Corinne Nazaret

Rat NKCC2/NKCC1 cotransporter selectivity for loop diuretic drugs

Abstract. It is generally assumed that bumetanide possesses some selectivity for the renal Na-K-Cl cotransporter NKCC2, although the results are scarce in the literature and comparisons were done with extra-renal NKCC1 at its basal, almost silent state. Here we investigated NKCC2/NKCC1 selectivity of loop diuretic drugs (bumetanide, piretanide and furosemide) as a function of the NKCC1 activated state (NKCC1 was activated by hypertonic media). NKCC2 activity was measured in isolated rat medullary thick ascending limb (mTAL) and NKCC1 in rat thymocytes and erythrocytes. When NKCC2 was compared with NKCC1at its activated state, all three diuretic drugs inhibited NKCC2 and NKCC1 with the same potency (bumetanide pIC50=6.48, 6.48 and 6.47; piretanide pIC50=5.97, 5.99 and 6.29; and furosemide pIC50=5.15, 5.04 and 5.21 for mTAL NKCC2, erythrocyte NKCC1 and thymocyte NKCC1, respectively). Basal NKCC1 exhibited a lower diuretic sensitivity, although with marked differences depending on the diuretic drug and the cell type in consideration and with the notable exception of furosemide in erythrocytes. Molecular modelling showed that bumetanide and piretanide possess four potentially active groups, of which three are shared with furosemide at similar intergroup distances. Of these three common groups, one should not bind to basal NKCC1 in thymocytes. The fourth (phenoxy) group (absent in furosemide) confers higher lipophilicity and should not bind to basal NKCC1 in erythrocytes. In conclusion, loop diuretics had no NKCC2/NKCC1 selectivity, when NKCC1 is measured at its activated state. Basal NKCC1 has a reduced diuretic sensitivity, of very different magnitude depending on the diuretic drug and cell type in consideration.

Positive regulatory control loop between gut leptin and intestinal GLUT2/GLUT5 transporters links to hepatic metabolic functions in rodents.

Background and Aims The small intestine is the major site of absorption of dietary sugars. The rate at which they enter and exit the intestine has a major effect on blood glucose homeostasis. In this study, we determine the effects of luminal leptin on activity/expression of GLUT2 and GLUT5 transporters in response to sugars intake and analyse their physiological consequences. Methodology Wistar rats, wild type and AMPKα2 −/− mice were used. In vitro and in vivo isolated jejunal loops were used to quantify transport of fructose and galactose in the absence and the presence of leptin. The effects of fructose and galactose on gastric leptin release were determined. The effects of leptin given orally without or with fructose were determined on the expression of GLUT2/5, on some gluconeogenesis and lipogenic enzymes in the intestine and the liver. Principal Findings First, in vitro luminal leptin activating its receptors coupled to PKCβII and AMPKα, increased insertion of GLUT2/5 into the brush-border membrane leading to enhanced galactose and fructose transport. Second in vivo, oral fructose but not galactose induced in mice a rapid and potent release of gastric leptin in gastric juice without significant changes in plasma leptin levels. Moreover, leptin given orally at a dose reproducing comparable levels to those induced by fructose, stimulated GLUT5-fructose transport, and potentiated fructose-induced: i) increase in blood glucose and mRNA levels of key gluconeogenesis enzymes; ii) increase in blood triglycerides and reduction of mRNA levels of intestinal and hepatic Fasting-induced adipocyte factor (Fiaf) and iii) increase in SREBP-1c, ACC-1, FAS mRNA levels and dephosphorylation/activation of ACC-1 in liver. Conclusion/Significance These data identify for the first time a positive regulatory control loop between gut leptin and fructose in which fructose triggers release of gastric leptin which, in turn, up-regulates GLUT5 and concurrently modulates metabolic functions in the liver. This loop appears to be a new mechanism (possibly pathogenic) by which fructose consumption rapidly becomes highly lipogenic and deleterious.

