72(SLC7A6) 2. 4F2hc (SLC3A2) Tyrosine, phenylalanine BLM 26 BBM, brush border membrane; BLM, basolateral membranes; TEA, tetraethyl ammonium. Transporters cloned from human intestinal cDNA libraries, or cloned from other human libraries and known to be expressed in the small intestine. The human gene nomenclature used in the table (www.gene.ucl.ac.uk/nomenclature/) is that employed by the database Online Mendelian Inheritance in Man (OMIM) for human genetic disorders (www.ncbi.nlm.nih.gov/Omim). 80 HUMAN INTESTINAL NUTRIENT TRANSPORTERS was used. This rested on the finding that Xenopus laevis oocytes have the remarkable ability to efficiently translate foreign complementary RNA (cRNA) from virtually any source and, in the case of membrane proteins, correctly target the functional protein to the plasma membrane. The rate of sodium-dependent sugar uptake into native oocytes is extraordinarily small (< 0.25 pmol/h for 50 |xM aMDG), but this increases by more than an order of magnitude after injecting 50 ng of intestinal polyA+ RNA into the oocytes (30). Then, this radioactive tracer assay was used to isolate a clone from a rabbit intestinal cDNA that coded for the Na + /glucose cotransporter. Injection of cRNA from the plasmid pMJC 429 into oocytes increased sodium-dependent uptake more than 1,000 times above background (31), and the characteristics of this cloned transport were very similar to those of the native brush border transporter (28,32). The human transporter was cloned soon after using the rabbit cDNA to screen a human intestinal cDNA library (3). Expression cloning is now the method of choice to identify genes coding for membrane transporters. Of the 23 human nutrient transporter clones listed in Table 1, more than half were directly or indirectly isolated by expression cloning and their function characterized by expression in oocytes. Generally, the transporter clones were first isolated from animal cDNA libraries by expression cloning and then from human cDNA libraries by homology screening. In some instances, the cDNA clones were isolated from nonintestinal libraries (e.g., renal or liver libraries) and these genes were subsequently shown to be expressed in the human intestine by Northern blot or reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The func- tional characteristics of these cloned transporters closely match the properties of the transporters in intestinal brush border and basolateral membranes. HUMAN INTESTINAL TRANSPORTERS Thus far, some 23 transporters belonging to 15 gene families have been shown to be expressed in the human intestine (Table 1). Given the pace of research and the advances of the human genome project, this number is expected to grow rapidly. Ion and water channels, pumps, and ion exchangers expressed in the intestine are not included in Table 1. The list is divided into the secondary active transporters expressed in the brush border, and the uniporters expressed in