M. P. Lostao

Distribution of the long leptin receptor isoform in brush border, basolateral membrane, and cytoplasm of enterocytes

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.

Phenylglucosides and the Na+/glucose cotransporter (SGLT1): analysis of interactions.

Phenylglucosides are transported by the intestinal Na+/glucose cotransporter (SGLT1) and phlorizin, the classical competitive inhibitor of SGLT1, is also a phenylglucoside. To investigate the structural requirements for binding of substrates to SGLT1, we have studied the interactions between phenylglucosides and the cotransporter expressed in Xenopus oocytes using tracer uptake and electrophysiological methods. Some phenylglucosides inhibited the Na+-dependent uptake of 14C-α-methyl-d-glucopyranoside (αMDG) with apparent Kis in the range 0.1 to 20 mm, while others had no effect. Electrophysiological experiments indicated that phenylglucosides can act either as: (1) transported substrates, e.g., arbutin; (2) nontransported inhibitors, e.g., glucosylphenyl-isothiocyanate; or (3) noninteracting sugars, e.g., salicin. The transported substrates (glucose, arbutin, phenylglucoside and helicin) induced different maximal currents, and computer simulations showed that this may be explained by a difference in the translocation rates of the sugar and Na+-loaded transporter. Computational chemistry indicated that all these β-phenylglucosides have similar 3-D structures. Analysis showed that among the side chains in the para position of the phenyl ring the -OH group (arbutin) facilitates transport, but the-NCS (glucosylphenyl-isothiocyanate) inhibits transport. In the ortho position, -CH2OH (salicin) prevents interaction, but the aldehyde (helicin) permits the molecule to be transported. Studies such as these may help to understand the geometry and nature of glucoside binding to SGLT1.

Glycoside binding and translocation in Na(+)-dependent glucose cotransporters: comparison of SGLT1 and SGLT3.

Abstract. Using cotransporters as drug delivery vehicles is a topic of continuing interest. We examined glucose derivatives containing conjugated aromatic rings using two isoforms of the Na+/glucose cotransporter: human SGLT1 (hSGLT1) and pig SGLT3 (pSGLT3, SAAT1). Our studies indicate that there is similarity between SGLT1 and SGLT3 in the overall architecture of the vestibule leading to the sugar-binding site but differences in translocation pathway interactions. Indican was transported by hSGLT1 with higher affinity (K0.5 0.06 mm) and 2-naphthylglucose with lower affinity (K0.5 0.5 mm) than α-methyl-d-glucopyranoside (αMDG, 0.2 mm). Both were poorly transported (maximal velocities, Imax, 14% and 8% of αMDG). Other compounds were inhibitors (Kis 1–13 mm). In pSGLT3, indican and 2-naphthylglucose were transported with higher affinity than αMDG (K0.5s 0.9, 0.2 and 2.5 mm and relative Imaxs of 80, 25 and 100%). Phenylglucose and arbutin were transported with higher Imaxs (130 and 120%) and comparable K0.5s (8 and 1 mm). Increased affinity of indican relative to αMDG suggests that nitrogen in the pyrrole ring is favorable in both transporters. Higher affinity of 2-naphthylglucose for pSGLT3 than hSGLT1 suggests more extensive hydrophobic/aromatic interaction in pSGLT3 than in hSGLT1. Our results indicate that bulky hydrophobic glucosides can be transported by hSGLT1 and pSGLT3, and discrimination between them is based on steric factors and requirements for H-bonding. This provides information for design of glycosides with potential therapeutic value.

Compound missense mutations in the sodium/D-glucose cotransporter result in trafficking defects

BACKGROUND & AIMS Defects in the Na+-dependent glucose transporter (SGLT1) are associated with the disorder glucose-galactose malabsorption, characterized by severe diarrhea. This study focused on a unique proband with glucose-galactose malabsorption who was investigated 30 years ago, and the aims of the study were to identify mutations in the SGLT1 gene and to determine the defect in sugar transport. METHODS Mutations were identified by sequencing, and each mutant protein was then studied using a Xenopus oocyte heterologous expression system. Analysis included Western, freeze fracture, radiotracer uptake, and electrophysiological assays. RESULTS Two heterozygous missense mutations (Cys355Ser and Leu147Arg) were identified that entirely eliminated Na+/sugar cotransport activity. Western blot analysis showed that the levels of both mutant proteins in the oocyte were comparable to wild-type SGLT1, but no complex glycosylation was detected. No SGLT1 charge movements were observed with the mutant proteins, and freeze fracture data showed that neither mutant protein reached the plasma membrane. CONCLUSIONS The Cys355Ser and Leu147Arg mutations eliminate the Na+/sugar cotransport by blocking the transfer of SGLT1 protein from the endoplasmic reticulum to the plasma membrane. This is consistent with earlier studies on phlorizin binding to the brush border membrane of duodenal biopsy specimens from this patient.