Reduced intestinal absorption of dipeptides via PepT1 in mice with diet-induced obesity is associated with leptin receptor down-regulation.

Leptin is a major determinant of energy homeostasis, acting both centrally and in the gastrointestinal tract. We previously reported that acute leptin treatment enhances the absorption of di- and tripeptides via the proton-dependent PepT1 transporter. In this study, we investigated the long term effect of leptin on PepT1 levels and activity in Caco2 cell monolayers in vitro. We then assessed the significance of the regulation of PepT1 in vivo in a model of diet-induced obesity. We demonstrated that 1) leptin regulated PepT1 at the transcriptional level, via the MAPK pathway, and at the translational level, via ribosomal protein S6 activation, in Caco2 cells and 2) this activation was systematically followed by a time- and concentration-dependent loss of leptin action reflecting desensitization. Deciphering this desensitization, we demonstrated that leptin induced a down-regulation of its own receptor protein and mRNA expression. More importantly, we showed, in mice with diet-induced obesity, that a 4-week hypercaloric diet resulted in a 46% decrease in PepT1-specific transport, because of a 30% decrease in PepT1 protein and a 50% decrease in PepT1 mRNA levels. As shown in Caco2 cells, these changes in PepT1 were supported by a parallel 2-fold decrease in leptin receptor expression in mice. Taken together, these results indicate that during induction of obesity, leptin resistance may also occur peripherally in the gastrointestinal tract, disrupting the absorption of oligopeptides and peptidomimetic drugs.

Resistin-like molecule-β inhibits SGLT-1 activity and enhances GLUT2-dependent jejunal glucose transport

OBJECTIVE An increased expression of RELM-β (resistin-like molecule-β), a gut-derived hormone, is observed in animal models of insulin resistance/obesity and intestinal inflammation. Intestinal sugar absorption is modulated by dietary environment and hormones/cytokines. The aim of this study was to investigate the effect of RELM-β on intestinal glucose absorption. RESEARCH DESIGN AND METHODS Oral glucose tolerance test was performed in mice and rats in the presence and the absence of RELM-β. The RELM-β action on glucose transport in rat jejunal sacs, everted rings, and mucosal strips was explored as well as downstream kinases modulating SGLT-1 and GLUT2 glucose transporters. RESULTS Oral glucose tolerance test carried out in rodents showed that oral administration of RELM-β increased glycemia. Studies in rat jejunal tissue indicated that mucosal RELM-β promoted absorption of glucose from the gut lumen. RELM-β had no effect on paracellular mannitol transport, suggesting a transporter-mediated transcellular mechanism. In studies with jejunal mucosa mounted in Ussing chamber, luminal RELM-β inhibited SGLT-1 activity in line with a diminished SGLT-1 abundance in brush border membranes (BBMs). Further, the potentiating effect of RELM-β on jejunal glucose uptake was associated with an increased abundance of GLUT2 at BBMs. The effects of RELM-β were associated with an increased amount of protein kinase C βII in BBMs and an increased phosphorylation of AMP-activated protein kinase (AMPK). CONCLUSIONS The regulation of SGLT-1 and GLUT2 by RELM-β expands the role of gut hormones in short-term AMPK/protein kinase C mediated control of energy balance.

Regulation of renal Na-K-Cl cotransporter NKCC2 by humoral natriuretic factors : Relevance in hypertension

A furosemide-sensitive Na-K-Cl cotransporter (NKCC2 isoform) accounts for almost all luminal NaCl reabsorption in the thick ascending limb of Henles loop (TALH). The activity of this transport protein is regulated by humoral factors (CIF: cotransport inhibitory factors). One family of CIF compounds is represented by the urinary phytoestrogens equol and genistein, which inhibit cotransport fluxes at similar concentrations as furosemide. Moreover, they possess similar salidiuretic potency as furosemide in the isolated perfused rat kidney, but are less potent than furosemide in vivo. Thus, dietary phytoestrogens can be responsible, at least in part, for the low blood pressure of vegetarians. A second type of CIF is represented by a circulating and urinary factor which is evoked by salt-loading. This, which is not a ouabain-like factor, appears to be a new retropituitary natriuretic compound. Endogenous CIF is increased in hypertensive Dahl salt-sensitive rats, probably as a compensatory mechanism against the enhanced NaCl reabsorption in the TALH, which characterizes this model of hypertension. Finally, chronic excess of circulating CIF inhibits and induces up-regulation of erythrocyte Na-K-Cl cotransporter NKCC1.

