Background and aim: Leptin, a hormone mainly produced by fat cells, acts primarily on the hypothalamus regulating energy expenditure and food intake. Leptin receptors are expressed in several tissues and the possible physiological role of leptin is being extensively investigated, with the result that important peripheral actions of the hormone in the organism are being discovered. Recent studies have demonstrated leptin and leptin receptor expression in gastric epithelial cells. In the present study, we report the presence of the long leptin receptor isoform (OB-Rb) in human, rat, and mouse small intestine, supporting the hypothesis of leptin as a hormone involved in gastrointestinal function. Methods: The presence of the leptin receptor was determined by immunocytochemical methods using antibodies against the peptide corresponding to the carboxy terminus of the long isoform of the leptin receptor. Human duodenal biopsies from normal individuals undergoing gastrointestinal endoscopy, and intestinal fragments of Wistar rats and Swiss mice were processed for the study. Results: Immunoreactivity for the long leptin receptor isoform was observed in the three studied species. Staining was located throughout the cytoplasm of the enterocytes, of both villi and crypts, and in the basolateral plasma membrane. Immunolabelling for OB-Rb protein was also found in the brush border of human enterocytes of formol and paraformaldehyde fixed samples. Conclusion: This report demonstrates the presence of the long leptin receptor isoform in the absorptive cells of rat, mouse, and human small intestine, suggesting that leptin could have a physiological role in the regulation of nutrient absorption.

The membrane topology of the human Na/glucose cotransporter SGLT1 has been probed using N-glycosylation scanning mutants and nested truncations. Functional analysis proved essential for establishment of signal-anchor topology. The resultant model diverges significantly from previously held suppositions of structure based primarily on hydropathy analysis. SGLT1 incorporates 14 membrane spans. The N terminus resides extracellularly, and two hydrophobic regions form newly recognized membrane spans 4 and 12; the large charged domain near the C terminus is cytoplasmic. This model was evaluated further using two advanced empirically-based algorithms predictive of transmembrane helices. Helix ends were predicted using thermodynamically-based algorithms known to predict x-ray crystallographically determined transmembrane helix ends. Several considerations suggest the hydrophobic C terminus forms a 14th transmembrane helix, differentiating the eukaryotic members of the SGLT1 family from bacterial homologues. Our data inferentially indicate that these bacterial homologues incorporate 13 spans, with an extracellular N terminus. The model of SGLT1 secondary structure and the predicted helix ends signify information prerequisite for the rational design of further experiments on structure/function relationships.

Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H+-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na+-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na+-and H+-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues. For example, we have identified a charged residue which dramatically changes the kinetics of charge transfer. (iii) We have examined the kinetics of sugar and Na+ analogs. The Vmax of sugar transport decreases dramatically with bulky phenyl glucosides and increases when H+ replaces Na+. These results permit us to extend and refine our model for transport. The model has been useful in the analysis of mutant SGLT1 proteins: in the case of a D176A mutant, the primary effect is to alter rates of conformational changes of the unloaded protein, and with the glucose/galactose malabsorption syndrome mutant D28N SGLT1, the mutation disrupts the delivery of SGLT1 glycosylated protein from the endoplasmic reticulum to the plasma membrane.