Intestinal d -Galactose Transport in an Endotoxemia Model in the Rabbit

Lipopolysaccharide (LPS) is an endotoxin causing sepsis. Studies from our laboratory revealed impaired intestinal absorption of l-leucine and d-fructose in LPS-treated rabbits. The aim of this study was to examine intestinal d-galactose transport following intravenous administration of LPS in the rabbit and to identify the cellular mechanisms driving this process. Endotoxin treatment diminished the buildup of d-galactose in intestinal tissue, the mucosal to serosal transepithelial flux of the sugar and its uptake by brush border membrane vesicles (BBMVs). Intracellular signaling pathways associated with protein kinase C (PKC), protein kinase A (PKA), p38 mitogen-activated protein kinase (p38MAPK), Jun N-terminal kinase (JNK), MAPK/extracellular signal-regulated kinases 1 and 2 (MEK1/2) and proteasome were found to be involved in this reduction in sugar uptake. Na+/glucose cotransporter 1 (SGLT1) protein levels in BBMVs were lower for LPS-treated animals than control animals. These findings indicate that LPS inhibits the intestinal absorption of d-galactose via a complex cellular mechanism that could involve posttranscriptional regulation of the SGLT1 transporter.

Luminal leptin inhibits intestinal sugar absorption in vivo.

Aim:  We have previously demonstrated that leptin inhibits galactose absorption in rat intestinal everted rings and that leptin receptors are present in the apical membrane of the enterocytes. This adipocyte‐derived hormone is also secreted by gastric mucosal cells and is able to reach the intestinal lumen. The goal of the present study was to prove whether luminal leptin acts on intestinal sugar absorption in vivo both at low (basal state) and high sugar concentration (post‐prandial state).

Luminal leptin inhibits l-glutamine transport in rat small intestine: involvement of ASCT2 and B0AT1

L-glutamine is the primary metabolic fuel for enterocytes. Glutamine from the diet is transported into the absorptive cells by two sodium-dependent neutral amino acid transporters present at the apical membrane: ASCT2/SLC1A5 and B(0)AT1/SLC6A19. We have demonstrated that leptin is secreted into the stomach lumen after a meal and modulates the transport of sugars after binding to its receptors located at the brush border of the enterocytes. The present study was designed to address the effect of luminal leptin on Na(+)-dependent glutamine (Gln) transport in rat intestine and identify the transporters involved. We found that 0.2 nM leptin inhibited uptake of Gln and phenylalanine (Phe) (substrate of B(0)AT1) using everted intestinal rings. In Ussing chambers, 10 mM Gln absorption followed as Na(+)-induced short-circuit current was inhibited by leptin in a dose-dependent manner (maximum inhibition at 10 nM; I(C50) = approximately 0.1 nM). Phe absorption was also decreased by leptin. Western blot analysis after 3-min incubation of the intestinal loops with 10 mM Gln, showed marked increase of ASCT2 and B(0)AT1 protein in the brush-border membrane that was reduced by rapid preincubation of the intestinal lumen with 1 nM leptin. Similarly, the increase in ASCT2 and B(0)AT1 gene expression induced by 60-min incubation of the intestine with 10 mM Gln was strongly reduced after a short preincubation period with leptin. Altogether these data demonstrate that, in rat, leptin controls the active Gln entry through reduction of both B(0)AT1 and ASCT2 proteins traffic to the apical plasma membrane and modulation of their gene expression.

Inhibitory effect of TNF-α on the intestinal absorption of galactose

Sepsis is a systemic response to infection in which toxins, such as bacterial lipopolysaccharide (LPS), stimulate the production of inflammatory mediators like the cytokine tumor necrosis factor alpha (TNF‐α). Previous studies from our laboratory have revealed that LPS inhibits the intestinal absorption of L‐leucine and D‐fructose in rabbit when it was intravenously administered, and that TNF‐α seems to mediate this effect on amino acid absorption. To extend this work, the present study was designed to evaluate the possible effect of TNF‐α on D‐galactose intestinal absorption, identify the intracellular mechanisms involved and establish whether this cytokine mediates possible LPS effects. Our findings indicate that TNF‐α decreases D‐galactose absorption both in rabbit intestinal tissue preparations and brush‐border membrane vesicles. Western blot analysis revealed reduced amounts of the Na+/glucose cotransporter (SGLT1) protein in the plasma membrane attributable to the cytokine. On the contrary, TNF‐α increased SGLT1 mRNA levels. Specific inhibitors of the secondary messengers PKC, PKA, the MAP kinases p38 MAP, JNK, MEK1/2 as well as the proteasome, diminished the TNF‐α‐evoked inhibitory effect. LPS inhibition of the uptake of the sugar was blocked by a TNF‐α antagonist. In conclusion, TNF‐α inhibits D‐galactose intestinal absorption by decreasing the number of SGLT1 molecules at the enterocyte plasma membrane through a mechanism in which several protein‐like kinases are involved. J. Cell. Biochem. 101: 99–111, 2007.