Inhibition of Na-K-Cl cotransport fluxes and salidiuretic action by an urinary extract of salt-loaded rats

We previously found a potent inhibitor of the Na-K-Cl cotransport system in urines from salt-loaded rats (C.I.F. = cotransport inhibitory factor, ref. 1). Here we extracted an urinary fraction (≈ 1 ‰ urine dry weight), free from immunoreactive A.N.P. and digoxine activity, which: (i) potently inhibited cotransport fluxes in MDCK (Madin and Darby canine kidney] cells and in human erythrocytes, (ii) inhibited Na+-dependent chloride/bicarbonate exchange with 2–3 times less potency than cotransport and (iii) strongly increased natriuresis and diuresis after i.v. infusion in rats with no significant change in kaliuresis (salidiuretic action reduced by probenecid). Therefore, C.I.F. seems to be a new natriuretic factor with part, but not all the biological profile of loop diuretic drugs.

PO41 La leptine digestive contrôle l’absorption intestinale du fructose dans une boucle de régulation couplée au métabolisme hépatique

Introduction L’activite des transporteurs intestinaux de sucre a un impact immediat sur la glycemie et l’homeostasie glucidique. Comprendre les mecanismes controlant l’activite et l’expression de ces transporteurs est importante dans le contexte d’une alimentation humaine de plus en plus riche en fructose. Nous avons etudie l’effet de la leptine secretee dans la lumiere digestive sur l’activite/expression des transporteurs GLUT2 et GLUT5 et analyse les consequences physiologiques. Materiels et Methodes Le transport transepithelial de fructose ou de galactose en presence ou en absence de leptine luminale etait quantifie dans le modele d’anses intestinales isolees chez des rats Wistar et des souris sauvages. L’expression des transporteurs etait etudiee par western blot (WB) et par qPCR. Les effets du fructose etaient determines sur la secretion de leptine gastrique ainsi que les parametres biologiques et l’expression (ARNm) d’enzymes cles du metabolisme intestinal et hepatique. Resultats La leptine luminale augmentait de facon dose-dependante le transport de fructose et de galactose dans les anses jejunales isolees requerant l’activation de la sous-unite α2 de l’AMPK et la phosphorylation de la PKCbetaII. L’administration orale de fructose -mais pas de galactose- stimulait apres 15 min la secretion de leptine dans la lumiere gastrique sans modifier la leptine plasmatique. In vivo , la leptine augmentait le transport intestinal de fructose GLUT5-dependent, associe 1) a une augmentation rapide de la glycemie et une potentialisation des effets fructose sur l’expression hepatique de SREBP-1c, ACC-1, FAS ; 2) a une augmentation des triglycerides circulants et une activation de la lipogenese hepatique. Conclusion Le fructose augmente la secretion de leptine dans la lumiere intestinale favorisant son absorption specifique par GLUT5, avec pour consequence une elevation de la glycemie et des lipides disponibles. Cette boucle de regulation positive peut renforcer le caractere lipogenique du fructose, et rendre compte des effets de la consommation excessive de ce sucre dans la pathogenie de la steatose hepatique associee a certaines obesites.

