Carles Garriga

Hexose transport in the apical and basolateral membranes of enterocytes in chickens adapted to high and low NaCl intakes

1 The effect of a low‐NaCl diet (LS diet) on the properties of hexose transport across the brush‐border and basolateral membranes of enterocytes from jejunum, ileum and rectum of the chicken was investigated. 2 In the brush‐border membrane, LS adaptation had no effect on Km for α‐methyl‐d‐glucoside while Vmax values were significantly reduced in the ileum and in the rectum. All Scatchard plots of specific [3H]phlorizin binding give a straight line, consistent with a single population of binding sites. Phlorizin binding vs.α‐methyl‐d‐glucoside maximal transport rates showed a linear correlation. 3 In the basolateral membrane, the LS diet did not modify the Km for d‐glucose but reduced the Vmax in the ileum and in the rectum. Scatchard plots of [3H]cytochalasin B binding support the view that there is a single transport system in this membrane. There was a linear correlation between cytochalasin B binding and d‐glucose Vmax values. 4 The response of the chicken intestine to LS intake consists of a dramatic reduction in the number of glucose transporters in both apical and basolateral membranes of the rectum, an intermediate response in the ileum and no significant effects in the jejunum.

Expression of Na+-d-glucose cotransporter in brush-border membrane of the chicken intestine

We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564-575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 ( antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402-420 of the same protein ( antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only when antibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.

Effects of Resalination on Intestinal Glucose Transport in Chickens Adapted to Low Na+ Intakes

In chickens, we have shown that intestinal absorption of glucose via apical SGLT1 and basolateral GLUT2 transport systems is affected by dietary Na+; low‐Na+ adapted birds show a dramatic reduction of glucose transporters in both membranes in the rectum, an intermediate response in the ileum and no effects in the jejunum. We have now studied the effect of resalination of low‐Na+ adapted chickens on glucose kinetics across SGLT1 (using α‐methyl‐D‐glucoside as substrate) and GLUT2 (using D‐glucose) and on the specific binding of phlorizin and cytochalasin B, respectively. Twelve‐week‐old male Leghorn chickens were fed wheat and barley with drinking water containing either 150 mM NaCl (high‐Na+ group) or 0.015 mM (low‐Na+ group) for 14 days (serum aldosterone: 242 ± 6 pg ml‐1 in the low‐Na+ and 46 ± 4 pg ml‐1 in the high‐Na+ group). On day 14, the low‐Na+ group was either resalinated with an oral dose of NaCl (9 g (kg body wt)‐1) or switched to the high‐Na+ condition, for 1 week. Serum aldosterone measured 4 h, 1 day and 7 days after the change in NaCl intake fell to between 30 and 39 pg ml‐1. The changes in apical α‐methyl‐D‐glucoside and basolateral D‐glucose transport observed in the ileum and rectum of low‐Na+ adapted animals were completely reversed by resalination within 4 h of NaCl administration to the level of values observed for high‐Na+ adapted birds. The good correlation between the α‐methyl‐D‐glucoside and D‐glucose Vmax and the SGLT1 and GLUT2 density, respectively, supports the view that the increase in apical and basolateral hexose transport found in the ileum and rectum of both groups of resalinated birds is due to an increase in the number of protein transporters. The rapid changes in the number of glucose transporters observed suggest that the target of the regulatory signal(s) involved are the mature enterocytes present in the villi rather than the developing enterocytes in the crypt.

Aldosterone mediates the changes in hexose transport induced by low sodium intake in chicken distal intestine

1 In chickens, low Na+ diets markedly decrease the hexose transport in the rectal segment of the large intestine; transport in the ileum shows a lower, but significant reduction and transport in the jejunum is unaffected. These effects involve both apical (SGLT1) and basolateral (GLUT2) hexose transporters. 2 The role of the renin‐angiotensin‐aldosterone axis (RAAS) in the epithelial response to Na+ intake was studied in chickens fed high‐NaCl (HS) and low‐NaCl (LS) diets. The Vmax of α‐methyl‐D‐glucoside and D‐glucose were determined in vesicles from the brush‐border (BBMVs) and basolateral (BLMVs) membranes, respectively. The binding of phlorizin to BBMV and cytochalasin B to BLMV were used as indicators of the abundance of SGLT1 and GLUT2, respectively. 3 In HS‐adapted chickens, the serum concentration of aldosterone (means ± S.E.M.) was 35 ± 5 pg ml−1 (n= 6) and that of renin was 20 ± 2 ng ml−1 (n= 3). In LS‐fed birds, these values were 166 ± 12 pg ml−1 (n= 6) and 122 ± 5 ng ml−1 (n= 3), respectively. Administration of captopril, the inhibitor of the angiotensin‐converting enzyme (ACE), to LS‐chickens lowered the aldosterone serum concentration without affecting the renin concentration. Captopril also prevented the reduction of apical and basolateral hexose transport in ileum and rectum characteristic of the intestinal response to LS adaptation. 4 Administration of the aldosterone antagonist spironolactone to LS‐adapted chickens did not affect the serum concentrations of aldosterone, but prevented the effects of LS intake on hexose transport in both apical and basolateral membranes. This suggests that the effects of aldosterone are mediated by cytosolic mineralcorticoid receptors. 5 Administration of exogenous aldosterone to HS‐fed birds induced hexose transport and binding properties typical of the LS‐adapted animals. These findings support the view that aldosterone, besides its primary role in controlling intestinal Na+ absorption, can also modulate the expression of apical and basolateral glucose transporters in the chicken distal intestine.

Kinetic characterization of apical D-fructose transport in chicken jejunum

In mammals, D-fructose transport takes place across the brush-border membrane of the small intestine through GLUT5, a member of the facilitative glucose transporter family. In the present paper, we describe and characterize for the first time the apical transport of D-fructose in chicken intestine. Brush-border membrane vesicles (BBMV) were obtained from jejunum of 5- to 6-wk-old chickens. D-Fructose uptake by BBMV from chicken jejunum comprises a saturable component and a simple diffusion process. The maximal rate of transport (Vmax) for D-fructose was 2.49 nmol·(mg prot)−1·s−1, the Michaelis constant (Km) was 29 mM, and the diffusion constant (Kd) was 25 nl·(mg prot)−1·s−1. The apical transport of D-fructose was Na+-independent, phlorizin-, phloretin-, and cytochalasin B-insensitive, and did not show cis-inhibition by D-glucose or D-galactose. These properties, together with the detection of specific GLUT5 mRNA, indicate the presence of a low-affinity high-capacity GLUT5-type carrier in the chicken jejunum, responsible for the entry of D-fructose across the brush-border membrane of enterocytes.