Further Characterization of the Electrogenicity and pH Sensitivity of the Human Organic Anion-Transporting Polypeptides OATP1B1 and OATP1B3

Organic anion-transporting polypeptides (OATPs) are involved in the liver uptake of many endogenous and xenobiotic compounds, such as bile acids and drugs, respectively. Using Xenopus laevis oocytes and Chinese hamster ovary (CHO) cells expressing rat Oatp1a1, human OATP1B1, or OATP1B3, the sensitivity of these transporters to extracellular/intracellular pH (pHo/pHi) and changes in plasma membrane potential (ΔΨ) was investigated. In X. laevis oocytes, nonspecific plasma membrane permeability increased only at pHo below 4.5. Above this value, both using oocytes and CHO cells, extracellular acidification affected differently the specific transport of taurocholic acid (TCA) and estradiol 17β-d-glucuronide (E217βG) by Oatp1a1 (stimulation), OATP1B1 (inhibition), and OATP1B3 (stimulation). Changes in substrate uptake in the presence of valinomycin (K+-ionophore), carbonyl cyanide 3-chlorophenylhydrazone and nigericin (protonophores), and amiloride (Na+/H+-inhibitor) and cation replacement in the medium were studied with fluorescent probes for measuring substrate uptake (cholylglycyl amidofluorescein) and changes in pHi (SNARF-4F) and ΔΨ [DilC1(5)]. The results suggest that activity of these three carriers is sodium/potassium-independent and affected differently by changes in pHo and ΔΨ: Oatp1a1 was confirmed to be an electroneutral anion exchanger, whereas the function of both OATP1B1 and OATP1B3 was markedly affected by the magnitude of ΔΨ. Moreover, electrophysiological measurements revealed the existence of a net anion influx associated to OATP1B1/OATP1B3-mediated transport of TCA, E217βG, and estrone-3-sulfate. Furthermore, a leakage of Na+ through OATP1B1 and OATP1B3, which is not coupled to substrate transport, was found. In conclusion, these results suggest that OATP1B1 and OATP1B3 are electrogenic transporters whose activity may be strongly affected under circumstances of displacement of local pH.

Nucleoside transporters in absorptive epithelia

There are two families of nucleoside transporters, concentrative (termed CNTs) and equilibrative (called ENTs). The members of both families mediate the transmembrane transport of natural nucleosides and some drugs whose structure is based on nucleosides. CNT transporters show a high affinity for their natural substrates (with Km values in the low micromolar range) and are substrate selective. In contrast, ENT transporters show lower affinity and are more permissive regarding the substrates they accept. Both types of transporters are tightly regulated in all cell types studied so far, both by endocrine and growth factors and by substrate availability. The degree of cell differentiation and the proliferation status of a cell also affect the pattern of expressed transporters. Although the presence of both types of transporters in the cells of absortive epithelia suggested the possibility of a transepithelial flux of nucleosides, their exact localization in the different plasma membrane domains of epithelial cells had not been demonstrated until recently. Concentrative transporters are found in the apical membrane while equlibrative transporters are located in the basolateral membrane, thus strengthening the hypothesis of a transepithelial flux of nucleosides.ResumenExisten dos familias de transportadores de nucleósidos, la de los concentrativos (denominada CNT) y la de equilibrativos (o ENT). Los miembros de ambas familias median el transporte transmembrana de los nucleósidos naturales y de algunos fármacos cuya estructura deriva de aquéllos. Los transportadores CNT presentan una alta afinidad por sus sustratos naturales (sus Km están en el rango micromolar bajo) y una cierta selectividad por sustrato. Los ENT, por el contrario, muestran menor afinidad y son más permisivos en cuanto a los sustratos que aceptan. Ambos tipos de transportadores están estrechamente regulados en los diversos tipos celulares estudiados, tanto por factores endocrinos o de crecimiento como por la disponibilidad de sus sustratos. También el grado de diferenciación y el estado de proliferación de una célula influyen sobre el patrón de transportadores expresado por esa célula. Aunque la presencia de ambos tipos de transportadores en las células de los epitelios absorbentes parecía sugerir la posibilidad de un flujo transepitelial de nucleósidos, hasta hace muy poco no se ha podido demostrar su localización en los diferentes dominios de la membrana plasmática de las células epiteliales; así, los transportadores concentrativos se encuentran en la membrana apical mientras que los equilibrativos se localizan en la membrana basolateral, lo que refuerza la tesis de un flujo transepitelial de nucleósidos.