592 The Facilitative Glucose Transporter GLUT-2 Is Up-Regulated By Mucosal Leptin Through AMPK Involved Mechanisms in the Small Intestine

produce IL-10 in response to TLR signals and regulate IL-10 secretion during antigenspecific activation of naive CD4+ T cells. T cells expressing IL-10 were found to be a subset of Foxp3+ Tregs which emerged in the lamina propria during the recovery from colitis. Depletion of pDCs did not impact the recruitment of Foxp3+ Tregs, but impaired IL-10 secretion by Foxp3+ Tregs and delayed mucosal repair. The regulatory function of pDCs during mucosal inflammation also included the control of intestine-specific secretion of IL17 but not IFN-γ by CD4+Foxp3T cells. CONCLUSION: During colitis, pDCs become a major DC subset in the lamina propria and direct mucosa-specific T cell functions essential for the resolution of inflammation. Our results demonstrate that pDCs display a distinct function in the colon in defining intestine-specific adaptations of T effectors and Foxp3+ Tregs to mucosal inflammation.

Modulation of Dextran Sulphate Sodium-Induced Colonic Inflammation by Local Supplementation of Leptin~!2008-08-10~!2008-10-20~!2008-12-05~!

Background & Aims: Leptin is overproduced in gastrointestinal mucosa during inflammatory processes, sug- gesting that mucosal cells represent sources of secreted leptin active in the lumen. The effects of leptin, acting apically from colonic epithelial cells, were analysed on dextran sulphate sodium-induced colonic inflammation in rats. Methods: We determined the effects of intracolonic leptin on the phosphorylation of STAT3, MAPkinase and on colon mucosa-derived inflammation-related genes (IL-8, IL1ufffb, TNFufffb, COX-2). Colitis was induced by administering dextran sodium sulphate. The effects of intracolonic leptin on DSS colitis was evaluated based on disease symptoms, cytokine ex- pression and PPAR ufffb , ufffb . Results: In vivo, intracolonic leptin rapidly stimulated STAT-3 and p42-MAPK phosphorylation. We also detected a 3- fold increase in COX-2 induction, and a dose-dependent increase in mucosal PGE2 content (EC50 0.89 nM). Intracolonic leptin reduced the severity of DSS-induced colitis, whereas intraperitoneal leptin exacerbated colitis. Intracolonic leptin decreased DSS-induced inflammatory IL-8 (-75%; P<0.01 vs DSS) and IL-1ufffb (-60%; P<0.01 vs DSS); it also prevented a DSS-induced decrease in the levels of mucosa PPARufffb mRNA and increased the levels of PPARmRNA two-fold (P<0.01 vs DSS). Conclusion: Leptin activates its apical receptor and this mechanism coupled to the activation of STAT-3 and MAPKinase signalling pathways may have a beneficial effect on the integrity of the epithelium upon mucosa injury. These data shed further light on the role of gastrointestinal luminally acting leptin in the modulation of intestinal inflammation.

948–53 A New Natriuretic Factor Acting Like Loop Diuretic Drugs in Heart Failure. Relation to the Severity of Left Ventricular Dysfunction

We have previously found in urine from salt-loaded rats and in patients with heart failure, a new natriuretic factor which has, in part, the biological profile of loop diuretic drugs. In patients with congestive heart failure, it is not known whether the activation of this factor is related to the impairment in systolic contractility. The aim of this study was to determine the differences in Cotransport Inhibitory Factor (C.I.F.) response between patients with or without left ventricular dysfunction. Twenty one patients were included in this study (16 men and 5 women, mean age 53, range 38 to 72). All treatments were maintained except loop diuretics. Left ventricular ejection fraction (LVEF) was measured by contrast or isotopic angiography (range 14–74%). Plasma and urine C.I.F. levels were measured by potency of the samples to inhibit cotransport fluxes in Madin and Darby canine kidney (MDCK) cells, and in human erythrocytes. Cotransport inhibition in urine and plasma was correlated with LVEF as shown in figure (*and **xa0=xa0pxa0<xa00.0002). n n n n n n nFigure options n n n nDownload full-size image nDownload high-quality image (75 K) nDownload as PowerPoint slide n n n n n n n nThis study shows that the degree of C.I.F activation is related to the level of LV dysfunction and that besides ANF, C.I.F. could playa key role in diuresis and natriuresis control in congestive heart failure.